阅读说明：本技术 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 (Frame structure-based nano-particles for mass flow cytometry and preparation method thereof ) 是由 陈缘 赵俊杰 罗英武 曾浔 于 2021-07-26 设计创作，主要内容包括：本发明公开了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,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。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。抗体通过接枝剂与有机配体或者连接子连接。本发明能够负载多种金属同位素,并且负载量高,能够在流式质谱检测中提供高灵敏度、可用金属同位素通道数多的金属检测信号,实现更多细胞参数同时测量的单细胞蛋白分析检测,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。(The invention discloses a frame structure-based nanoparticle for mass flow cytometry and a preparation method thereof, wherein the nanoparticle comprises metal ions, an antibody and a coordination network frame formed by connecting organic ligands with each other through a linker, wherein the metal ions are combined on the organic ligands. The linker is an organic linker with a functionality of n or a metal cluster with a coordination number of n, n being 2 or more. The antibody is linked to an organic ligand or linker by a grafting agent. The invention can load various metal isotopes, has high load capacity, can provide metal detection signals with high sensitivity and a plurality of available metal isotope channels in flow mass spectrometry detection, realizes single-cell protein analysis and detection with more cell parameters measured simultaneously, and provides an important analysis tool for accurate diagnosis of diseases at the gene level and research and development and detection of biomedicine.)

1. A framework-based nanoparticle for mass cytometry, wherein the nanoparticle comprises a metal ion, an antibody, and a coordination network framework formed by interconnecting organic ligands via linkers, wherein the metal ion is bound to the organic ligands. 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 linked to an organic ligand or linker by a grafting agent.

2. The nanoparticle of claim 1, wherein the organic ligand is composed of one or more of the following structures:

wherein, the substituent A is a functional group participating in connection to form a coordination network, and R1-R8 are other substituents.

3. The nanoparticle according to claim 1, wherein when the linker is a metal cluster, the A substituent of the organic ligand terminates with a carboxyl group, a catechol hydroxyl group, a pyridyl group, a pyrazinyl group, an imidazolyl group, a triazolyl group, a tetrazolyl group, a pyrazolyl group, or the like.

4. The nanoparticle according to claim 1, wherein the organic ligand comprises amino + aldehyde group, catechol hydroxyl + boronic acid group, amino + nitro group, aldehyde + o-phenylenediamine group, acid chloride + amino group, phthalic anhydride group + amino group, cyano + aldehyde group, o-quinonyl + o-phenylenediamine group, catechol hydroxyl + o-difluorophenyl group, and the like, in combination through the paired reactive groups at the end of the a substituent and the end of the organic linker.

5. The nanoparticle of claim 1, wherein the metal ion has an atomic weight of 75-210.

6. The nanoparticle of claim 1, wherein the grafting agent is a bifunctional linear molecule, one end of the head is bonded to a corresponding substituent group on an organic ligand or a linker through a click reaction, and the other end is bonded to an amino group on an antibody or a thiol group reduced by a disulfide bond through a click reaction.

7. The nanoparticle as claimed in claim 6, wherein 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, chlorosiloxane group, etc.

8. The nanoparticle of claim 7, wherein the grafting agent head is combined with an organic ligand or linker group as

Substituents of organic ligands or linkers Grafting agent head Amino group N-hydroxysuccinimide, pentafluorophenol ester, carboxyl and aldehyde group Mercapto group Maleimido, vinylsulfonyl Carboxyl group Amino group Azido group Alkynyl radical Alkynyl radical Azido group Hydroxy radical Chlorosiloxyalkyl Cl-Si (CH)₃)₂-

9. The nanoparticle of claim 1, wherein the coordination network framework has a diameter of 20-200 nm.

10. A method for preparing nanoparticles according to any one of claims 1 to 9, comprising the following steps:

(1) synthesizing a nano framework structure: mixing 1-30 parts by weight of organic ligand, 0.5-10 parts by weight of linker and 3000 parts by weight of organic solvent of 500-3000 parts by weight, heating to 35-150 ℃, stirring and reacting for 0.5-24 hours to obtain dispersion liquid containing a nano-framework structure, adding 0.1-1 part by weight of metal salt into the synthesized nano-framework dispersion liquid, heating to 35-120 ℃, stirring and reacting for 0.5-24 hours to obtain nano-framework material dispersion liquid containing metal;

or directly mixing 1-30 parts by weight of organic ligand containing metal ions, 0.5-10 parts by weight of linker and 3000 parts by weight of organic solvent, heating to 35-150 ℃, and stirring for reaction for 0.5-24 hours to obtain the metal-containing nano-framework material dispersion liquid.

(2) Linking the antibody: centrifuging dispersion liquid of the metal-containing nano-framework material to remove organic solvent, adding 20-100 parts by weight of deionized water for redispersion, adding 0.2-5 parts by weight of grafting agent into the aqueous dispersion liquid of the metal-containing nano-framework material, reacting at room temperature for 0.5-12 hours, dialyzing to separate free grafting agent, adding 0.01-0.1 part by weight of antibody, reacting at room temperature for 0.5-2 hours, and dialyzing to separate free antibody to obtain the nano-particles.

Technical Field

The invention relates to the technical field of cell biology, in particular to a nanoparticle material of a metal-bound framework structure for labeling an antibody based on a single-cell protein detection technology of flow-type binding ICP-MS.

Background

The Mass spectrometry flow technology (Mass Cytometry) integrates the principles of Mass spectrometry and flow Cytometry, and performs specific identification on cell surface antigen proteins through metal-labeled antibodies, so that multiparameter and high-flux qualitative analysis on the types and the quantities of the antigen proteins expressed by each cell is realized, and an important analysis tool is further provided for accurate diagnosis of diseases at the gene level and research and development and detection of biomedicine. The method inherits the characteristics of high-speed analysis of the traditional flow cytometer, and has the technical advantages of high resolution capability of mass spectrometry detection, diversified data processing and the like, and because the mass spectrometry cytometer has more parameters and no interference among channels compared with the traditional fluorescence flow cytometer, and does not need fluorescence compensation calculation, the method greatly expands the data output and detection range of a single sample, enables the data to be more comprehensive and the result to be more reliable, becomes a new direction of single-cell protein expression analysis, and is increasingly widely applied to the fields of clinical medicine and biological research.

However, the current flow mass spectrometry technology has the problems of few available metal isotope channels and insufficient sensitivity, and the main reason is that on one hand, the number of metal types which can be loaded by the existing commercial metal antibody label is less than 40 lanthanide metal isotopes, and one metal isotope corresponds to the combination of a labeled antibody and an antigen, so that cells with more characteristic protein types cannot meet the requirement of high-level multi-parameter detection; on the other hand, because the number of metal atoms of the existing single metal antibody label is only about 100, one antibody is combined with 2-4 metal labels, and the detection of a flow mass spectrometer needs to reach tens of thousands of metal atoms to have good sensitivity, the existing metal label only has good detection effect on cells with high protein expression (the number of certain protein antigens on the surface of one cell reaches hundreds), and the detection of the label of the antigen protein with low expression is limited.

Disclosure of Invention

The invention aims to provide novel metal antibody labeled nanoparticles for mass spectrometry flow cytometry and a preparation method thereof, wherein the nanoparticles are nanoparticles with a framework structure of surface grafted antibodies and combined metal elements, metal elements other than lanthanide can be combined at present, and the metal ion load capacity of a single nanoparticle can reach 10³-10⁴And the problems that the number of available metal channels of the existing commercial metal antibody label is small and the sensitivity of a single metal label is not high enough are solved, and the number of detection channels is expanded to 135 channels with the atomic weight of 75-210.

The technical scheme adopted by the invention is as follows:

a framework-based nanoparticle capable of linking antibodies for use in mass cytometry comprising a metal ion, an antibody and a coordination network framework formed by interconnecting organic ligands via linkers, wherein the metal ion is bound to the organic ligands. 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 linked to an organic ligand or linker by a grafting agent.

The coordination network framework of the nano-particle is a porous network formed by self-assembling an organic ligand and a linker through covalent bonds or coordination bonds, each structural unit has a functional group capable of being combined with metal, the number of the structural units can ensure high loading capacity of metal atoms, and the variability of the structural units can be suitable for loading various different types of metal elements in the framework; in addition, the surface of the framework can be grafted by chemically modifying the grafting functional group to form a bond with a corresponding group on the antibody.

Further, the organic ligand is formed by mixing one or more structures in the following structures:

the organic ligand is sequentially of structures such as o-dihydroxybenzol, bipyridine, 2' -diphenol hydroxyl-biphenyl, disalicylaldehyde diamine, porphyrin, dehydro triphenylcyclo-olefin and the like, wherein A substituent groups participate in connection to form a coordination network, and R1-R8 are other substituent groups.

When the linker is a metal cluster, the terminal of the substituent a 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, a pyrazolyl group, or the like.

Specifically, the following structure is preferred:

further, the combination of the terminal of the substituent A of the organic ligand and the paired reaction group at the terminal of the organic linker comprises amino + aldehyde group, catechol hydroxyl group + boric acid group, amino + nitro group, aldehyde group + o-phenylenediamine group, acid chloride group (-COCl) + amino group, phthalic anhydride group + amino group, cyano group + aldehyde group, catechol group + o-phenylenediamine group, catechol hydroxyl group + o-difluorophenyl group and the like.

Wherein the structure of the phthalic anhydride group, the o-benzoquinone group, the o-diaminobenzene group and the o-difluorophenyl group is as follows:

specific group combinations and bonding connection forms are shown in table 1, and the combination forms shown in table 1 can form a stable nano framework structure.

TABLE 1 reactive group pairing of the A substituent end of the organic ligand with the linker end

Further, the atomic weight of the metal ion is 75-210.

Furthermore, the grafting agent is a bifunctional linear structure molecule, one end of the head part is combined with a corresponding substituent group on an organic ligand or a linker through click reaction to form a bond, and the other end of the head part is combined with an amino group on an antibody or a sulfhydryl group reduced by a disulfide bond through click reaction to form a bond. The click reaction is a reaction with mild reaction conditions and high reaction efficiency in a short time,

further, a group at one end of the tail of the grafting agent is an N-hydroxysuccinimide group, a pentafluorophenol ester group, a carboxyl group, an aldehyde group, a maleimide group, a vinyl sulfone group and the like. Wherein, the structures of the N-hydroxysuccinimide group, the pentafluorophenol ester group and the maleimide group and the vinyl sulfone group are as follows:

further, the grafting agent head in combination with the group of organic ligands or linkers is shown in table 2.

TABLE 2 group combinations of the grafting agent head and framework surface click reactions

Substituents of organic ligands or linkers	Grafting agent head
		Amino group	N-hydroxysuccinimide, pentafluorophenol ester, carboxyl and aldehyde group
Mercapto group	Maleimido, vinylsulfonyl
		Carboxyl group	Amino group
Azido group	Alkynyl radical
		Alkynyl radical	Azido group
Hydroxy radical	Chlorosiloxyalkyl Cl-Si (CH)3)2-

The combination of groups of the grafting reagent head and the antibody click reaction is shown in Table 3

TABLE 3 grafting agent tails in combination with groups for antibody click reactions

Antibody groups	Grafting agent tail group
		Amino group	N-hydroxysuccinimide, pentafluorophenol ester, carboxyl and aldehyde group
Mercapto group	Maleimido, vinylsulfonyl

Further, the diameter of the coordination network framework is 20-200 nm.

The invention prepares the frame structure nano-particle material which can be combined with metal through rational design and synthesis, and the nano-particle material is used as a metal marker of an antibody and a marking material for single-cell protein detection of flow mass spectrometry.

The invention also provides a preparation method of the nano-particles, which comprises the following steps:

(1) synthesizing a nano framework structure: mixing 1-30 parts by weight of organic ligand, 0.5-10 parts by weight of linker and 3000 parts by weight of organic solvent of 500-3000 parts by weight, heating to 35-150 ℃, stirring and reacting for 0.5-24 hours to obtain dispersion liquid containing a nano-framework structure, adding 0.1-1 part by weight of metal salt into the synthesized nano-framework dispersion liquid, heating to 35-120 ℃, stirring and reacting for 0.5-24 hours to obtain nano-framework material dispersion liquid containing metal;

or directly mixing 1-30 parts by weight of organic ligand containing metal ions, 0.5-10 parts by weight of linker and 3000 parts by weight of organic solvent, heating to 35-150 ℃, and stirring for reaction for 0.5-24 hours to obtain the metal-containing nano-framework material dispersion liquid. Wherein the organic solvent is a reagent capable of dissolving the organic ligand and the linker participating in the reaction.

(2) Linking the antibody: centrifuging dispersion liquid of the metal-containing nano-framework material to remove organic solvent, adding 20-100 parts by weight of deionized water for redispersion, adding 0.2-5 parts by weight of grafting agent into the aqueous dispersion liquid of the metal-containing nano-framework material, reacting at room temperature for 0.5-12 hours, dialyzing to separate free grafting agent, adding 0.01-0.1 part by weight of antibody, reacting at room temperature for 0.5-2 hours, and dialyzing to separate free antibody to obtain the nano-particles.

The invention has the advantages that:

1. high loading capacity: through the metal loaded by the nano-frame, compared with the existing metal carrier of the polymer linear chain, the metal loading capacity can be greatly improved, and the detection sensitivity is further improved. At present, a commercial polymer antibody label can only load about 100 metal ions on one chain, and one antibody is averagely connected with 3 chains, according to the detection limit of flow mass spectrometry, signals of at least ten thousand metal ions are required on one cell, and the labeling effect can be realized only by combining at least one hundred antibodies corresponding to one cell, which means that the detection of low-expression protein is greatly limited; after the three-dimensional framework structure greatly improves the metal loading, the metal loading of a single label is improved by at least one order of magnitude, the same sensitivity can be obtained for a cell combined with more than ten or even less framework structure antibody labels, and convenience is provided for the detection of the expression of low-abundance protein.

2. Facilitating the loading of a wider variety of metalloids: the frame material prepared by the invention has the advantages that firstly, the type combination of the frame structure is flexible and changeable, and the structural unit containing proper functional groups can be adopted aiming at different metal designs, so that the frame material is suitable for various metal elements; secondly, because the metal is combined in the frame structure, cross-linking points cannot be formed between the nano particles, thereby avoiding the problem of cross-linking aggregation caused by the combination of metal carriers of the existing polymer linear chains and certain metal ions.

In conclusion, the structure of the internal organic ligand of the frame structure nanoparticle has strong binding capacity for various metal elements, so that the metal loading capacity can be greatly increased to improve the signal sensitivity, and on the other hand, different metal isotopes, especially metal elements except lanthanide series, can be conveniently loaded by one frame structure, so that the existing available detection channels of the metal isotopes can be greatly expanded, and a key new material is provided for the multichannel high-precision detection of the flow mass spectrum single-cell protein.

Drawings

FIG. 1 is a schematic diagram of a process for preparing a metal antibody tag capable of binding to a metal framework structure according to the present invention;

FIG. 2 is a schematic diagram of an organic ligand-bound metal of a phthalic acid dimercapto structure;

FIG. 3 is a schematic diagram of an organic ligand-bound metal of bipyridine structure;

FIG. 4 is a schematic representation of an organic ligand-bound metal of the 2,2' -biphenol hydroxy-biphenyl structure;

FIG. 5 is a schematic diagram of organic ligand-bound metal of disalicylaldehyde diethylamide structure;

FIG. 6 is a schematic diagram of an organic ligand-bound metal of porphyrin structure;

FIG. 7 is a schematic diagram of an organic ligand-bound metal of a dehydrohexahydrotriphenylcycloalkene structure;

FIG. 8 is a schematic diagram of the chemical structure of the repeating structural units of the organic ligand, grafting agent, framework nanoparticle of example 1;

FIG. 9 is a schematic diagram of the chemical structure of the repeating structural units of the organic ligand, grafting agent, framework nanoparticle of example 2;

FIG. 10 is a schematic chemical structure diagram of the repeating structural units of the organic ligand, grafting agent, framework nanoparticle of example 3;

FIG. 11 is a schematic chemical structure diagram of the repeating structural units of the organic ligand, linker, grafting agent, framework nanoparticle of example 4;

FIG. 12 is a schematic chemical structure diagram of the repeating structural units of the organic ligand, linker, grafting agent, framework nanoparticle of example 5;

FIG. 13 is a schematic chemical structure diagram of the repeating structural units of the organic ligand, linker, grafting agent, framework nanoparticle of example 6;

FIG. 14 is a graph showing the effect of the Pt-195 labeled antibody PD-1 on staining and clustering of both yin and yang cells in example 1;

FIG. 15 is a graph showing the effect of Sn-120-labeled antibody CD-4 on staining and clustering of both yin and yang cells in example 2;

FIG. 16 is a graph showing the effect of Zr-90-labeled antibody lgD on staining and clustering of both yin and yang cells in example 3;

FIG. 17 is a graph showing effects of staining groups of yin and yang cells by ICOS, an antibody labeled with Tm-169, in example 4;

FIG. 18 is a graph showing the effect of Pd-104-labeled antibody TCR Va7.2 on staining and clustering of yin and yang cells in example 5;

FIG. 19 is a graph showing the effect of the Yb-171 labeled antibody CCR7 on staining and clustering of both the positive and negative cells of example 6.

Detailed Description

The metal element-containing nano-particle material with the framework structure can firstly change the structural units of the framework nano-particles flexiblyThe functional group realizes the effective loading of various metal isotopes, so that the available detection channels are expanded to the mass spectrum detectable range, namely over one hundred isotope channels with the molecular weight of 75-210, and the higher-level multi-parameter synchronous labeling detection is realized. Meanwhile, the coupled antibody is chemically modified on the surface, so that the antibody can be grafted efficiently and firmly, and the specific recognition of the target cell group by the antibody is not influenced. In addition, the number of structural units can be adjusted by controlling the size of the nano-particles, so that the number of metal ions loaded by one nano-particle can be adjusted to 10³-10⁴And the metal signal intensity of a single metal label is greatly improved, and the sensitive detection of the combined low-expression antigen protein label on the cell is favorably realized.

Specifically, the nano-framework material provided by the invention is synthesized by a two-step method, as shown in fig. 1, the first step is that an organic ligand and a linker are coordinated and self-assembled to form a framework structure, and then metal is loaded, or the first step is that the organic ligand containing metal and the linker are directly self-assembled to obtain the framework structure; and the second step is to introduce grafting agent to the surface of the frame structure and graft antibody to the end of the grafting agent to obtain the nanometer particle as the metal antibody label.

Wherein the organic ligand in the first step, the part of which mainly binds to the metal is shown in FIGS. 2-7, and the end of the A substituent is bonded to the end group of the linker in various combinations as shown in Table 1, thereby obtaining various self-assembled framework structures; and the metal ions may be all metals having an atomic weight of 75-210.

And in the second step, a grafting agent is introduced to the surface of the framework, the grafting agent is bonded through click reaction between a head group of the grafting agent and an organic ligand substituent group or a substituent group of a linker on the framework, and then a tail group of the grafting agent is bonded through click reaction with a group on the antibody, so that the metal-bonded and antibody-connected nano framework material is finally obtained. There are also many combinations of two-step click-reacted groups, as shown in Table 2, which can provide a wide variety of ligation strategies.

The invention will be further illustrated with reference to the following specific examples and the accompanying drawings:

example 1

Based on a frame material (1) of o-benzene dimercapto combined metal, organic ligands of 2, 3-dimercapto-6-azido-1, 4-terephthalic acid and a linker of a zirconium metal cluster are coordinated and self-assembled to form frame structure nano particles, and PtCl is used₄As the metal reagent, Pt is loaded in a frame, and CH ≡ C-CH is used₂-CH₂-O-CH₂And the-MAH is used as a grafting agent and is bonded and connected with the azido group on the organic ligand substituent through a head group carbon-carbon triple bond, and is bonded and connected with the sulfhydryl group obtained by reducing a disulfide bond on the antibody through the-MAH of a tail group. The organic ligand, grafting agent and repeating structural unit for obtaining the framework nano-particle are shown in figure 8. The preparation method comprises the following steps:

1) adding 50mg of 2, 3-dimercapto-6-azido-1, 4-terephthalic acid and 150mg of zirconium octahydrate into 50g of DMF solution, fully stirring and dissolving, heating to 90 ℃, reacting for 2 hours, centrifuging the mixed solution at 15000Xg for 30min, and removing supernatant by suction to obtain the frame-structured nanoparticles.

2) Adding 5g DMF, resuspending, dispersing, centrifuging, collecting the nanoparticles, and adding 500 μ L PtCl with concentration of 20mg/ml₄Heating the DMF solution to 120 ℃, reacting for 24 hours, centrifuging the mixed solution at 15000Xg for 20min, and absorbing and removing supernatant to obtain the metal Pt-loaded framework structure nano-particles.

3) Adding 2g of deionized water to resuspend and disperse the collected Pt-loaded frame structure nanoparticles, dialyzing in 400ml of deionized water for 3 times by using a dialysis bag with the molecular weight cutoff of 300KD, wherein each time lasts for at least 2 hours, and completely separating free organic solvent molecules and metal ions; 10mg of the grafting agent CH ≡ C-CH are then added₂-CH₂-O-CH₂MAH, reacting for 1 hour at room temperature after fully mixing and dissolving; dialyzing with dialysis bag with molecular weight cut-off of 300KD in 400ml deionized water for 3 times, each time for at least 2 hours; the reduction of the disulfide bonds of the antibodies was prepared at the same time as the dialysis: mu.g of the antibody was reacted in PBS buffer for 0.5 hour by adding 100. mu.L of 4mM TCEP, and then centrifuged in a 3KD stop tube 12000Xg for 30min to separate TCEP, followed by addition of 300Kmu.L of PBS buffer was resuspended.

4) Mixing the framework structure nanoparticles which are dialyzed to remove the redundant grafting agent and the antibody which reduces the disulfide bond, reacting for 1 hour at room temperature, finally dialyzing for 3 times in 400ml of deionized water by using a dialysis bag with the molecular weight cutoff of 300KD, separating free antibody for at least 2 hours each time, and finally obtaining the Pt-loaded framework structure nanoparticles connected with the antibody.

Example 2

Based on a bipyridine combined metal framework material (2), 2 '-bipyridine-5, 5' -p-dibenzoic acid and a linker of a zirconium metal cluster are coordinated and self-assembled to form a framework structure nano particle, and SnCl is used₂·2H₂O as a metal reagent to support Sn in a frame, Cl-Si (CH)₃)₂-CH₂-CH₂-O-CH₂-NHS as grafting agent via the head group Cl-Si (CH)₃)₂Bonding connection with a hydroxyl group on the zirconium metal cluster, and bonding connection with an amino group on the antibody through a-NHS group of the tail group. The organic ligand, grafting agent and repeating structural unit for obtaining the framework nano-particle are shown in figure 9. The preparation method comprises the following steps:

1) adding 30mg of 2,2 '-bipyridyl-5, 5' -p-dibenzoic acid and 150mg of zirconium oxychloride octahydrate into 50g of DMF solution, fully stirring and dissolving, heating to 90 ℃, reacting for 5 hours, centrifuging the mixed solution at 15000Xg for 30min, and sucking and removing supernatant to obtain the frame-structured nanoparticles.

2) Adding 5g DMF, resuspending, dispersing, centrifuging, collecting the nanoparticles, adding 500 μ L SnCl with concentration of 20mg/ml₂.2H₂And heating the DMF solution of O to 35 ℃, reacting for 0.5 hour, centrifuging the mixed solution for 20min at 15000Xg, and absorbing and removing supernatant to obtain the metal Sn loaded framework structure nano-particles.

3) Adding 2g of deionized water to resuspend and disperse the collected Sn-loaded frame structure nanoparticles, dialyzing in 400ml of deionized water for 3 times by using a dialysis bag with the molecular weight cutoff of 300KD, wherein each time lasts for at least 2 hours, and completely separating free organic solvent molecules and metal ions; 10mg of the grafting agent Cl-Si (CH) are then added₃)₂-CH₂-CH₂-O-CH₂NHS, fully mixing and dissolving, and reacting at room temperature for 12 hours; then dialyzing in 400ml deionized water for 3 times with a dialysis bag with molecular weight cut-off of 300KD for at least 2 hours each time; .

4) Mixing the framework structure nanoparticles which are dialyzed to remove the redundant grafting agent and 100 mu g of antibody in 100 mu L of PBS buffer, reacting for 1 hour at room temperature, finally dialyzing for 3 times in 400ml of deionized water by using a dialysis bag with the molecular weight cutoff of 300KD, separating free antibody for at least 2 hours each time, and finally obtaining the Sn-loaded framework structure nanoparticles connected with the antibody.

Example 3

The 2,2' -dihydroxyl hydroxyl-biphenyl combined metal-based framework material (3) is formed by the coordination self-assembly of 2, 2-dihydroxyl-5, 5-dialkynyl-1, 1 ' -biphenyl-4, 4 ' -bipyridine and zinc ions to form nanoparticles with a framework structure, and ZrCl is used₄As a metal reagent, Zr was supported on a frame, and N was used₃-CH₂-CH₂-O-CH₂NHS as grafting agent, the amino group of the antibody is bonded and connected with the carbon-carbon triple bond of the organic ligand substituent through the head group azido group and the amino group of the antibody through the N-hydroxysuccinimide group of the tail group. The organic ligand, grafting agent and repeating structural unit for obtaining the framework nano-particle are shown in figure 10. The preparation method comprises the following steps:

1) adding 50mg of 2, 2-dihydroxyl-5, 5-dialkynyl-1, 1 '-biphenyl-4, 4' -bipyridine and 150mg of zinc nitrate into 50ml of methanol solution, fully stirring and dissolving, heating to 35 ℃, reacting for 0.5 hour, centrifuging the mixed solution at 15000Xg for 30min, and removing supernatant by suction to obtain the frame-structured nanoparticles.

2) Adding 5g DMSO to resuspend, disperse and centrifuge the collected nanoparticles, and adding 500 μ L ZrCl with concentration of 20mg/ml₄Heating the DMF solution to 45 ℃, reacting for 12 hours, centrifuging the mixed solution for 20min at 15000Xg, and absorbing and removing supernatant to obtain the metal Zr-loaded framework structure nano-particles.

3) Zr-loaded framework structure sodium obtained by adding 2g of deionized water for heavy suspension and dispersion collectionDialyzing the rice particles in 400ml of deionized water for 3 times with a dialysis bag with the molecular weight cutoff of 300KD for at least 2 hours each time to completely separate free organic solvent molecules and metal ions; then 10mg of grafting agent N are added₃-CH₂-CH₂-O-CH₂NHS, fully mixing and dissolving, and reacting at room temperature for 2 hours; then dialyzing in 400ml deionized water for 3 times with a dialysis bag with molecular weight cut-off of 300KD for at least 2 hours each time;

mixing the framework structure nanoparticles which are dialyzed to remove the redundant grafting agent and 100 mu g of antibody in 100 mu L of PBS buffer, reacting for 1 hour at room temperature, finally dialyzing for 3 times in 400ml of deionized water by using a dialysis bag with the molecular weight cutoff of 300KD, separating free antibody for at least 2 hours each time, and finally obtaining the Zr-loaded framework structure nanoparticles connected with the antibody.

Example 4

The frame material (4) based on disalicylaldehyde diamine combined metal is prepared by self-assembling tetra (3-aldehyde-4-phenolic hydroxyl phenyl) methane and 4-mercapto-1, 2 diphenylamine through condensation reaction of aldehyde and amino to obtain nano particles with a frame structure, and TmCl is used₃Tm is loaded in a frame as a metal reagent, and MAH-CH is used₂-CH₂-O-CH₂-COOH as grafting agent, through head group-MAH to bond with sulfhydryl on the substituent of linker, and through carboxyl of tail group to bond with amino of antibody under catalysis of N, N' -diisopropylcarbodiimide. The organic ligand, linker, grafting agent used, and the repeating structural unit to obtain the framework nanoparticle are shown in fig. 11. The preparation method comprises the following steps:

1) adding 50mg of tetra (3-aldehyde-4-phenolic hydroxyl phenyl) methane and 120mg of 4-mercapto-1, 2 diphenylamine into 100g of DMF solution, fully stirring and dissolving, heating to 120 ℃, reacting for 12 hours, centrifuging the mixed solution at 15000Xg for 30min, and sucking and removing supernatant to obtain the nano-particles with the framework structure.

2) Adding 5g of water to resuspend, disperse and centrifuge the collected nanoparticles, and adding 500 mu L of TmCl with the concentration of 20mg/ml₃Heating the aqueous solution to 60 ℃, reacting for 6 hours, and centrifuging the mixed solution at 15000Xg for 20mAnd in, absorbing and removing the supernatant to obtain the frame structure nano-particles loaded with the metal Tm.

3) Adding 2g of deionized water to resuspend and disperse the collected Tm-loaded frame structure nanoparticles, dialyzing in 400ml of deionized water for 3 times by using a dialysis bag with the molecular weight cutoff of 300KD, wherein each time lasts for at least 2 hours, and completely separating free organic solvent molecules and metal ions; then 10mg of grafting agent MAH-CH is added₂-CH₂-O-CH₂-COOH, after thoroughly mixing and dissolving, reacting at room temperature for 1 hour; then dialyzed in 400ml of deionized water for 3 times, each for at least 2 hours, using a dialysis bag having a molecular weight cut-off of 300 kD.

Mixing the framework structure nanoparticles which are dialyzed to remove the redundant grafting agent and 100 mu g of antibody in 100 mu L of PBS buffer, adding 2 mu L of 0.2M DMSO solution of N, N' -diisopropylcarbodiimide, uniformly mixing, reacting at room temperature for 2 hours, finally dialyzing in 400ml deionized water for 3 times with at least 2 hours each time by using a dialysis bag with the molecular weight cut-off of 300KD, separating free antibody, and finally obtaining the Tm-loaded framework structure nanoparticles connected with the antibody.

Example 5

The framework material (5) based on porphyrin-bonded metal is prepared by self-assembling palladium-supported 5,10,15, 20-tetra (4-aminophenyl) porphyrin and tetra (3-alkynyl-4-aldehyde phenyl) methane to obtain nanoparticles with a framework structure, and Pd (NO) is used₃)₃Pd supported on a frame as a metal reagent, using N₃-CH₂-CH₂-O-CH₂And the-MAH is used as a grafting agent, is bonded and connected with a carbon-carbon triple bond on a substituent of a linker through a head group azido group, and is bonded and connected with a sulfhydryl group obtained by reducing a disulfide bond on an antibody through a tail group-MAH. The organic ligand, linker, grafting agent used, and the repeating structural unit to obtain the framework nanoparticle are shown in fig. 12. The preparation method comprises the following steps:

1) adding 50mg of palladium-supported 5,10,15, 20-tetra (4-aminophenyl) porphyrin and 120mg of tetra (3-alkynyl-4-aldehyde phenyl) methane into 100ml of DMF solution, fully stirring and dissolving, heating to 150 ℃, reacting for 24 hours, centrifuging the mixed solution at 15000Xg for 30min, and removing the supernatant by suction to obtain the metal Pd-supported nano-particles with the framework structure.

2) Adding 2g of deionized water to resuspend and disperse the collected Pd-loaded nano particles with the frame structure, dialyzing the nano particles with the cut-off molecular weight of 300KD for 3 times in 400ml of deionized water for at least 2 hours each time, and completely separating free organic solvent molecules and metal ions; 10mg of grafting agent N are then added₃-CH₂-CH₂-O-CH₂MAH, reacting for 1 hour at room temperature after fully mixing and dissolving; then dialyzing in 400ml deionized water for 3 times with a dialysis bag with molecular weight cut-off of 300KD for at least 2 hours each time; the reduction of disulfide bonds of the antibodies was prepared simultaneously with dialysis, 100. mu.l of 4mM TCEP was added to 100. mu.g of the antibodies and reacted for half an hour in PBS buffer, and then the TCEP was separated by centrifugation in 12000Xg of a 3KD retention tube for 30min and resuspended in 300. mu.l PBS buffer.

Mixing the framework structure nanoparticles which are dialyzed to remove the redundant grafting agent and the antibodies which reduce the disulfide bonds, reacting for 1 hour at room temperature, finally dialyzing for 3 times in 400ml of deionized water by using a dialysis bag with the molecular weight cutoff of 300KD, separating free antibodies for at least 2 hours each time, and finally obtaining the Tm-loaded framework structure nanoparticles connected with the antibodies.

Example 6

The framework material (6) based on dehydrohexahydrotriphenylcycloalkene combined metal is prepared by self-assembling Dehydrobenzocyclopentene (DBA) and tetra- (3-mercapto-4-dihydroxyborophenyl) methane (TBPM) through condensation reaction of phenolic hydroxyl and boric acid group to obtain nanoparticles with a framework structure, and YbCl is used₃As a metal reagent, Yb was supported on a frame, and MAH-CH was used₂-CH₂-O-CH₂the-NHS is used as a grafting agent and is bonded and connected with a substituent sulfhydryl group on a linker through an-MAH group of a head group and is bonded and connected with an amino group on an antibody through an epoxy group of a tail group. The organic ligand, linker, grafting agent used, and the repeating structural unit to obtain the framework nanoparticle are shown in fig. 13. The preparation method comprises the following steps:

1) adding 50mg of Dehydrobenzocyclopentene (DBA) and 100mg of tetra- (2-mercapto-4-dihydroxyborylphenyl) methane (TBPM) into 150g of DMF solution, fully stirring and dissolving, heating to 150 ℃, reacting for 24 hours, centrifuging the mixed solution at 15000Xg for 30min, and sucking and removing supernatant to obtain the nano-particles with the framework structure.

2) Adding 5g of water to resuspend, disperse and centrifuge the collected nanoparticles, and adding 500 mu L of YbCl with the concentration of 20mg/ml₃Heating the aqueous solution to 60 ℃, reacting for 6 hours, centrifuging the mixed solution at 15000Xg for 20min, and absorbing and removing supernatant to obtain the metal Yb-loaded framework-structured nano-particles.

3) Adding 2g of deionized water to resuspend and disperse the collected Yb-loaded nano particles with the frame structure, dialyzing the Yb-loaded nano particles in 400ml of deionized water for 3 times by using a dialysis bag with the molecular weight cutoff of 300KD, wherein each time lasts for at least 2 hours, and completely separating free organic solvent molecules and metal ions; then 10mg of grafting agent MAH-CH is added₂-CH₂-O-CH₂NHS, fully mixing and dissolving, and reacting at room temperature for 1 hour; then dialyzed in 400ml of deionized water for 3 times, each for at least 2 hours, using a dialysis bag having a molecular weight cut-off of 300 kD.

4) Mixing the framework structure nanoparticles which are dialyzed to remove the redundant grafting agent and 100 mu g of antibody in 100 mu L of PBS buffer, reacting for 1 hour at room temperature, finally dialyzing for 3 times in 400ml of deionized water by using a dialysis bag with the molecular weight cutoff of 300KD, wherein each time lasts for at least 2 hours, separating free antibody, and finally obtaining the Yb-loaded framework structure nanoparticles connected with the antibody.

The particle size and particle size distribution of the nanoparticles obtained in examples 1 to 6 were measured by a nanoparticle size potential analyzer Zetasizer Nano-ZS, and the aqueous dispersion of the nanoparticles obtained in examples 1 to 6 was left to stand for six months, and the presence or absence of aggregation and sedimentation of the aqueous dispersion was observed to determine whether the stability was good or not, and the results are shown in table 4 and show: the nanoparticles at each particle size have a narrow size distribution and good dispersion stability.

Table 4 particle size, distribution and dispersion stability of the frame-structured nanoparticles

Examples	Average particle diameter/nm	Particle size distribution (Cv/%)	Stability of dispersion
				1	100	6％	Good taste
2	40	8％	Good taste
				3	60	7％	Good taste
4	80	6％	Good taste
				5	20	10％	Good taste
6	200	8％	Good taste

The metal ion concentration in the sample solution was measured by ICP-MS, and the metal loading of individual nanoparticles was calculated, the results are shown in table 5, which indicates that the nanoparticles can successfully load the target metal ion and increase the loading number to 10 by increasing the particle size³-10⁴And (4) respectively.

Table 5 metal loading of nanoparticle metal antibody tags of framework structure

Using 6 isotope detection channels with 6 extracellular antibodies in table 6, the signal was detected by the pair 1: 1 the mixed cells were subjected to marker staining to detect the clustering effect.

TABLE 6 Experimental groups for Metal antibody tag labeling in conjunction with cell staining

Examples	Antibodies	Clone number	Marking metal isotopes
				1	PD-1	EH12.2H7	Pt-195
2	CD4	RPA-T4	Sn-120
				3	lgD	lA6-2	Zr-90
4	TCR Va7.2	3C10	Tm-169
				5	ICOS	C398.4A	Pd-104
6	CCR7	G043H7	Yb-171

The labeled cell staining method is as follows:

1. fresh normal human peripheral blood is prepared and immune cells are extracted.

2. The immune cells are divided equally into two groups, each of about 2 x 10^6 cells, a control group of 2 x 10^6 negative cells not expressing the target protein, and an experimental group of 1 x 10^6 positive cells expressing the target protein mixed with 1 x 10^6 negative cells not expressing the target protein. Resuspend with PBS respectively, adjust the volume to 1mL, add Rh-103, stain for 5min at room temperature, differentiate cell death and viability.

3. 2mL of a 0.5-5mg/mL bovine serum albumin solution was added to each group, centrifuged at 500Xg for 5min, the supernatant was aspirated, and 50. mu.L of a blocking solution (0.5. mu.L of a 0.5-1.5mg/mL human immunoglobulin solution, 0.5. mu.L of a 0.5-1.5mg/mL mouse immunoglobulin solution, 0.5. mu.L of a 0.5-1.5mg/mL rat immunoglobulin solution, 0.5. mu.L of a 0.5-1.5mg/mL hamster immunoglobulin solution, 48. mu.L of a 0.5-5mg/mL bovine serum albumin solution) was added thereto and blocked on ice for 20 min.

4. The control group was added with 50. mu.L of 0.5-5mg/ml bovine serum albumin solution as a blank control, and the experimental group was added with 50. mu.L of extracellular antibody mixture (the nanoparticles of the present invention, with an antibody concentration of 0.1-1mg/ml), resuspended cells, and stained on ice for 30 min.

5. Adding 2ml of 0.5-5mg/ml bovine serum albumin solution, centrifuging at 500Xg for 5min, sucking out the supernatant, adding 1ml of fixed-membrane rupture mixed liquor (fix and perm buffer, fluidigm, 201067) containing 0.5v/v ‰ single cell indicator 191/193Ir (201192B, fluidigm), resuspending the cells, and standing at 4 ℃ overnight.

6. Adding 2ml of 0.5-5mg/ml bovine serum albumin solution, centrifuging at 800Xg for 5min, sucking off the supernatant, and repeating for 2 times.

7.2 mL of deionized water was added, centrifuged at 800Xg for 5min, the supernatant aspirated, and repeated 2 times.

8. And filtering the sample, counting cells, adjusting the volume, preparing a machine, and performing mass spectrometry flow detection.

The results of the staining effect of the 6 kinds of antibodies are shown in FIGS. 14 to 19, and the abscissa Ir191 represents the case of staining DNA, which is confirmed to be a cell signal; the ordinate is the corresponding isotope signal intensity, and the intensity of the ordinate of the background signal is obtained according to the control group, so as to obtain the boundary line of the yin and yang cells, namely the percentage of the positive cell population of each antibody in the total number of the cells is circled in the frame. Compared with a control group, the immune cells added with the antibody are divided into a negative group and a positive group, which proves that the antibody marking effect is good, the target cells and the non-target cells can be separated, and the antibody can be used for subsequent experiments.

The above examples are only for illustrating the present invention, but are not intended to limit the scope of the present invention.