阅读说明：本技术 基于稀土掺杂纳米金属有机骨架材料的金属标签 (Metal label based on rare earth doped nano metal organic framework material ) 是由 刘志周 何良 王彤 南雪燕 白鹏利 王辉 胡玮 于 2020-12-28 设计创作，主要内容包括：本发明公开了一种基于稀土掺杂纳米金属有机骨架材料的金属标签,该金属标签通过以下方法制备得到：1)制备稀土金属配位物,其结构如下式(Ⅰ)所示：2)制备多孔结构的Ln@ZIF-8纳米颗粒；3)制备Ln@ZIF-8@SiO-2；4)制备NH-2-Ln@ZIF-8@SiO-2；5)制备PEG-Ln@ZIF-8@SiO-2。本发明能将单个颗粒稀土含量提高到10～5-10～6,并进一步在其表面进行SiO-2包覆,提高了稳定性,并进一步采用PEG进行表面修饰,研制成为PEG-Ln@ZIF-8@SiO-2复合纳米颗粒,在可以被质谱流式灵敏检测到的同时还具备良好的生物相容性,合成工艺操作简单。(The invention discloses a metal tag based on a rare earth doped nano metal organic framework material, which is prepared by the following method: 1) preparing a rare earth metal complex having the structure shown in formula (I): 2) preparing Ln @ ZIF-8 nano particles with porous structures; 3) preparation of Ln @ ZIF-8@ SiO 2 (ii) a 4) Preparation of NH 2 ‑Ln@ZIF‑8@SiO 2 (ii) a 5) Preparation of PEG-Ln @ ZIF-8@ SiO 2 . The invention can increase the rare earth content of single particle to 10 5 ‑10 6 And further carrying out SiO on the surface 2 Coating, improving the stability, further adopting PEG to carry out surface modification, and developing into PEG-Ln @ ZIF-8@ SiO 2 The composite nano-particles can be sensitively detected by mass spectrum flow, and simultaneously have good biocompatibility, and the synthesis process is simple to operate.)

1. A metal label based on a rare earth doped nano metal organic framework material is characterized by being prepared by the following method:

1) preparing a rare earth metal complex having the structure shown in formula (I):

2) preparing Ln @ ZIF-8 nano particles with porous structures by using the rare earth metal coordination compound prepared in the step 1);

3) the Ln @ ZIF-8 nano particles are coated with silicon dioxide to prepare the Ln @ [email protected] SiO₂；

4) For Ln @ [email protected] SiO₂Carrying out amination modification to prepare NH₂[email protected]@SiO₂；

5) To NH₂[email protected]@SiO₂PEG modification is carried out to prepare PEG-Ln @ [email protected] SiO₂I.e. the metal label.

2. The metal tag based on rare earth doped nanometal-organic framework material according to claim 1, characterized in that the step 1) comprises in particular:

1-1) dissolving 2-thenoyltrifluoroacetone in ethanol, and dropwise adding LnCl under the condition of stirring₃Adding ammonia water after the dropwise addition of the aqueous solution is finished, adjusting the pH value to 9, continuously stirring, adding water, and stirring again; wherein, the rare earth element Ln is any one of Tb, Ho and Tm;

1-2) performing rotary evaporation to remove part of ethanol, filtering and drying, dissolving the obtained solid in ethanol, dropwise adding an ethanol solution containing 1, 10-phenanthroline, heating to boiling for concentration after dropwise adding is completed, and recrystallizing the cooled solid with a hot mixed solution of acetone and ethanol to obtain the rare earth metal coordination compound.

3. The metal tag based on the rare earth doped nano metal organic framework material as claimed in claim 2, wherein the volume ratio of acetone to ethanol in the mixed solution of acetone and ethanol is 1: 2.

4. The metal tag based on rare earth doped nanometal-organic framework material according to claim 2, characterized in that the step 2) comprises in particular:

2-1) preparing two groups of solutions:

dissolving 2-methylimidazole and rare earth complex in methanol to obtain solution A; dissolving zinc nitrate hexahydrate and hexadecyl trimethyl ammonium bromide in methanol to obtain a solution B;

2-2) adding the solution B into the solution A, stirring at normal temperature, and centrifugally cleaning a product by using ethanol; finally, the Ln @ ZIF-8 nano-particles with the porous structure containing the rare earth metal coordination compound are obtained.

5. The metal tag based on rare earth doped nanometal-organic framework material according to claim 4, characterized in that the step 3) comprises in particular:

dissolving Ln @ ZIF-8 nano particles in ethanol, adding an aqueous solution of sodium hydroxide, uniformly stirring, adding tetraethyl orthosilicate, stirring at room temperature, centrifuging the obtained product, washing with ethanol, and drying to obtain Ln @ [email protected] SiO₂。

6. The metal tag based on rare earth doped nanometal-organic framework material according to claim 5, characterized in that the step 4) comprises in particular: mixing Ln @ [email protected] SiO₂Dissolving in ethanol, ultrasonic dispersing, adding APTES, stirring at room temperature, centrifuging, washing with ethanol, and vacuum drying to obtain NH₂[email protected]@SiO₂。

7. The metal tag based on rare earth doped nanometal-organic framework material according to claim 6, characterized in that the step 5) comprises in particular: reacting NH₂[email protected]@SiO₂Mixing with PEG solution, adding EDC and NHS, stirring at room temperature for reaction, washing the obtained product with ethanol and deionized water, and drying to obtain PEG-Ln @ [email protected] SiO₂。

8. An antibody tag for mass cytometry by the use of PEG-Ln @ [email protected] SiO as claimed in any one of claims 1 to 7₂Coupling with monoclonal antibody to obtainAnd (4) obtaining.

9. The antibody tag for mass cytometry according to claim 8, wherein the antibody tag is prepared by a method comprising the steps of:

a. taking PEG-Ln @ [email protected] SiO₂Adding into borate buffer solution, adding monoclonal antibody, EDC and NHS, and incubating at 37 deg.C;

b. and c, centrifugally washing the product obtained in the step a by using a PBS (phosphate buffer solution) at room temperature to obtain the antibody label.

10. The antibody tag for mass cytometry according to claim 9, wherein said monoclonal antibody is CD-3, CD-4, CD-8 or CD-45.

Technical Field

The invention relates to the technical field of biological imaging, in particular to a metal label based on a rare earth doped nano metal organic framework material.

Background

The mass cytometry combines the traditional flow cytometry analysis method and the mass spectrometry detection method together, and is an emerging high-throughput and multidimensional single-cell biological detection technology. The traditional flow cytometry method adopts a method that cells are stained by an antibody marked by a fluorescent dye, and a fluorescent signal is detected by a fluorescence detector to realize the identification and detection of the cells. The mass flow cytometry is to couple cells through a stable heavy metal element labeled antibody, and further detect a metal signal through inductively coupled plasma mass spectrometry to achieve the purpose of cell identification. The technology overcomes the defects of overlapping fluorescence signals and few detection channels in the traditional flow cytometry, can simultaneously detect more than 40 parameters of the same cell at most, and greatly improves the detection efficiency.

Despite the advantages described above, there are still some areas where improvements are needed in the application of current mass flow detection technology, such as the detection kits used in current mass flow detection technology, which are based on commercially available Maxpar sizing polymers (MCP) reagents polymeric metal chelate tags, where about 20-30 rare earth metal atoms can be attached to each polymer chain, and about 2-3 polymer chains can be attached to each antibody during the antibody labeling process, so that about only 60-90 rare earth metals can be attached to each antibody. However, mass spectrometry can only detect one of 104 metal signals, so at least a hundred antibodies need to be attached to each cell to achieve the detection effect. On the other hand, labels on the market are usually imported from foreign countries and are expensive, and the price of a set of standard kits (containing the rare earth coordination polymer 40 x 100 micrograms, which can be used for 40 marking experiments) is usually more than 50000 RMB.

Therefore, a more reliable solution is now needed.

Disclosure of Invention

The technical problem to be solved by the present invention is to provide a metal tag based on a rare earth doped nanometal organic framework material, aiming at the defects in the prior art.

In order to solve the technical problems, the invention adopts the technical scheme that: a metal label based on a rare earth doped nano metal organic framework material is prepared by the following steps:

1) preparing a rare earth metal complex having the structure shown in formula (I):

2) preparing Ln @ ZIF-8 nano particles with porous structures by using the rare earth metal coordination compound prepared in the step 1);

3) the Ln @ ZIF-8 nano particles are coated with silicon dioxide to prepare the Ln @ [email protected] SiO₂；

4) For Ln @ [email protected] SiO₂Carrying out amination modification to prepare NH₂[email protected]@SiO₂；

5) To NH₂[email protected]@SiO₂PEG modification is carried out to prepare PEG-Ln @ [email protected] SiO₂I.e. the metal label.

Preferably, the step 1) specifically includes:

1-1) dissolving 2-thenoyltrifluoroacetone in ethanol, and dropwise adding LnCl under the condition of stirring₃Adding ammonia water after the dropwise addition is finished, adjusting the pH value to 9, and thenContinuously stirring, adding water, and stirring; wherein, the rare earth element Ln is any one of Tb, Ho and Tm;

1-2) performing rotary evaporation to remove part of ethanol, filtering and drying, dissolving the obtained solid in ethanol, dropwise adding an ethanol solution containing 1, 10-phenanthroline, heating to boiling for concentration after dropwise adding is completed, and recrystallizing the cooled solid with a hot mixed solution of acetone and ethanol to obtain the rare earth metal coordination compound.

Preferably, the volume ratio of acetone to ethanol in the mixture of acetone and ethanol is 1: 2.

Preferably, the step 2) specifically includes:

2-1) preparing two groups of solutions:

dissolving 2-methylimidazole and rare earth complex in methanol to obtain solution A; dissolving zinc nitrate hexahydrate and hexadecyl trimethyl ammonium bromide in methanol to obtain a solution B;

2-2) adding the solution B into the solution A, stirring at normal temperature, and centrifugally cleaning a product by using ethanol; finally, the Ln @ ZIF-8 nano-particles with the porous structure containing the rare earth metal coordination compound are obtained.

Preferably, the step 3) specifically includes:

dissolving Ln @ ZIF-8 nano particles in ethanol, adding an aqueous solution of sodium hydroxide, uniformly stirring, adding tetraethyl orthosilicate, stirring at room temperature, centrifuging the obtained product, washing with ethanol, and drying to obtain Ln @ [email protected] SiO₂。

Preferably, the step 4) specifically includes: mixing Ln @ [email protected] SiO₂Dissolving in ethanol, ultrasonic dispersing, adding APTES, stirring at room temperature, centrifuging, washing with ethanol, and vacuum drying to obtain NH₂[email protected]@SiO₂。

Preferably, the step 5) specifically includes: reacting NH₂[email protected]@SiO₂Mixing with PEG solution, adding EDC and NHS, stirring at room temperature for reaction, washing the obtained product with ethanol and deionized water, and drying to obtain PEG-Ln @ [email protected] SiO₂。

The invention also provides an antibody label for mass cytometryTabs prepared by PEG-Ln @ [email protected] SiO as described above₂Coupling with monoclonal antibody.

Preferably, the preparation method of the antibody tag comprises the following steps:

a. taking PEG-Ln @ [email protected] SiO₂Adding into borate buffer solution, adding monoclonal antibody, EDC and NHS, and incubating at 37 deg.C;

b. and c, centrifugally washing the product obtained in the step a by using a PBS (phosphate buffer solution) at room temperature to obtain the antibody label.

Preferably, the monoclonal antibody is CD-3, CD-4, CD-8 or CD-45.

The invention has the beneficial effects that:

the invention is different from the existing mass flow polymer metal chelating label, skillfully dopes the rare earth metal into the stable ZIF-8 hollow porous structure, and can improve the rare earth content of a single particle to 10⁵-10⁶And further carrying out SiO on the surface₂Coating, improving the stability, further adopting PEG to carry out surface modification, and developing into PEG-Ln @ [email protected] SiO₂The composite nano particles can be sensitively detected by mass flow, have good biocompatibility, are simple in synthesis process operation and low in cost, and can greatly reduce the use cost of the mass flow detection reagent. After the PEG-Ln @ [email protected] Sio2 composite nano-particles are combined with different antibodies, different cell populations can be specifically identified, and a reliable detection means is provided for mass spectrum flow type researches in the aspects of cell biology immunology, hematology, drug screening research and development, clinical diagnosis and the like.

Drawings

FIG. 1 is a synthetic route of a metal tag based on a rare earth doped nanometal organic framework material of the present invention;

FIG. 2 is a TEM image of Tb @ ZIF-8 in an example of the present invention;

FIG. 3 is a PEG-Tb @ [email protected] SiO in an embodiment of the present invention₂-CD4 versus T cell assay results.

Detailed Description

The present invention is further described in detail below with reference to examples so that those skilled in the art can practice the invention with reference to the description.

It will be understood that terms such as "having," "including," and "comprising," as used herein, do not preclude the presence or addition of one or more other elements or groups thereof.

The invention provides a metal tag based on a rare earth doped nano metal organic framework material, which is prepared by the following method:

1) preparing a rare earth metal complex having the structure shown in formula (I):

2) preparing Ln @ ZIF-8 nano particles with porous structures by using the rare earth metal coordination compound prepared in the step 1), 2-methylimidazole, methanol, zinc nitrate hexahydrate and other raw materials; among them, ZIF-8, 2-methylimidazolium zinc salt MAF-4, is a porous coordination polymer, also called metal organic framework, and has the characteristics of high pore volume, high hydrophobicity, high thermal stability and chemical stability;

3) TEOS (tetraethyl orthosilicate) reacts with Ln @ ZIF-8 nano-particles to coat the Ln @ ZIF-8 nano-particles with silicon dioxide to prepare the Ln @ [email protected] SiO₂；

4) APTES (3-aminopropyltriethoxysilane) is used for Ln @ [email protected] SiO₂Carrying out amination modification to prepare NH₂[email protected]@SiO₂；

5) To NH₂[email protected]@SiO₂PEG modification is carried out to prepare PEG-Ln @ [email protected] SiO₂I.e. the metal label.

The synthesis route of the metal tag is shown in FIG. 1.

The invention also provides an antibody label for mass cytometry, which is prepared by the PEG-Ln @ [email protected] SiO₂Coupling with monoclonal antibody.

More specific examples are provided below to further illustrate the invention.

Example 1

A metal label based on a rare earth doped nano metal organic framework material is prepared by the following steps:

1. preparation of rare earth metal complexes

In this embodiment, the lanthanide rare-earth element Ln is specifically selected as Tb, and the specific steps are as follows:

1-1, dissolving 440mg of 2-thenoyl trifluoroacetone in 15mL of ethanol, and rapidly stirring;

1-2, 0.05M of TbCl is dripped₃15mL of aqueous solution, adding a few drops of ammonia water after the dropwise addition is finished, and adjusting the pH value to 9;

1-3, stirring for 1 hour, adding 25mL of water, and stirring for 1 hour;

1-4, removing most ethanol by rotary evaporation, filtering and drying to obtain solid 0.44 g;

1-5, dissolving the obtained solid in 15mL of ethanol, dropwise adding 6mL of ethanol solution containing 105mg of 1, 10-phenanthroline, heating to boiling and concentrating after dropwise adding is finished, and recrystallizing the cooled solid with hot acetone/ethanol mixed solution (volume ratio is 1:2) to obtain a rare earth metal coordination compound: a Tb complex.

2. Preparation of Tb @ ZIF-8 nanoparticles with porous structure

2-1, preparing two groups of solutions:

dissolving 820mg of 2-methylimidazole and 28mg of Tb complex in 40mL of methanol to obtain a solution A; 714mg of zinc nitrate hexahydrate and 72.9mg of cetyltrimethylammonium bromide were dissolved in 40mL of methanol to obtain a solution B;

2-2, quickly adding the solution B into the solution A, and stirring for 24 hours at normal temperature;

2-3, centrifuging at 8000rpm for 15min to remove supernatant

And 2-4, centrifuging and cleaning the obtained solid with ethanol, centrifuging at 8000rpm for 15min, repeating the steps for three times, and drying to obtain Tb @ ZIF-8 nanoparticles.

Referring to FIG. 2, a transmission electron microscope photograph of Tb @ ZIF-8 shows that Tb @ ZIF-8 has a particle size of about 50 nm and a uniform size distribution.

3. Preparation of Tb @ [email protected] SiO₂

3-1, dispersing 40mgTb @ ZIF-8 nano particles in 100mL ethanol, and violently stirring;

3-2, adjusting the pH value of the solution obtained in the step to 8 by using a 0.1M NaOH solution;

3-3, adding 20% TEOS (tetraethyl orthosilicate) ethanol solution for three times, wherein 1.2mL of the ethanol solution is added every time, and the interval of each time is half an hour;

3-4, stirring for 18 hours at room temperature, and centrifuging for 15min at the rotating speed of 8000rpm to remove supernatant;

3-5, centrifuging the obtained solid by using ethanol, centrifuging at 8000rpm for 15min, and repeating for three times to obtain Tb @ [email protected] SiO₂。

4. Preparation of NH₂[email protected]@SiO₂

4-1, 50mgTb @ [email protected] SiO₂Dispersing in 100mL of ethanol, and stirring vigorously;

4-2, adding 50 mu L of APTES (3-aminopropyltriethoxysilane), and stirring for 6 hours at room temperature;

4-3, centrifuging at 8000rpm for 15min to remove supernatant;

4-4, centrifuging and cleaning the obtained solid with ethanol, centrifuging at 8000rpm for 15min, repeating for three times, and vacuum drying to obtain NH2-Tb @ [email protected] SiO₂。

5. Preparation of EG-Tb @ [email protected] SiO₂

5-1, 50mgNH₂[email protected]@SiO₂Dispersing in 100mL of ethanol, and stirring vigorously;

5-2, adding 100mg of PEG (polyethylene glycol) solution (with carboxyl groups at two ends, wt,6000) and stirring;

5-3, adding EDC and NHS (N-hydroxysuccinimide), and stirring at room temperature for 12 hours;

5-4, centrifuging at 8000rpm for 15min to remove supernatant, washing the obtained solid with ethanol and deionized water, and drying to obtain PEG-Tb @ [email protected] SiO₂。

Wherein by NH₂[email protected]@SiO₂Can be mixed with PEG solution with different molecular weights according to different proportions (such as 1:2, 1:3, etc.) to obtain NH with different parameters₂[email protected]@SiO₂。

Example 2

The present embodiment provides an antibody tag for mass cytometry, and the preparation method of the antibody tag comprises the following steps:

100ug of PEG-Tb @ [email protected] SiO prepared in example 1 was taken₂Adding into 50mM borate buffer solution with pH 8.2, adding monoclonal antibody (specifically selected as CD-45 in this example) and EDC and NHS (both 150gL concentration) as crosslinking agent^-1) Incubating at 37 ℃ for a period of time, centrifuging and washing the obtained product with 0.01M PBS (pH 7.4) at room temperature, dispersing into PBS (0.01M, pH 7.4, 0.5% BSA), and storing at 4 ℃ for later use to obtain the antibody label (PEG-Tb @ [email protected] SiO @)₂-CD 4). 1uL antibody tag was applied to human peripheral blood mononuclear cells (10)⁶Individually) were stained and tested on a mass cytometer.

Referring to FIG. 3, PEG-Tb @ [email protected] SiO₂The detection result of the T cells by the-CD 4 shows that PEG-Tb @ [email protected] SiO₂The CD4 antibody tag enables efficient clustering of T cells (two cell populations left and right), wherein cells labeled with CD4 antibody account for 58.36% of the total number of cells. In fig. 3 can be understood as: all cells were uniformly labeled on the ordinate and then used for clustering on the abscissa, with 159TbCD4 on the right and none on the left.

While embodiments of the invention have been disclosed above, it is not limited to the applications listed in the description and the embodiments, which are fully applicable in all kinds of fields of application of the invention, and further modifications may readily be effected by those skilled in the art, so that the invention is not limited to the specific details without departing from the general concept defined by the claims and the scope of equivalents.