Mass spectrum flow type general cell coding reagent based on UIO-66 nano material and application thereof

Abstract

The invention discloses a mass spectrum flow type general cell coding reagent based on a UIO-66 nano material and application of the mass spectrum flow type general cell coding reagent. The cell coding reagent is a mixture of a UIO-66 (Zr) nano material and a UIO-66 (Hf) nano material. The UIO-66 (Hf) is prepared by the following steps: respectively dissolving anhydrous hafnium chloride and terephthalic acid in a solvent, mixing the two prepared solutions, transferring the obtained mixture into a reaction kettle with a polytetrafluoroethylene lining, adding formic acid, reacting while heating, cooling to room temperature after the reaction is finished, centrifuging, and cleaning the obtained solid to obtain the product. The electropositive UIO-66 nano material is adsorbed to the surface of an electronegative cell in an electrostatic adsorption manner, does not depend on specific molecules on the surface of the cell, and can be used as a universal coding reagent for coding different types of cells; at most 84 cell samples can be coded at the same time, which is far higher than that of the existing commercialized mass spectrum flow type coding product.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of nanomaterials, and particularly to a mass spectrometry flow universal cell coding reagent based on UIO-66 nanomaterials and application thereof. BACKGROUND

[0002] Mass cytometry is an emerging high-throughput multi-parameter single-cell analysis technology, combining the advantages of flow cytometry and inductively coupled plasma time-of-flight mass spectrometry (ICP-TOFMS). By using heavy atom isotopes (most of which are isotopes of lanthanides and cadmium) as mass tags, mass cytometry overcomes the fluorescence overlap and autofluorescence phenomena faced by traditional flow cytometry, and can detect the expression of more than 50 proteins in a single cell without any spectral compensation algorithm. Therefore, mass cytometry is favored by biologists in various research fields involving single-cell analysis, and is considered to be the next generation of flow cytometry. However, as a newly developed technology, mass cytometry still faces many problems, especially in large sample measurement. For example, the throughput of mass cytometry is about 300-500 cells per second, which is much lower than that of flow cytometry (>10000 cells per second). The lower throughput not only takes longer analysis time, but also leads to unpredictable changes in mass signal intensity, resulting in bias between different cell samples. In addition, heavy atom isotope tags are much more expensive than mature flow cytometer tags.

[0003] Encoding techniques have been widely applied in multiplexing technologies to solve these problems. By encoding each test sample separately and pooling them in one tube for mass cytometry staining and detection, the sample-to-sample variation and instrument bias can be effectively eliminated. This approach can also reduce the consumption of expensive mass cytometry reagents and time cost. Lanthanides were first used for mass cytometry encoding, but due to their occupation of the detection channel of commercial mass cytometry polymer (MCP) mass tags, the multiplexing detection capability of mass cytometry was limited. For example, Newell et al. reported a mass cytometry encoding strategy by unique lanthanide isotope (10 choose 3) combinations, which achieved up to 120 encodings and was used for high-dimensional analysis of antigen-specific T cells. To avoid occupying the MCP detection channel, the US company Fluidigm developed a new encoding strategy using 6 palladium isotopes instead of lanthanides, providing 20 unique encodings (6-choose-3). This is also the most commonly used commercial encoding product (Cell-ID 20-Plex PD encoding kit). However, this encoding strategy is based on intracellular labeling, which requires cell fixation and penetration through the cell membrane, limiting its application. Some encoding reagents coupled with specific molecules on the cell surface have also been developed. For example, Chiu reported a proportional encoding strategy by coupling an anti-CD45 antibody with lanthanide-coordinated semiconductor polymer dots, which achieved 16 encodings occupying 3 lanthanide isotope channels. Compared with intracellular encoding strategies, these encoding reagents can only encode cells with common surface molecules, but not different cells. Therefore, new mass cytometry cell surface encoding strategies with more encoding combinations, less lanthanide channel occupation, and higher universality are still challenging. SUMMARY

[0004] The technical problem solved by the present application is to provide a mass spectrometry flow universal cell coding reagent based on UIO-66 nanomaterial and application thereof in view of the above-mentioned deficiencies in the prior art. The present application aims to design a mass spectrometry flow cell surface coding reagent based on UIO-66(Zr) and UIO-66(Hf) metal organic framework materials (MOFs). The size of the two MOFs is controlled within 40 nanometers by adjusting the type and amount of the template agent, and the MOF nanoparticles with different Zr and Hf isotopes are adsorbed to the cell surface by electrostatic action to realize mass coding of the cells. The present application designs a mass spectrometry flow coding strategy based on the nanometer-sized UIO-66(Zr) and UIO-66(Hf) metal organic framework materials (MOFs), which has nine stable isotopes, only one of which coincides with the rare earth lanthanide series channel, and the strategy is combined with the cells by electrostatic adsorption and is independent of specific molecules on the cell surface, so that coding of all kinds of cells can be realized, and at most 84 mass spectrometry flow coding can be realized; after the MOF surface is modified with a fluorescein molecule, mass spectrometry flow and fluorescence flow coding and cell imaging can be realized at the same time.

[0005] To achieve the above-mentioned object, the technical scheme adopted by the present application is as follows: In a first aspect, the present application provides a mass spectrometry flow universal cell coding reagent based on UIO-66 nanomaterial, which is a mixture of UIO-66(Zr) nanomaterial and UIO-66(Hf) nanomaterial;

[0006] The UIO-66(Hf) nanomaterial is prepared by the following steps:

[0007] The anhydrous hafnium chloride and terephthalic acid are dissolved in a solvent respectively, the two prepared solutions are mixed, the obtained mixture is transferred to a polytetrafluoroethylene-lined reaction kettle, formic acid is added, and the reaction is carried out under heating, after the reaction is completed, the temperature is cooled to room temperature, centrifugation is carried out, and the obtained solid is washed to obtain the UIO-66(Hf) nanomaterial.

[0008] Preferably, the UIO-66(Zr) nanomaterial is prepared by the following steps:

[0009] ZrCl4 and terephthalic acid are dissolved in a solvent respectively, the two prepared solutions are mixed, the obtained mixture is transferred to a polytetrafluoroethylene-lined reaction kettle, formic acid is added, and the reaction is carried out under heating, after the reaction is completed, the temperature is cooled to room temperature, centrifugation is carried out, and the obtained solid is washed to obtain the UIO-66(Zr) nanomaterial.

[0010] Preferably, the solvent is N,N-dimethylformamide.

[0011] Preferably, the UIO-66(Hf) nanomaterial is prepared by the following steps:

[0012] S1-1, 0.75-3 mmol of anhydrous hafnium chloride and 2.25-9 mmol of terephthalic acid were respectively dissolved in 15-60 mL of N,N-dimethylformamide, and the resulting two solutions were mixed after ultrasonic treatment until complete dissolution;

[0013] S1-2, the resulting mixture was transferred to a reaction kettle with a polytetrafluoroethylene liner, 0.9-2.4 mol / L of formic acid was added, and the reaction was carried out at 80-100°C for 6-18 hours;

[0014] S1-3, after the reaction was completed, the temperature was cooled to room temperature, and centrifugation was carried out at 10000-40000 g for 15-60 minutes, and the supernatant was removed;

[0015] S1-4, 17.5-70 mL of N,N-dimethylformamide was added, vortexed, ultrasonicated, resuspended, washed, and this step was repeated at least once;

[0016] S1-5, the resulting solid was washed several times with ethanol and several times with deionized water to obtain the UIO-66(Hf) nanomaterial.

[0017] Preferably, the UIO-66(Hf) nanomaterial is prepared by the following steps:

[0018] S1-1, 1.5 mmol of anhydrous hafnium chloride and 4.5 mmol of terephthalic acid were respectively dissolved in 30 mL of N,N-dimethylformamide, and the resulting two solutions were mixed after ultrasonic treatment until complete dissolution;

[0019] S1-2, the resulting mixture was transferred to a reaction kettle with a polytetrafluoroethylene liner, 0.9 mol / L of formic acid was added, and the reaction was carried out at 90°C for 6 hours;

[0020] S1-3, after the reaction was completed, the temperature was cooled to room temperature, and centrifugation was carried out at 20000 g for 30 minutes, and the supernatant was removed;

[0021] S1-4, 35 mL of N,N-dimethylformamide was added, vortexed, ultrasonicated, resuspended, washed, and this step was repeated once;

[0022] S1-5, the resulting solid was washed once with ethanol and twice with deionized water to obtain the UIO-66(Hf) nanomaterial.

[0023] Preferably, the UIO-66(Zr) nanomaterial is prepared by the following steps:

[0024] S2-1, 0.75-3mmol ZrCl4 and 2.25-9mmol terephthalic acid are respectively dissolved in 15-60mL N,N-dimethylformamide, and the prepared two solutions are mixed under ultrasonic until completely dissolved;

[0025] S2-2, the obtained mixture is transferred to a reaction kettle with a polytetrafluoroethylene lining, 0.9-2.4mol / L formic acid is further added, and the reaction is carried out at 80-100℃ for 6-18 hours;

[0026] S2-3, after the reaction is completed, cooling to room temperature, centrifugation at 10000-40000g for 15-60 minutes, and removing the supernatant;

[0027] S2-4, 17.5-70mL N,N-dimethylformamide is added, vortexed, ultrasonic, resuspended, washed, and the step is repeated at least once;

[0028] S2-5, the obtained solid is washed several times with ethanol and several times with deionized water to obtain the UIO-66(Zr) nanomaterial.

[0029] Preferably, the UIO-66(Zr) nanomaterial is prepared by the following steps:

[0030] S2-1, 1.5mmol ZrCl4 and 4.5mmol terephthalic acid are respectively dissolved in 30mL N,N-dimethylformamide, and the prepared two solutions are mixed under ultrasonic until completely dissolved;

[0031] S2-2, the obtained mixture is transferred to a reaction kettle with a polytetrafluoroethylene lining, 0.9mol / L formic acid is further added, and the reaction is carried out at 90℃ for 6 hours;

[0032] S2-3, after the reaction is completed, cooling to room temperature, centrifugation at 20000g for 30 minutes, and removing the supernatant;

[0033] S2-4, 35mL N,N-dimethylformamide is added, vortexed, ultrasonic, resuspended, washed, and the step is repeated once;

[0034] S2-5, the obtained solid is washed once with ethanol and twice with deionized water to obtain the UIO-66(Zr) nanomaterial.

[0035] In a second aspect of the present application, another universal cell coding reagent based on UIO-66 nanomaterial is provided, which is a mixture of the above-mentioned UIO-66 nanomaterial modified by fluorescein, specifically, a mixture of fluorescein-modified UIO-66(Zr) nanomaterial and fluorescein-modified UIO-66(Hf) nanomaterial;

[0036] The fluorescein-modified UIO-66(Zr) nanomaterial is prepared by the following method:

[0037] 1-1) 0.5-2 mg of the UIO-66(Zr) nanomaterial according to any one of claims 1-7 is dispersed in 0.5-2 mL of deionized water, and centrifuged and washed;

[0038] 1-2) 5-20 μL of 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride aqueous solution with a concentration of 5-20 mg / mL and 5-20 μL of N-hydroxysuccinimide aqueous solution with a concentration of 5-20 mg / mL are added to the product of step 1), and incubated in the dark for 15-60 minutes;

[0039] 1-3) After washing several times with deionized water, 25-100 μg of 5-amino fluorescein is added, and incubated in the dark for 1.5-6 hours;

[0040] 1-4) Centrifuged and washed several times with deionized water to obtain the fluorescein-modified UIO-66(Zr) nanomaterial;

[0041] The fluorescein-modified UIO-66(Hf) nanomaterial is prepared by the following method:

[0042] 1-1) 0.5-2 mg of the UIO-66(Hf) nanomaterial according to any one of claims 1-7 is dispersed in 0.5-2 mL of deionized water, and centrifuged and washed;

[0043] 1-2) 5-20 μL of 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride aqueous solution with a concentration of 5-20 mg / mL and 5-20 μL of N-hydroxysuccinimide aqueous solution with a concentration of 5-20 mg / mL are added to the product of step 1), and incubated in the dark for 15-60 minutes;

[0044] 1-3) After washing several times with deionized water, 25-100 μg of 5-amino fluorescein is added, and incubated in the dark for 1.5-6 hours;

[0045] 1-4) Centrifuged and washed several times with deionized water to obtain the fluorescein-modified UIO-66(Hf) nanomaterial.

[0046] Preferably, the fluorescein-modified UIO-66(Zr) nanomaterial is prepared by the following method:

[0047] 1-1) 1 mg of the UIO-66(Zr) nanomaterial according to any one of claims 1-7 is dispersed in 1 mL of deionized water, and centrifuged and washed twice at 20000 g;

[0048] 1-2) To the product of step 1), 10 μL of 1-ethyl-(3-dimethylaminopropyl) carbodiimide aqueous solution with a concentration of 10 mg / mL and 10 μL of N-hydroxysuccinimide aqueous solution with a concentration of 10 mg / mL were added, and incubated in dark for 30 minutes;

[0049] 1-3) After washing twice with deionized water, 50 μg of 5-amino fluorescein was added, and incubated in dark for 3 hours;

[0050] 1-4) Centrifugation and washing twice with deionized water to obtain the fluorescein-modified UIO-66(Zr) nanomaterial;

[0051] The fluorescein-modified UIO-66(Hf) nanomaterial was prepared by the following method:

[0052] 1-1) 1 mg of the UIO-66(Hf) nanomaterial as described in any one of claims 1-7 was dispersed in 1 mL of deionized water, and centrifuged and washed twice at 20000 g;

[0053] 1-2) To the product of step 1), 10 μL of 1-ethyl-(3-dimethylaminopropyl) carbodiimide aqueous solution with a concentration of 10 mg / mL and 10 μL of N-hydroxysuccinimide aqueous solution with a concentration of 10 mg / mL were added, and incubated in dark for 30 minutes;

[0054] 1-3) After washing twice with deionized water, 50 μg of 5-amino fluorescein was added, and incubated in dark for 3 hours;

[0055] 1-4) Centrifugation and washing twice with deionized water to obtain the fluorescein-modified UIO-66(Hf) nanomaterial.

[0056] In the present application, in order to make comparison, the preparation methods of the functional group-modified UIO-66 nanomaterials are also provided, i.e. the preparation method of carboxylated polyethylene glycol-modified UIO-66 nanomaterial and the preparation method of polyacrylic acid and carboxylated polyethylene glycol complex-modified UIO-66 nanomaterial, which are specifically as follows:

[0057] The preparation method of carboxylated polyethylene glycol-modified UIO-66 nanomaterial is as follows:

[0058] A certain amount of UIO-66 nanomaterial (UIO-66(Hf) nanomaterial or UIO-66(Zr) nanomaterial) was dispersed in deionized water and washed twice, and then appropriate amounts of EDC aqueous solution and NHS aqueous solution with a concentration of 10 mg / mL were added, and incubated in dark for 30 minutes. After washing twice with deionized water, 5 mg of polyethylene glycol with amino and carboxyl groups at both ends and a chain length of 5000 (NH2-PEG 5000-COOH), incubated overnight at room temperature, centrifuged and washed twice with deionized water, and stored in deionized water.

[0059] The preparation method of the polyacrylic acid and carboxylated polyethylene glycol composite modified UIO-66 nanomaterial is as follows:

[0060] A certain amount of UIO-66 nanomaterial (UIO-66 (Hf) nanomaterial or UIO-66 (Zr) nanomaterial) is dispersed in deionized water and washed twice, dispersed in 1 mL of deionized water, 1.5 mg of PAA is added, quickly vortexed and mixed, and placed on a shaker at 1000 revolutions per minute at room temperature for 20 minutes, then washed once with deionized water, and then an appropriate amount of 10 mg / mL EDC aqueous solution and sulfo-NHS aqueous solution are added, respectively, and activated at room temperature for 20 minutes, then washed once with deionized water, and finally 5 mg of NH2-PEG 5000 -COOH room temperature reaction overnight, washed twice with deionized water, to obtain a preparation method of a carboxylated polyethylene glycol modified UIO-66 nanomaterial, and a polyacrylic acid and carboxylated polyethylene glycol composite modified UIO-66 nanomaterial, which is stored in deionized water.

[0061] In a third aspect, the application provides a use of any one of the cell coding reagents as described above in mass cytometry cell coding.

[0062] In a preferred embodiment, the application method is as follows:

[0063] After fixing a plurality of cell samples in different flow tubes using a 1.6% paraformaldehyde solution, the cells are washed with 5 mL of cell staining buffer at 400g, and blocked with blocking agent for 10 minutes. Then the cell samples are incubated with UIO-66 prepared using different Zr and Hf isotopes for 45 minutes. After washing away the excess MOF with cell staining buffer, all cell samples are combined in one flow tube, and the UIO-66 coded cells are incubated with MCP antibody tags according to the Fluidigm antibody labeling scheme, then the cells are washed twice with cell staining buffer, and the cell nuclei are stained with Fluidigm cell Ir-DNA intercalator at room temperature for 1 hour (or overnight at 4°C). After washing with deionized water, mass cytometry detection is performed according to the Fluidigm Mass Cytometry protocol. 90 Zr, 91 Zr, 92 Zr, 94 Zr, 174 Hf, 176 Hf, 177 Hf, 178 Hf, 179 Hf, 180Hf channel carries out cluster decoding on the cells, and then analyzes surface protein expression of each decoded cell sample according to the isotope element species of the antibody tag.

[0064] The present application has the following advantages:

[0065] The present application provides a mass spectrometry flow cytometry universal cell coding reagent based on UIO-66 nanomaterials, which is different from existing mass spectrometry flow cytometry coding technology. The present application uses electrostatic adsorption to adsorb positively charged UIO-66 nanomaterials to the surface of negatively charged cells, and does not depend on specific molecules on the surface of the cells, and can be used as a universal coding reagent to code different types of cells. Zr and Hf have nine stable isotopes, and using three different isotopes to code a cell sample can simultaneously code up to 84 cell samples, which is much higher than the existing commercial mass spectrometry flow cytometry coding product (Cell-ID 20-Plex PD coding kit). At the same time, the sensitivity of UIO-66 coding is 16 times higher than that of the commercial MCP coding reagent, and has high compatibility with the MCP antibody tag, and does not interfere with the expression level of the cell protein marker. The UIO-66 nanomaterials modified by the fluorescent dye can also be used for fluorescence coding or fluorescence imaging. BRIEF DESCRIPTION OF DRAWINGS

[0066] Figure 1 A flow chart for the synthesis and surface modification of UIO-66 nanomaterials;

[0067] Figure 2 A flow chart for mass spectrometry flow cytometry coding and decoding of UIO-66 nanomaterials;

[0068] Figure 3 UIO-66(Zr) (top) and UIO-66(Hf) (bottom) nanomaterials transmission electron microscopy, surface potential and EDX mapping diagrams;

[0069] Figure 4 UIO-66 mixed with mass spectrometry flow cytometry tuning fluid for CyTOF mass spectrometry detection schematic diagram;

[0070] Figure 5 UIO-66(Hf) and UIO-66(Hf)-FITC after cell staining flow cytometry and confocal microscope test diagram;

[0071] Figure 6 (A) 0.1 μg, 0.5 μg, 1 μg UIO-66(Zr); (B) 0.1 μg, 0.5 μg, 1 μg UIO-66(Hf); (C) 1 μL 151-Eu-MCP_CD45 after cell staining mass spectrometry flow cytometry scatter plot; (D) corresponding signal intensity heat map;

[0072] Figure 7 Surface potential and mass cytometry flow scatter plots of UIO-66(Hf), PEG-COOH@UIO-66(Hf) and PEG-COOH@PAA@UIO-66(Hf) and after incubation with cells;

[0073] Figure 8 Mass cytometry flow scatter plots of two groups of cells after encoding and mixing using UIO-66(Zr) / 151Eu-MCP_CD45 and UIO-66(Hf) / 154Sm-MCP_CD45 respectively;

[0074] Figure 9 Mass cytometry flow scatter plots of three groups of cells after encoding and mixing using UIO-66(Zr) / 150Sm-MCP_CD45, UIO-66(Hf) / 154Sm-MCP_CD45 and UIO-66(Zr / Hf) / 151Eu-MCP_CD45 respectively. DETAILED DESCRIPTION

[0075] The application will be further described in conjunction with the examples below, so that those skilled in the art can implement the application according to the description and the examples.

[0076] It should be understood that the terms such as "have", "contain" and "include" used herein do not exclude the presence or addition of one or more other elements or combinations thereof.

[0077] The test methods used in the following examples are conventional methods unless otherwise specified. The materials and reagents used in the following examples, unless otherwise specified, can be obtained commercially. The specific conditions not specified in the following examples are carried out according to the conventional conditions or the conditions recommended by the manufacturer. The reagents or instruments used, if not specified by the manufacturer, are conventional products that can be obtained by commercial purchase.

[0078] Example 1 Preparation of UIO-66(Hf) nanomaterial

[0079] S1-1, 1.5 mmol of anhydrous hafnium chloride and 4.5 mmol of terephthalic acid were dissolved in 30 mL of N,N-dimethylformamide (DMF) respectively, and the resulting two solutions were mixed after ultrasonic treatment until completely dissolved;

[0080] S1-2, the obtained mixture was transferred to a polytetrafluoroethylene lined reaction kettle, and 0.9 mol / L of formic acid was added, and the reaction was carried out at 90°C for 6 hours;

[0081] S1-3, after the reaction was completed, it was cooled to room temperature, centrifuged at 20000g for 30 minutes, and the supernatant was removed;

[0082] S1-4, 35 mL of N,N-dimethylformamide was added, vortexed, ultrasonic, resuspended, washed, and this step was repeated once;

[0083] S1-5, the obtained solid was washed with ethanol once, deionized water twice, to obtain the UIO-66(Hf) nanomaterial, ultrasonic dispersion in water storage.

[0084] Reference Figure 3 B, the test analysis results of this embodiment, from the test results can be seen, UIO-66(Hf) nanomaterial particle size is about 40 nm, the surface potential is about 42 mV, Hf element is evenly distributed on the nanomaterial.

[0085] Example 2 preparation of UIO-66(Zr) nanomaterial

[0086] S2-1, 1.5 mmol of ZrCl4 and 4.5 mmol of terephthalic acid were dissolved in 30 mL of N,N-dimethylformamide respectively, and ultrasonic was used until completely dissolved, the two prepared solutions were mixed;

[0087] S2-2, the obtained mixture was transferred to a polytetrafluoroethylene lined reactor, 0.9 mol / L formic acid was added, and reacted at 90℃ for 6 hours;

[0088] S2-3, after the reaction was completed, it was cooled to room temperature, centrifuged at 20000g for 30 minutes, and the supernatant was removed;

[0089] S2-4, 35 mL of N,N-dimethylformamide was added, vortexed, ultrasonic, resuspended, washed, and this step was repeated once;

[0090] S2-5, the obtained solid was washed with ethanol once, deionized water twice, to obtain the UIO-66(Zr) nanomaterial, ultrasonic dispersion in water storage.

[0091] Reference Figure 3 A, the test analysis results of this embodiment, from the test results can be seen, UIO-66(Hf) nanomaterial particle size is about 40 nm, the surface potential is about 32 mV, Zr element is evenly distributed on the nanomaterial.

[0092] Example 3

[0093] (1) 2 mg / mL of UIO-66(Zr) and UIO-66(Hf) suspension was prepared using deionized water, and ultrasonic dispersion was performed;

[0094] (2) 1 μL of the above solution was dispersed in 1 mL of deionized water to prepare a 2 μg / mL suspension;

[0095] (3) Take 1 μL of the above solution and disperse it in 1 mL of mass spectrometry flow tuning liquid to configure a 2 μg / L suspension;

[0096] (4) Test in mass spectrometry flow solution mode, and record all channel signal intensities.

[0097] Reference Figure 4 For the test analysis results of the present embodiment, it can be seen from the test results that the UIO-66(Zr) and UIO-66(Hf) nanomaterials can form stable metal signals in mass spectrometry flow, and each isotope channel is obviously distinguished from the rare earth isotope channel in the mass spectrometry flow tuning liquid.

[0098] Example 4: Preparation of fluorescein-modified UIO-66(Hf) nanomaterial

[0099] 1-1) Disperse 1 mg of the UIO-66(Hf) nanomaterial prepared in Example 1 in 1 mL of deionized water, and centrifuge and wash twice at 20000 g;

[0100] 1-2) Add 10 μL of 1-ethyl-(3-dimethylaminopropyl) carbodiimide aqueous solution with a concentration of 10 mg / mL and 10 μL of N-hydroxysuccinimide aqueous solution with a concentration of 10 mg / mL to the product of step 1), and incubate in the dark for 30 minutes;

[0101] 1-3) After washing twice with deionized water, add 50 μg of 5-amino fluorescein, and incubate in the dark for 3 hours;

[0102] 1-4) Centrifuge and wash twice with deionized water to obtain the fluorescein-modified UIO-66(Hf) nanomaterial, which is recorded as UIO-66(Hf)-FITC, and is stored by dispersing in 1 mL of deionized water.

[0103] Example 5: Preparation of carboxylated polyethylene glycol-modified UIO-66(Hf) nanomaterial

[0104] (1) Disperse 1 mg of the UIO-66(Hf) nanomaterial prepared in Example 1 in 1 mL of deionized water, and centrifuge and wash twice at 20000 g;

[0105] (2) Respectively add 10 μL of 1-ethyl-(3-dimethylaminopropyl) carbodiimide (10 mg / mL) aqueous solution and N-hydroxysuccinimide (10 mg / mL) aqueous solution, and incubate in the dark for 30 minutes;

[0106] (3) After washing twice with deionized water, add 10 mg of NH2-PEG 5000 -COOH, and incubate in the dark for 3 hours;

[0107] (4) centrifugation and washing with deionized water twice to obtain carboxylated polyethylene glycol modified UIO-66(Hf) nanomaterial, recorded as: PEG-COOH@UIO-66(Hf), dispersed in 1 mL deionized water for storage.

[0108] Example 6 Preparation of polyacrylic acid and carboxylated polyethylene glycol complex modified UIO-66(Hf) nanomaterial

[0109] (1) 1 mg of UIO-66(Hf) nanoparticles prepared in Example 1 was dispersed in 1 mL deionized water, and washed twice at 20000 g;

[0110] (2) 1.5 mg of PAA was added and mixed quickly by vortex;

[0111] (3) The reaction solution was placed on a shaker at 1000 rpm at room temperature for 20 minutes, and washed once with deionized water;

[0112] (4) 10 μL of 1-ethyl-(3-dimethylaminopropyl) carbodiimide (10 mg / mL) aqueous solution and N-hydroxysuccinimide (10 mg / mL) aqueous solution were added respectively, and incubated in the dark for 30 minutes;

[0113] (5) After washing twice with deionized water, 10 mg of NH2-PEG 5000 -COOH was added and incubated in the dark for 3 hours;

[0114] (6) Centrifugation and washing with deionized water twice to obtain polyacrylic acid and carboxylated polyethylene glycol complex modified UIO-66(Hf) nanomaterial, recorded as: PAA@COOH@UIO-66(Hf), dispersed in 1 mL deionized water for storage.

[0115] Example 7 Mass spectrometry flow coding reagent based on UIO-66 nanomaterial for cell coding detection

[0116] (1) Take 10^6 white blood cells and wash with Fluidigm cell staining buffer;

[0117] (2) Add 5 μL of Fluidigm blocking solution and block for 10 minutes;

[0118] (4) Add 1 μg of UIO-66(Hf)-FITC prepared in Example 3 to the cell suspension and incubate for 45 minutes;

[0119] (5) Wash twice with Fluidigm cell staining buffer;

[0120] (6) Take a photo of the cells under the FITC channel using a confocal microscope;

[0121] (7) The cell suspension was detected on the flow cytometer with the FITC channel and cell grouping was performed.

[0122] Referring to Figure 5 For the test analysis results of the present embodiment, it can be seen from the test results that the white blood cells have no signal on the flow cytometer FITC channel, and the cells after incubation with UIO-66(Hf)-FITC generate obvious FITC signals on the FITC channel (Fig. A, B); all cells after incubation with UIO-66(Hf)-FITC have green fluorescence under the FITC field of view of the confocal microscope (Fig. C, D), proving that UIO-66(Hf)-FITC can be effectively adsorbed to the cell surface.

[0123] Example 8

[0124] (1) Take 7 x 10^6 white blood cells, and wash with Fluidigm cell staining buffer;

[0125] (2) Add 10 μL of Fluidigm blocking solution and block for 10 minutes;

[0126] (3) The cell suspension was equally divided into 7 parts;

[0127] (4) Add 0.1, 0.5, 1 μg of UIO-66(Zr), 0.1, 0.5, 1 μg of UIO-66(Hf), and 1 μL of commercial Fluidigm 151Eu-MCP_CD45 label, respectively, and incubate at room temperature for 45 minutes;

[0128] (5) Stain the cell nucleus with Fluidigm cell Ir-DNA intercalator for 1 hour;

[0129] (6) Wash twice with Fluidigm cell staining buffer and once with deionized water;

[0130] (7) Disperse the cells in 1 mL of an aqueous solution containing 10% Fluidigm EQ Beads;

[0131] (8) Test the cell sample using mass cytometry;

[0132] (9) Analyze the test results using Cytobank software, and analyze the 90Zr, 180Hf, and 151Eu positive cells.

[0133] Referring to Figure 6For the test analysis results of this embodiment, it can be seen from the test results that the UIO-66(Zr), UIO-66(Hf) and commercial 151Eu-MCP_CD45 label can all distinguish the cells from the background signal, and with the increase of the amount of UIO-66 added, the metal signal intensity on the cells also increases and is much higher than the signal intensity of the commercial 151Eu-MCP_CD45.

[0134] Example 9

[0135] (1) Take 3x10^6 white blood cells, and wash with Fluidigm cell staining buffer;

[0136] (2) Add 10 μL of Fluidigm blocking solution and block for 10 minutes;

[0137] (3) Equally divide the cell suspension into 3 parts;

[0138] (4) Add 0.5 μg of UIO-66(Hf) to one part of the cells, 0.5 μg of PEG-COOH@UIO-66(Hf) to another part, and 0.5 μg of PEG-COOH@PAA@UIO-66(Hf) to the other part, and incubate at room temperature for 45 minutes;

[0139] (5) Stain the cell nucleus with Fluidigm cell Ir-DNA intercalator for 1 hour;

[0140] (6) Wash twice with Fluidigm cell staining buffer and once with deionized water;

[0141] (7) Disperse the cells in 1 mL of an aqueous solution containing 10% Fluidigm EQ Beads;

[0142] (8) Test the cell sample using mass spectrometry flow cytometry;

[0143] (9) Analyze the test results using Cytobank software, gate the 180Hf positive cells, and compare and analyze the cell coding ability of different surface modified UIO-66 labels.

[0144] Reference Figure 7 For the test analysis results of this embodiment, it can be seen from the test results that the PEG-COOH and PAA modification can greatly reduce the surface potential of UIO-66(Hf), thereby reducing the adsorption strength of the nanomaterials and the cells, and further reducing the metal signal intensity on the cells.

[0145] Example 10

[0146] (1) Take 2x10^6 white blood cells, wash with Fluidigm cell staining buffer;

[0147] (2) Add 5 μL of Fluidigm blocking solution, block for 10 minutes;

[0148] (3) Equally divide the cell suspension into 2 parts;

[0149] (4) Add 1 μg of UIO-66(Zr) and 1 μL of 151Eu-MCP_CD45 to one part of the cells, and add 0.5 μg of UIO-66(Hf) and 1 μL of 154Sm-MCP_CD45 to the other part of the cells, and incubate at room temperature for 45 minutes;

[0150] (5) Stain the cell nuclei with Fluidigm cell Ir-DNA intercalator for 1 hour;

[0151] (6) Wash twice with Fluidigm cell staining buffer and once with deionized water;

[0152] (7) Disperse the cells in 1 mL of an aqueous solution containing 10% Fluidigm EQ Beads;

[0153] (8) Test the cell sample using a mass cytometer;

[0154] (9) Analyze the test results using Cytobank software, gate 90Zr positive, 180Hf positive, 151Eu positive and 154Sm positive cells, and compare and analyze the cell coding ability of UIO-66 labels and MCP labels.

[0155] Reference Figure 8 The test and analysis results of this example show that the proportion of each cell population after coding with a single UIO-66 nanomaterial is consistent with the proportion of coding with commercial MCP labels, and no Hf element signal is detected on the cells coded with UIO-66(Zr), and no Zr element signal is detected on the cells coded with UIO-66(Hf), proving that UIO-66 can be firmly adsorbed on the coded cells without falling off to affect the coding results.

[0156] Example 11

[0157] (1) Take 3x10^6 white blood cells, wash with Fluidigm cell staining buffer;

[0158] (2) Add 10 μL of Fluidigm blocking solution, block for 10 minutes;

[0159] (3) The cell suspension was equally divided into 3 parts;

[0160] (4) 1 μg UIO-66(Zr) and 1 μL 150Sm-MCP_CD45 were added to one part of the cells, 1 μg UIO-66(Zr) and 1 μL 154Sm-MCP_CD45 were added to another part of the cells, and 1 μg UIO-66(Zr), 0.5 μg UIO-66(Hf) and 1 μL 151Eu-MCP_CD45 were added to the third part of the cells, and incubated at room temperature for 45 minutes;

[0161] (5) The cell nuclei were stained for 1 hour using Fluidigm cell Ir-DNA intercalator;

[0162] (6) The cells were washed twice using Fluidigm cell staining buffer and once using deionized water;

[0163] (7) The cells were dispersed in 1 mL of water solution containing 10% Fluidigm EQ Beads;

[0164] (8) The cell samples were tested using a mass cytometry flow cytometer;

[0165] (9) The test results were analyzed using Cytobank software, and 90Zr positive / 180Hf negative, 90Zr negative / 180Hf positive, 150Sm positive, 151Eu positive and 154Sm positive cells were gated, and the encoding ability of UIO-66 labels and MCP labels on cells was compared and analyzed.

[0166] Reference Figure 9 For the test and analysis results of the present embodiment, it can be seen from the test results that the use of two kinds of nanomaterials, UIO-66(Zr) and UIO-66(Zr), to simultaneously encode a kind of cell can also effectively distinguish it from other cells, and therefore it is feasible to prepare a plurality of UIO-66 nanomaterials using different isotopes (e.g. 90 Zr, 91 Zr, 92 Zr, 94 Zr, 174 Hf, 176 Hf, 177 Hf, 178 Hf, 179 Hf, 180 Hf) and to use a plurality of UIO-66 nanomaterials to combine encoding of cells.

[0167] While embodiments of the application have been disclosed in connection with the preferred embodiments of the application, as illustrated in the drawings and described above, those skilled in the art will readily appreciate that yet other modifications can be made to the application without departing from the concept and scope of the application as set forth in the claims and equivalents thereof.

Claims

1. A mass spectrometry flow cytometry universal cell coding reagent based on UIO-66 nanomaterial, characterized in that, It is a mixture of UIO-66(Zr) nanomaterial and UIO-66(Hf) nanomaterial; The UIO-66(Hf) nanomaterial is prepared by the following steps: The anhydrous hafnium chloride and terephthalic acid are respectively dissolved in a solvent, the prepared two solutions are mixed, the obtained mixture is transferred to a polytetrafluoroethylene-lined reaction kettle, formic acid is added, and the reaction is carried out under heating, after the reaction is completed, it is cooled to room temperature, centrifuged, and the obtained solid is washed to obtain the UIO-66(Hf) nanomaterial.

2. The UIO-66 nanomaterial-based mass spectrometric flow cytometric universal cell coding reagent according to claim 1, characterized in that, The UIO-66(Zr) nanomaterial is prepared by the following steps: The ZrCl4 and terephthalic acid are respectively dissolved in a solvent, the prepared two solutions are mixed, the obtained mixture is transferred to a polytetrafluoroethylene-lined reaction kettle, formic acid is added, and the reaction is carried out under heating, after the reaction is completed, it is cooled to room temperature, centrifuged, and the obtained solid is washed to obtain the UIO-66(Zr) nanomaterial.

3. The UIO-66 nanomaterial-based mass spectrometric flow cytometric universal cell coding reagent according to claim 2, characterized in that, The solvent is N,N-dimethylformamide.

4. The UIO-66 nanomaterial-based mass spectrometric flow cytometric universal cell coding reagent according to claim 3, characterized in that, The UIO-66(Hf) nanomaterial is prepared by the following steps: S1-1, 0.75-3mmol of anhydrous hafnium chloride and 2.25-9mmol of terephthalic acid are respectively dissolved in 15-60mL of N,N-dimethylformamide, and ultrasonic is carried out until complete dissolution, the prepared two solutions are mixed; S1-2, the obtained mixture is transferred to a polytetrafluoroethylene-lined reaction kettle, 0.9-2.4mol / L of formic acid is added, and the reaction is carried out at 80-100℃ for 6-18 hours; S1-3, after the reaction is completed, it is cooled to room temperature, centrifuged at 10000-40000g for 15-60 minutes, and the supernatant is removed; S1-4, 17.5-70mL of N,N-dimethylformamide is added, vortexed, ultrasonic, resuspended, washed, and the step is repeated at least once; S1-5, the obtained solid is washed with ethanol several times and deionized water several times to obtain the UIO-66(Hf) nanomaterial.

5. The UIO-66 nanomaterial-based mass spectrometric flow cytometric universal cell coding reagent according to claim 4, characterized in that, The UIO-66(Hf) nanomaterial is prepared by the following steps: S1-1, 1.5mmol of anhydrous hafnium chloride and 4.5mmol of terephthalic acid are respectively dissolved in 30mL of N,N-dimethylformamide, and ultrasonic is carried out until complete dissolution, the prepared two solutions are mixed; S1-2, the obtained mixture is transferred to a polytetrafluoroethylene-lined reaction kettle, 0.9mol / L of formic acid is added, and the reaction is carried out at 90℃ for 6 hours; S1-3, after the reaction is completed, it is cooled to room temperature, centrifuged at 20000g for 30 minutes, and the supernatant is removed; S1-4, 35mL of N,N-dimethylformamide is added, vortexed, ultrasonic, resuspended, washed, and the step is repeated once; S1-5, the obtained solid is washed with ethanol once and deionized water twice to obtain the UIO-66(Hf) nanomaterial.

6. The UIO-66 nanomaterial-based mass spectrometric flow cytometric universal cell coding reagent according to claim 3, characterized in that, The UIO-66(Zr) nanomaterial is prepared by the following steps: S2-1. Dissolve 0.75-3 mmol ZrCl4 and 2.25-9 mmol terephthalic acid in 15-60 mL N,N-dimethylformamide, respectively, and sonicate until completely dissolved. Mix the two solutions obtained. S2-2. The resulting mixture is transferred to a polytetrafluoroethylene-lined reactor, and 0.9-2.4 mol / L of formic acid is added. The mixture is reacted at 80-100℃ for 6-18 hours. S2-3. After the reaction is complete, cool to room temperature, centrifuge at 10000-40000g for 15-60 minutes, and discard the supernatant; S2-4. Add 17.5-70 mL of N,N-dimethylformamide, vortex, sonicate, resuspend, wash, and repeat this step at least once. S2-5. The obtained solid is washed several times with ethanol and several times with deionized water to obtain the UIO-66(Zr) nanomaterial.

7. The UIO-66 nanomaterial-based mass spectrometric flow cytometric universal cell coding reagent according to claim 6, characterized in that, The UIO-66(Zr) nanomaterial was prepared by the following steps: S2-1. Dissolve 1.5 mmol ZrCl4 and 4.5 mmol terephthalic acid in 30 mL N,N-dimethylformamide, respectively, and sonicate until completely dissolved. Mix the two solutions obtained. S2-2. The resulting mixture is transferred to a polytetrafluoroethylene-lined reactor, and 0.9 mol / L formic acid is added. The mixture is reacted at 90°C for 6 hours. S2-3. After the reaction is complete, cool to room temperature, centrifuge at 20000g for 30 minutes, and discard the supernatant; S2-4. Add 35 mL of N,N-dimethylformamide, vortex, sonicate, resuspend, wash, and repeat this step once. S2-5. The obtained solid is washed once with ethanol and twice with deionized water to obtain the UIO-66(Zr) nanomaterial.

8. A mass spectrometry flow cytometry universal cell coding reagent based on UIO-66 nanomaterials, characterized in that, It is a mixture of fluorescein-modified UIO-66(Zr) nanomaterials and fluorescein-modified UIO-66(Hf) nanomaterials; The fluorescein-modified UIO-66(Zr) nanomaterials were prepared by the following method: 1-1) Disperse 0.5-2 mg of the UIO-66(Zr) nanomaterial as described in any one of claims 1-7 in 0.5-2 mL of deionized water, and wash by centrifugation; 1-2) Add 5-20 μL of 1-ethyl-(3-dimethylaminopropyl)carbodiimide aqueous solution with a concentration of 5-20 mg / mL and 5-20 μL of N-hydroxysuccinimide aqueous solution with a concentration of 5-20 mg / mL to the product of step 1), and incubate in the dark for 15-60 minutes. 1-3) After washing several times with deionized water, add 25-100 μg of 5-aminofluorescein and incubate in the dark for 1.5-6 hours; 1-4) Centrifuge and wash several times with deionized water to obtain the fluorescein-modified UIO-66(Zr) nanomaterials; The fluorescein-modified UIO-66(Hf) nanomaterials were prepared by the following method: 1-1) Disperse 0.5-2 mg of the UIO-66(Hf) nanomaterial as described in any one of claims 1-7 in 0.5-2 mL of deionized water, and wash by centrifugation; 1-2) Add 5-20 μL of 1-ethyl-(3-dimethylaminopropyl)carbodiimide aqueous solution with a concentration of 5-20 mg / mL and 5-20 μL of N-hydroxysuccinimide aqueous solution with a concentration of 5-20 mg / mL to the product of step 1), and incubate in the dark for 15-60 minutes. 1-3) After washing several times with deionized water, add 25-100 μg of 5-aminofluorescein and incubate in the dark for 1.5-6 hours; 1-4) Centrifuge and wash several times with deionized water to obtain the fluorescein-modified UIO-66(Hf) nanomaterial.

9. The UIO-66 nanomaterial-based mass spectrometric flow cytometric universal cell coding reagent according to claim 8, characterized in that, The fluorescein-modified UIO-66(Zr) nanomaterials were prepared by the following method: 1-1) Disperse 1 mg of UIO-66(Zr) nanomaterial as described in any one of claims 1-7 in 1 mL of deionized water, and wash twice by centrifugation at 20000 g; 1-2) Add 10 μL of 1-ethyl-(3-dimethylaminopropyl)carbodiimide aqueous solution with a concentration of 10 mg / mL and 10 μL of N-hydroxysuccinimide aqueous solution with a concentration of 10 mg / mL to the product of step 1), and incubate in the dark for 30 minutes. 1-3) After washing twice with deionized water, add 50 μg of 5-aminofluorescein and incubate in the dark for 3 hours; 1-4) Centrifuge and wash twice with deionized water to obtain the fluorescein-modified UIO-66(Zr) nanomaterials; The fluorescein-modified UIO-66(Hf) nanomaterials were prepared by the following method: 1-1) Disperse 1 mg of UIO-66(Hf) nanomaterial as described in any one of claims 1-7 in 1 mL of deionized water, and wash twice by centrifugation at 20000 g; 1-2) Add 10 μL of 1-ethyl-(3-dimethylaminopropyl)carbodiimide aqueous solution with a concentration of 10 mg / mL and 10 μL of N-hydroxysuccinimide aqueous solution with a concentration of 10 mg / mL to the product of step 1), and incubate in the dark for 30 minutes. 1-3) After washing twice with deionized water, add 50 μg of 5-aminofluorescein and incubate in the dark for 3 hours; 1-4) Centrifuge and wash twice with deionized water to obtain the fluorescein-modified UIO-66(Hf) nanomaterial.

10. The use of a cell-encoding reagent as described in any one of claims 1-7 or any one of claims 8-9 in mass flow cytometry cell encoding.

Images