Rare earth metal coordination photo-graft polymer metal tag for mass spectrometry flow cytometry and preparation method of rare earth metal coordination photo-graft polymer metal tag

Abstract

The invention discloses a rare earth metal coordination photo-graft polymer metal tag for mass spectrometry flow cytometry and a preparation method thereof, photo-initiation monomers are polymerized to a main chain, active ester monomers are polymerized to a branched chain through photo-initiation, DOTA coordination groups are coupled to the branched chain, and the rare earth metal coordination photo-graft polymer metal tag for mass spectrometry flow cytometry is obtained. The number of DOTA coordination groups on a single polymer is greatly increased, so that the number of metal ions capable of being coordinated on a single label can be increased, and the mass spectrum flow type detection sensitivity is improved; the active ester monomer is polymerized through photo-initiation, the reaction time is short, heating is not needed, the preparation is faster, simpler and more convenient, and the water solubility of the polymer label is greatly improved through introduction of an ethylene glycol structural unit; the raw materials for preparing the photo-graft polymer are all domestic, so that the use cost of the mass spectrum flow technique can be greatly reduced, and the application of the mass spectrum flow technique in the fields of clinic, biomedical examination and life science is promoted.

Description

Technical Field

[0001] The invention relates to the technical field of biological imaging, and in particular to a rare earth metal coordinated photografted polymer metal label for mass spectrometry flow cytometry and a preparation method thereof. Background Art

[0002] At present, the internationally popular immunophenotyping method flow cytometry is based on the detection of fluorescence emission spectra, but the emission spectra of fluorescent groups are generally wide. When multi-parameter detection is performed, the emission spectra of different fluorescent elements often overlap, which will lead to two problems: ① limited detection flux; ② increased detection difficulty. In order to ensure the accuracy of the data, fluorescence compensation adjustment must be performed during multi-parameter detection, which greatly increases the complexity of the detection and is a technical problem that is difficult to overcome by traditional fluorescence flow cytometry. Mass spectrometry technology combines flow cytometry with mass spectrometry technology. It is based on attaching specially designed metal element labels to antibodies instead of fluorescent labels, and uses the high resolution, sensitivity and analysis speed of inductively coupled plasma time-of-flight mass spectrometry for analysis and determination. Mass spectrometry can use up to hundreds of stable metal isotopes as metal labels. By quantifying the types and intensities of stable metal isotopes bound to target biomarkers, it can achieve simultaneous detection of multiple proteins and gene transcripts at the single-cell level.

[0003] The metal labels commonly used in mass spectrometry are linear polymer labels, usually linear polyurethanes containing multiple oxygen- and nitrogen-donating ligands that can coordinate with trivalent metal ions, such as 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) and diethylenetriaminepentaacetic acid (DTPA). After binding with monoclonal antibodies, they can be used for single cell identification and detection. However, these linear polymers only have about 20-50 ligands per polymer chain. In other words, each polymer label can only bind to about 20-50 metal ions. The mass spectrometer detector counts 3 to 10 lanthanide ions injected into the instrument. Each antibody can carry about 100 metal atoms after coupling with the polymer label, so the antibody needs to bind to 100 labels to produce a detection signal. Therefore, polymer metal labels are currently mainly used to detect medium and high concentration biomarkers (10 4 -10 7 / per cell), it is impossible to detect low-abundance markers, so it is necessary to develop highly sensitive metal labels. However, simply increasing the number of metal coordination groups on the polymer chain by increasing the polymer chain length will lead to a decrease in the water solubility of the polymer, thereby affecting the polymer and antibody labeling efficiency.

[0004] Therefore, it is necessary to improve the existing technology to provide a more reliable solution. Summary of the invention

[0005] The technical problem to be solved by the present invention is to provide a rare earth metal coordinated photografted polymer metal label for mass spectrometry flow cytometry and a preparation method thereof in view of the deficiencies in the above-mentioned prior art. The present invention provides a photografted polymer with multiple DOTA ligands, one end of the polymer has a maleimide group, which can be used to couple antibodies, and the polymer contains multiple ethylene glycol units, which can improve the water solubility of the polymer. After the polymer label is coupled with the antibody, a large number of rare earth metal ions coordinated on the grafted polymer can provide the metal signal required for mass spectrometry flow detection.

[0006] In order to solve the above technical problems, the technical solution adopted by the present invention is: a method for preparing a rare earth metal coordinated photografted polymer metal label for mass spectrometry flow cytometry, comprising the following steps:

[0007] S1. Preparation of photoinitiator monomer polymer P1:

[0008] The main chain of the photoinitiator monomer polymer P1 used in the present invention is synthesized by reversible addition-fragmentation chain transfer polymerization. The monomers used are 2-(4-(2-hydroxy-2-methylpropionyl)phenoxy)ethyl acrylate (photoinitiator monomer) and N,N-dimethylacrylamide (comonomer, DMA), the initiator is azobisisobutyronitrile (AIBN) or azobisisovaleronitrile (AMBN), and the chain transfer agent is tert-butyl dithiobenzoate. The polymerization reaction solvent is anhydrous N,N-dimethylformamide (DMF) or 1,4-dioxane. Each polymer chain of the obtained polymer contains about 10 to 20 photoinitiator monomers (n=10 to 20) and 20 to 40 N,N-dimethylacrylamide (m=20 to 40), one end of the polymer chain is a dithiobenzoate group, and the other end is a tert-butyl group.

[0009] The synthetic route is as follows:

[0010]

[0011] The specific steps include:

[0012] S1-1, adding N,N-dimethylacrylamide, 2-(4-(2-hydroxy-2-methylpropionyl)phenoxy)ethyl acrylate, an initiator, and tert-butyl dithiobenzoate into a polymerization reaction solvent;

[0013] S1-2, heating and stirring the reaction under nitrogen protection;

[0014] S1-3. After the reaction is completed, the polymer is precipitated with ether, and the unreacted raw materials are washed away to obtain a photoinitiator monomer polymer P1.

[0015] S2. Preparation of photografted polymer P2:

[0016] The photo-grafted polymer P2 used in the present invention is prepared by irradiating the photo-monomer unit on the main chain (P1) of the photo-initiated monomer with ultraviolet light to generate free radicals, thereby initiating the polymerization of the polymerization monomer along the photo-monomer to generate a branched chain. The monomers used are N-hydroxysuccinimide acrylate (NAS), N,N-dimethylacrylamide and ethylene glycol methyl ether acrylate, the wavelength of the ultraviolet light is 200nm-400nm, and the polymerization reaction solvent is anhydrous N,N-dimethylformamide or 1,4-dioxane. Each branch of the obtained polymer contains about 10 to 40 succinimide structures (y=10 to 40), 10 to 20 N,N-dimethylacrylamide structures (x=10 to 20) and 10 to 20 ethylene glycol methyl ether acrylate structures (z=10 to 20), and the main polymer contains about 100 to 400 succinimide structures (n*y=100 to 400).

[0017] The synthetic route is as follows:

[0018]

[0019] The specific steps include:

[0020] S2-1, adding N,N-dimethylacrylamide, ethylene glycol methyl ether acrylate, photoinitiator monomer polymer P1, and N-hydroxysuccinimide acrylate into a polymerization reaction solvent;

[0021] S2-2, nitrogen protection, use ultraviolet light source to irradiate and stir the reaction at room temperature;

[0022] S2-3, after the reaction is completed, the polymer is precipitated with ether, and the unreacted raw materials are washed away to obtain the photografted polymer P2;

[0023] S3, preparation of rare earth metal coordinated photografted polymer P3:

[0024] S3-1, dissolving the photografted polymer P2 in a polymerization reaction solvent, adding triethylamine under nitrogen protection, adding a polymerization reaction solvent containing DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) under stirring, and stirring the reaction under nitrogen protection;

[0025] S3-2, after the reaction is completed, the polymerization solvent is removed by distillation under reduced pressure, trifluoroacetic acid is added to the obtained product, and the reaction is stirred under nitrogen;

[0026] S3-3, removing trifluoroacetic acid by distillation under reduced pressure, and extracting the product with deionized water;

[0027] S3-4, washing the product with water using an ultrafiltration tube, concentrating and then draining to obtain a rare earth metal coordinated photografted polymer P3;

[0028] S4. Preparation of antibody-conjugated photografted polymer P4:

[0029] S4-1, dissolving the rare earth metal coordinated photografted polymer P3 obtained in step S3 in a phosphate buffer containing DL-dithiothreitol, and heating and stirring to react;

[0030] S4-2, after the reaction is completed, the temperature is lowered to room temperature, the pH value of the solution is adjusted, and the product is washed using an ultrafiltration tube;

[0031] S4-3, dissolving the obtained concentrated solution in phosphate buffer, adding 150 mg of 1,8-di(maleimide)-3,6-dioxaoctane polymerization solvent, and stirring for reaction;

[0032] S4-14. Filter and remove the precipitate produced by the reaction. Wash the filtrate with water using an ultrafiltration tube. Drain the obtained concentrated solution and freeze-dry it to obtain the antibody-conjugated photografted polymer P4.

[0033] The synthetic routes of polymer P3 and polymer P4 are as follows:

[0034]

[0035] In the present invention, DOTA with an amino side chain is used to condense with an activated NHS ester group along the polymer side chain. The thioester derivative at one end of the polymer main chain will be cleaved into a primary thiol under the reaction conditions. The conjugate is treated with trifluoroacetic acid (TFA) to remove the protecting group to obtain a grafted polymer with a DOTA group on the side chain, and the total number of DOTA on the polymer is n*y=100 to 400. The grafted polymer reduces the disulfide bonds that may be generated in the above reaction to sulfhydryl groups in the presence of a reducing agent DL-dithiothreitol, and modifies the maleimide group to the grafted polymer main chain through a Michael addition reaction by adding a bismaleimide compound, so that it has the function of coupling with antibodies.

[0036] Preferably, step S1 specifically includes:

[0037] S1-1, add 0.24-0.96 g of N,N-dimethylacrylamide, 0.125-0.5 g of 2-(4-(2-hydroxy-2-methylpropionyl)phenoxy)ethyl acrylate, 4-16 mg of azobisisovaleronitrile, and 13.5-34 mg of tert-butyl dithiobenzoate to 10-40 mL of polymerization reaction solvent, and freeze and deoxygenate with liquid nitrogen;

[0038] S1-2, heat to 60-90°C under nitrogen protection, and stir to react for 1-16 hours.

[0039] S1-3. After the reaction is completed, cool to room temperature, add the reaction solution dropwise into 100-400 mL of ether, and filter; the obtained solid is redissolved in 5-20 mL of polymerization reaction solvent, and added dropwise into 100-400 mL of ether again, and filter; the obtained solid is vacuum dried overnight to obtain a photoinitiator monomer polymer P1.

[0040] Preferably, step S2 specifically includes:

[0041] S2-1, adding 0.48-1.92 g of N,N-dimethylacrylamide, 0.5-2 g of ethylene glycol methyl ether acrylate, 0.05-0.2 g of photoinitiator monomer polymer P1, and 1.23-4.92 g of N-hydroxysuccinimide acrylate to 22.5-90 mL of polymerization reaction solvent, and freezing with liquid nitrogen to deoxygenate;

[0042] S2-2, after deoxygenation is completed, nitrogen protection is used, and a 200nm-400nm ultraviolet light source is used for irradiation and stirring for 15-60 minutes at room temperature;

[0043] S2-3. After the reaction is completed, the reaction solution is added dropwise to 200-800 mL of ether and filtered. The obtained solid is redissolved in 5-20 mL of polymerization reaction solvent and added dropwise to 100-400 mL of ether again and filtered. The obtained solid is vacuum dried overnight to obtain the photografted polymer P2.

[0044] Preferably, step S3 specifically includes:

[0045] S3, preparation of rare earth metal coordinated photografted polymer P3:

[0046] S3-1, dissolving 50-200 mg of the photografted polymer P2 in 1.5-6 mL of a polymerization reaction solvent, adding 0.5-2 mL of triethylamine under nitrogen protection, adding 0.5-2 mL of a polymerization reaction solvent containing 100-400 mg of DOTA under stirring, and stirring under nitrogen protection for 6-24 hours;

[0047] S3-2, remove N,N-dimethylformamide by distillation under reduced pressure, add 1.5-6 mL of trifluoroacetic acid to the resulting oil, and stir under nitrogen for 6-24 hours;

[0048] S3-3, removing trifluoroacetic acid by distillation under reduced pressure, and extracting the product with deionized water;

[0049] S3-4. Wash the product with water using an ultrafiltration tube, concentrate it and then drain it to obtain the rare earth metal coordinated photografted polymer P3.

[0050] Preferably, step S4 specifically includes:

[0051] S4. Preparation of antibody-conjugated photografted polymer P4:

[0052] S4-1, dissolving the rare earth metal coordinated photografted polymer P3 obtained in step S3 in a phosphate buffer solution containing 10-40 mM DL-dithiothreitol, heating to 40-60° C. and stirring for 0.5-2 hours;

[0053] S4-2, after the reaction solution is cooled to room temperature, acetic acid is used to adjust the pH value of the solution to 3-5, and the product is washed with an ultrafiltration tube, and the washing liquid is acetic acid;

[0054] S4-3, dissolving the obtained concentrated solution in 2.5-10 mL of phosphate buffer, adding 0.5-2 mL of N,N-dimethylformamide solution containing 75-300 mg of 1,8-di(maleimide)-3,6-dioxaoctane, and stirring at room temperature for 0.5-2 hours;

[0055] S4-14. Filter and remove the precipitate produced by the reaction. Wash the filtrate with water using an ultrafiltration tube. Drain the obtained concentrated solution and freeze-dry it to obtain the antibody-conjugated photografted polymer P4.

[0056] Preferably, the initiator is azobisisobutyronitrile or azobisisovaleronitrile.

[0057] Preferably, the polymerization solvent is anhydrous N,N-dimethylformamide or 1,4-dioxane.

[0058] Preferably, the initiator is azobisisovaleronitrile and the polymerization solvent is anhydrous N,N-dimethylformamide.

[0059] Preferably, the method for preparing the rare earth metal coordinated photografted polymer metal label for mass spectrometry flow cytometry comprises the following steps:

[0060] S1. Preparation of photoinitiator monomer polymer P1:

[0061] S1-1, add 0.48 g of N,N-dimethylacrylamide, 0.25 g of 2-(4-(2-hydroxy-2-methylpropionyl)phenoxy)ethyl acrylate, 8 mg of azobisisovaleronitrile, and 27 mg of tert-butyl dithiobenzoate to 20 mL of dry anhydrous N,N-dimethylformamide, and perform liquid nitrogen freezing and deoxygenation cycles three times;

[0062] S1-2, heating to 60°C under nitrogen protection and stirring for 16 hours;

[0063] S1-3, after the reaction system is cooled to room temperature, the reaction solution is added dropwise to 200 mL of ether and filtered; the obtained solid is redissolved in 10 mL of N,N-dimethylformamide, and added dropwise to 200 mL of ether again and filtered; the obtained solid is vacuum dried overnight to obtain a photoinitiator monomer polymer P1;

[0064] S2. Preparation of photografted polymer P2:

[0065] S2-1, 0.96 g of N,N-dimethylacrylamide, 1.0 g of ethylene glycol methyl ether acrylate, 0.1 g of photoinitiator monomer polymer P1, and 2.46 g of N-hydroxysuccinimide acrylate were added to 45 mL of dry anhydrous N,N-dimethylformamide, and the system was frozen and deoxygenated with liquid nitrogen for three cycles;

[0066] S2-2, after deoxygenation, nitrogen protection was applied, and irradiation with a 400 nm ultraviolet light source and stirring were performed at room temperature for 1 hour;

[0067] S2-3, after the reaction is completed, the reaction solution is added dropwise to 400 mL of ether, filtered, the obtained solid is redissolved in 10 mL of N,N-dimethylformamide, and added dropwise to 200 mL of ether again, filtered, and the obtained solid is vacuum dried overnight to obtain a photografted polymer P2;

[0068] S3, preparation of rare earth metal coordinated photografted polymer P3:

[0069] S3-1, dissolve 100 mg of the photografted polymer P2 in 3 mL of dry N,N-dimethylformamide, add 1 mL of triethylamine under nitrogen protection, add 1 mL of N,N-dimethylformamide solution containing 200 mg of DOTA under stirring, and stir for 12 hours under nitrogen protection;

[0070] S3-2, remove N,N-dimethylformamide by distillation under reduced pressure, add 3 mL of trifluoroacetic acid to the resulting oil, and stir under nitrogen for 12 hours;

[0071] S3-3, removing trifluoroacetic acid by distillation under reduced pressure, and extracting the product with deionized water;

[0072] S3-4, washing the product three times with water using an ultrafiltration tube, concentrating and then draining to obtain a rare earth metal coordinated photografted polymer P3;

[0073] S4. Preparation of antibody-conjugated photografted polymer P4:

[0074] S4-1, dissolving the rare earth metal coordinated photografted polymer P3 obtained in step S3 in a phosphate buffer solution containing 20 mM DL-dithiothreitol, heating to 50° C. and stirring for 1 hour;

[0075] S4-2, after the reaction solution is cooled to room temperature, the pH value of the solution is adjusted to 4.0 using acetic acid, and the product is washed three times using an ultrafiltration tube, with the washing solution being 5 mM acetic acid;

[0076] S4-3, dissolving the obtained concentrated solution in 5 mL of phosphate buffer, adding 1 mL of N,N-dimethylformamide solution containing 150 mg of 1,8-di(maleimide)-3,6-dioxaoctane, and stirring at room temperature for 1 hour;

[0077] S4-14. Filter and remove the precipitate produced by the reaction. Wash the filtrate with water for 5 times using an ultrafiltration tube. Drain the obtained concentrated solution, freeze-dry it, and obtain the antibody-conjugated photografted polymer P4, which is stored at -20°C.

[0078] The present invention also provides a rare earth metal coordinated photografted polymer metal label for mass spectrometry flow cytometry, which is prepared by the method as described above.

[0079] The beneficial effects of the present invention are:

[0080] Different from the existing mass spectrometry linear polymer metal tags, the present invention polymerizes the photoinitiator monomer to the main chain, polymerizes the active ester monomer to the side chain through photoinitiation, and then couples the DOTA coordination group to the side chain, which greatly increases the number of DOTA coordination groups on a single polymer, thereby increasing the number of metal ions that can be coordinated on a single tag and improving the mass spectrometry detection sensitivity;

[0081] The active ester monomers in the present invention are polymerized by light initiation, with a short reaction time and no need for heating, making the preparation faster and easier. The introduction of the ethylene glycol structural unit greatly improves the water solubility of the polymer label.

[0082] In addition, a set of conventional metal label kits (containing 40*100 micrograms of linear polymers) is currently priced at 60,000 yuan and has a long delivery time, which limits the development of mass spectrometry technology. The raw materials for preparing the photografted polymers used in the present invention are all domestically produced, which can greatly reduce the cost of using mass spectrometry technology and promote the application of mass spectrometry technology in clinical, biomedical testing and life science fields. BRIEF DESCRIPTION OF THE DRAWINGS

[0083] Figure 1 is the 1H NMR spectrum of the photoinitiator monomer polymer P1;

[0084] Figure 2 is the 1H NMR spectrum of the photografted polymer P2;

[0085] Figure 3 1H NMR spectrum of the antibody-conjugated photografted polymer P4;

[0086] Figure 4 A roadmap for coupling antibody-conjugated photografted polymer P4 to antibodies;

[0087] Figure 5Results of rare earth metal coordinated photografted polymer metal tags for human peripheral blood mononuclear cell clustering. DETAILED DESCRIPTION

[0088] The present invention is further described in detail below in conjunction with embodiments so that those skilled in the art can implement the invention with reference to the description.

[0089] It should be understood that the terms such as “having”, “including” and “comprising” used herein do not exclude the existence or addition of one or more other elements or combinations thereof.

[0090] The test methods used in the following examples are conventional methods unless otherwise specified. The materials and reagents used in the following examples are all commercially available unless otherwise specified. In the following examples, if no specific conditions are specified, the experiments were carried out under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used, if the manufacturer is not specified, are all conventional products that can be purchased commercially.

[0091] Example 1 Preparation of Photoinitiator Monomer Polymer P1

[0092] S1-1, add 0.48 g of N,N-dimethylacrylamide, 0.25 g of 2-(4-(2-hydroxy-2-methylpropionyl)phenoxy)ethyl acrylate, 8 mg of azobisisovaleronitrile, and 27 mg of tert-butyl dithiobenzoate to 20 mL of dry anhydrous N,N-dimethylformamide, and perform liquid nitrogen freezing and deoxygenation cycles three times;

[0093] S1-2, heating to 60°C under nitrogen protection and stirring for 16 hours;

[0094] S1-3. After the reaction system was cooled to room temperature, the reaction solution was added dropwise to 200 mL of ether and filtered; the obtained solid was redissolved in 10 mL of N,N-dimethylformamide and added dropwise to 200 mL of ether again and filtered; the obtained solid was vacuum dried overnight to obtain 0.61 g of photoinitiator monomer polymer P1.

[0095] Reference Figure 1 , is the 1H NMR spectrum of the photoinitiated monomer polymer P1.

[0096] Example 2 Preparation of Photografted Polymer P2

[0097] S2-1, 0.96 g of N,N-dimethylacrylamide, 1.0 g of ethylene glycol methyl ether acrylate, 0.1 g of photoinitiator monomer polymer P1, and 2.46 g of N-hydroxysuccinimide acrylate were added to 45 mL of dry anhydrous N,N-dimethylformamide, and the system was frozen and deoxygenated with liquid nitrogen for three cycles;

[0098] S2-2, after deoxygenation, nitrogen protection was applied, and irradiation with a 400 nm ultraviolet light source and stirring were performed at room temperature for 1 hour;

[0099] S2-3. After the reaction was completed, the reaction solution was added dropwise to 400 mL of ether and filtered. The obtained solid was redissolved in 10 mL of N,N-dimethylformamide and added dropwise to 200 mL of ether again and filtered. The obtained solid was vacuum dried overnight to obtain 3.11 g of photografted polymer P2.

[0100] Reference Figure 2 , is the 1H NMR spectrum of the photografted polymer P2.

[0101] Example 3 Preparation of rare earth metal coordinated photografted polymer P3

[0102] S3-1, dissolve 100 mg of the photografted polymer P2 in 3 mL of dry N,N-dimethylformamide, add 1 mL of triethylamine under nitrogen protection, add 1 mL of N,N-dimethylformamide solution containing 200 mg of DOTA under stirring, and stir for 12 hours under nitrogen protection;

[0103] S3-2, remove N,N-dimethylformamide by distillation under reduced pressure, add 3 mL of trifluoroacetic acid to the resulting oil, and stir under nitrogen for 12 hours;

[0104] S3-3, removing trifluoroacetic acid by distillation under reduced pressure, and extracting the product with deionized water;

[0105] S3-4. Wash the product three times with water using an ultrafiltration tube, concentrate it and then drain it to obtain a yellow solid: rare earth metal coordinated photografted polymer P3.

[0106] Example 4 Preparation of Antibody-Conjugated Photografted Polymer P4

[0107] S4-1, dissolving the rare earth metal coordinated photografted polymer P3 obtained in Example 4 in a phosphate buffer solution (50 mM, pH=8.5) containing 20 mM DL-dithiothreitol, heating to 50° C. and stirring for 1 hour;

[0108] S4-2, after the reaction solution is cooled to room temperature, the pH value of the solution is adjusted to 4.0 using acetic acid, and the product is washed three times using a 5K MW CO ultrafiltration tube, with the washing solution being 5 mM acetic acid;

[0109] S4-3, dissolving the obtained concentrated solution in 5 mL of phosphate buffer (100 mM, pH 8.5), adding 1 mL of N,N-dimethylformamide solution containing 150 mg of 1,8-di(maleimide)-3,6-dioxaoctane, and stirring at room temperature for 1 hour;

[0110] S4-14. Filter and remove the precipitate produced by the reaction. Wash the filtrate with water for 5 times using a 5K MW CO ultrafiltration tube. Drain the obtained concentrate, freeze-dry and package it to obtain the antibody-conjugated photografted polymer P4, which is stored at -20°C.

[0111] Reference Figure 3 , is the 1H NMR spectrum of the antibody-conjugated photografted polymer P4.

[0112] Example 5 Photografted polymer (P4) labeled anti-CD45 antibody

[0113] (1) Take 100ug of anti-CD45 monoclonal antibody and add it to a 50k ultrafiltration tube containing 400uL PBS buffer and centrifuge at 12000g for 10 minutes.

[0114] (2) After centrifugation, add 100 μL of PBS buffer containing 4 mM tris(2-carboxyethyl)phosphine to the antibody concentrate, mix well by pipetting, and incubate at 37° C. for 30 minutes to open the disulfide bonds on the antibody.

[0115] (3) At the same time, 100 mg of the photografted polymer (P4) was dissolved in 400 uL PBS buffer, 5 mM 152GdCl3 aqueous solution was added, and incubated at 37°C for 30 minutes.

[0116] (4) After the incubation, use 300uL 20mM Tris-buffered saline buffer to wash the antibody and the photografted polymer labeled with 152Gd, respectively.

[0117] (5) The purified antibody and rare earth coordination polymer were mixed in a 50k ultrafiltration tube and incubated at 37°C for 90 minutes.

[0118] (6) Wash three times with 400uL 20mM Tris-buffered saline at 12000g.

[0119] (7) Collect the antibody labeled with 152Gd photografted polymer, detect the protein concentration, and dilute it to 0.5 mg / mL.

[0120] (8) Add 0.05% NaN3 as an antibody stabilizer and store at 4°C.

[0121] Example 6 Preparation of Antibody-Conjugated Photografted Polymer (P4) Labeled Anti-CD3 Antibody

[0122] (1) Take 100ug of anti-CD-3 monoclonal antibody and add it to a 50k ultrafiltration tube containing 400uL PBS buffer and centrifuge at 12000g for 10 minutes.

[0123] (2) After centrifugation, add 100 μL of PBS buffer containing 4 mM tris(2-carboxyethyl)phosphine to the antibody concentrate, mix well by pipetting, and incubate at 37° C. for 30 minutes to open the disulfide bonds on the antibody.

[0124] (3) At the same time, 100 mg of the photografted polymer (P4) was dissolved in 400 uL PBS buffer, 5 mM 156GdCl3 aqueous solution was added, and incubated at 37°C for 30 minutes.

[0125] (4) After the incubation, use 300uL 20mM Tris-buffered saline buffer to wash the antibody and the photografted polymer labeled with 156Gd, respectively.

[0126] (5) The purified antibody and rare earth coordination polymer were mixed in a 50k ultrafiltration tube and incubated at 37°C for 90 minutes.

[0127] (6) Wash three times with 400uL 20mM Tris-buffered saline at 12000g.

[0128] (7) Collect the antibody labeled with 156Gd photografted polymer, detect the protein concentration, and dilute it to 0.5 mg / mL.

[0129] (8) Add 0.05% NaN3 as an antibody stabilizer and store at 4°C.

[0130] Example 7 Preparation of Antibody-Conjugated Photografted Polymer (P4) Labeled Anti-CD8 Antibody

[0131] (1) Take 100ug of anti-CD-8 monoclonal antibody and add it to a 50k ultrafiltration tube containing 400uL PBS buffer and centrifuge at 12000g for 10 minutes.

[0132] (2) After centrifugation, add 100 μL of PBS buffer containing 4 mM tris(2-carboxyethyl)phosphine to the antibody concentrate, mix well by pipetting, and incubate at 37° C. for 30 minutes to open the disulfide bonds on the antibody.

[0133] (3) At the same time, 100 mg of the photografted polymer (P4) was dissolved in 400 uL PBS buffer, 5 mM 165 HoCl 3 aqueous solution was added, and incubated at 37°C for 30 minutes.

[0134] (4) After the incubation, use 300uL 20mM Tris-buffered saline buffer to wash the antibody and the photografted polymer labeled with 165Ho, respectively.

[0135] (5) The purified antibody and rare earth coordination polymer were mixed in a 50k ultrafiltration tube and incubated at 37°C for 90 minutes.

[0136] (6) Wash three times with 400uL 20mM Tris-buffered saline at 12000g.

[0137] (7) Collect the antibody labeled with 165Ho photo-grafted polymer, detect the protein concentration, and dilute it to 0.5 mg / mL.

[0138] (8) Add 0.05% NaN3 as an antibody stabilizer and store at 4°C.

[0139] Example 8 Mass spectrometry single cell detection

[0140] (1) Take 10 6 PBMC cells were washed twice with 0.5% BSA solution at 600 g.

[0141] (2) Resuspend the cells and add 2 μL of blocking solution for 10 minutes at room temperature.

[0142] (3) Add 1 μL 152Gd_CD45, 1 μL 156Gd_CD3, and 1 μL 165Ho_CD8 mixed antibody labels and incubate at room temperature for 45 minutes.

[0143] (4) Wash twice with 0.5% BSA solution at 600 g to remove excess antibody labels.

[0144] (5) Use Fluidigm Cell Ir-DNA Intercalator to stain the cell nuclei and place at room temperature for 1 hour.

[0145] (6) Wash once with 600 g of 0.5% BSA solution and twice with 600 g of deionized water.

[0146] (7) Resuspend the cells in 1 mL of deionized water containing 10% EQ beads and filter using a cell strainer.

[0147] (8) Computer testing, and the test results were analyzed using Cytobank.

[0148] Reference Figure 4, which is a roadmap for the coupling of antibody-conjugated photografted polymer P4 with antibodies. The DOTA group on the side chain of the P4 polymer can form a stable coordination with trivalent rare earth ions. The type of metal label depends on the relative atomic mass of the coordinated rare earth isotope, which is usually a rare earth element with a relative atomic mass between 138La-176Lu. Tri(2-carboxyethyl)phosphine is used to open the disulfide bond on the antibody to generate an active thiol group, which is chemically combined with the maleimide structure at the end of the P4 polymer main chain. The P4 polymer with rare earth isotopes is coupled to the corresponding antibody to prepare a rare earth isotope labeled antibody label. The antibody label is calibrated and stored at 4°C for subsequent cell labeling. Among them, the P4 polymer is used to label the anti-CD8 antibody with 165Ho isotope for testing, the Fluda polymer is used to label the anti-CD45 antibody with 152Gd isotope, and the anti-CD3 antibody is labeled with 156Gd isotope for T cell clustering.

[0149] According to the Fluida company's operating manual, the obtained antibody label was used to stain human peripheral blood mononuclear cells (PBMC). After the cells were washed and blocked, 1uL of antibody label was used for staining for 45 minutes per million cells. After washing away the excess antibody label, 1.6% was used for fixation and intracellular Ir staining. After staining, Fluida Helios mass spectrometer was used for detection, and single cell analysis was performed according to the type of rare earth isotopes and signal intensity on each cell, such as Figure 5 The three antibody labels CD45_152Gd, CD3_156Gd, and CD8_165Ho shown can effectively group PBMCs, among which 152Gd-positive cells are CD45+ cells, accounting for 99.83%, 152Gd and 156Gd double-positive cells are T cells, accounting for 54.89%, and 165Ho and 156Gd double-positive cells are CD8+T cells, accounting for 32.76% of the total T cells.

[0150] Although the embodiments of the present invention have been disclosed as above, they are not limited to the applications listed in the specification and the implementation modes. They can be fully applied to various fields suitable for the present invention. For those familiar with the art, additional modifications can be easily implemented. Therefore, without departing from the general concept defined by the claims and the scope of equivalents, the present invention is not limited to specific details.

Claims

1. A method for preparing a rare earth metal coordinated photografted polymer metal label for mass spectrometry flow cytometry, characterized in that: The following steps are involved: S1. Preparation of photoinitiator monomer polymer P1: S1-1, adding N,N-dimethylacrylamide, 2-(4-(2-hydroxy-2-methylpropionyl)phenoxy)ethyl acrylate, an initiator, and tert-butyl dithiobenzoate into a polymerization reaction solvent; S1-2, heating and stirring the reaction under nitrogen protection; S1-3, after the reaction is completed, ether is used to precipitate the polymer, and the unreacted raw materials are washed away to obtain a photoinitiator monomer polymer P1; S2. Preparation of photografted polymer P2: S2-1, adding N,N-dimethylacrylamide, ethylene glycol methyl ether acrylate, photoinitiator monomer polymer P1, and N-hydroxysuccinimide acrylate into a polymerization reaction solvent; S2-2, nitrogen protection, use ultraviolet light source to irradiate and stir the reaction at room temperature; S2-3, after the reaction is completed, the polymer is precipitated with ether, and the unreacted raw materials are washed away to obtain the photografted polymer P2; S3, preparation of rare earth metal coordinated photografted polymer P3: S3-1, dissolving the photografted polymer P2 in a polymerization reaction solvent, adding triethylamine under nitrogen protection, adding a polymerization reaction solvent containing DOTA under stirring, and stirring the reaction under nitrogen protection; S3-2, after the reaction is completed, the polymerization solvent is removed by distillation under reduced pressure, trifluoroacetic acid is added to the obtained product, and the reaction is stirred under nitrogen; S3-3, removing trifluoroacetic acid by distillation under reduced pressure, and extracting the product with deionized water; S3-4, washing the product with water using an ultrafiltration tube, concentrating and then draining to obtain a rare earth metal coordinated photografted polymer P3; S4. Preparation of antibody-conjugated photografted polymer P4: S4-1, dissolving the rare earth metal coordinated photografted polymer P3 obtained in step S3 in a phosphate buffer containing DL-dithiothreitol, and heating and stirring to react; S4-2, after the reaction is completed, the temperature is lowered to room temperature, the pH value of the solution is adjusted, and the product is washed using an ultrafiltration tube; S4-3, dissolving the obtained concentrated solution in phosphate buffer, adding 150 mg of 1,8-di(maleimide)-3,6-dioxaoctane polymerization solvent, and stirring for reaction; S4-14. Filter and remove the precipitate produced by the reaction. Wash the filtrate with water using an ultrafiltration tube. Drain the obtained concentrated solution and freeze-dry it to obtain the antibody-conjugated photografted polymer P4.

2. The method for preparing rare earth metal coordinated photografted polymer metal tags for mass spectrometry flow cytometry according to claim 1, characterized in that: Step S1 specifically includes: S1-1, add 0.24-0.96 g of N,N-dimethylacrylamide, 0.125-0.5 g of 2-(4-(2-hydroxy-2-methylpropionyl)phenoxy)ethyl acrylate, 4-16 mg of azobisisovaleronitrile, and 13.5-34 mg of tert-butyl dithiobenzoate to 10-40 mL of polymerization reaction solvent, and freeze and deoxygenate with liquid nitrogen; S1-2, heat to 60-90°C under nitrogen protection, and stir to react for 1-16 hours. S1-3. After the reaction is completed, cool to room temperature, add the reaction solution dropwise into 100-400 mL of ether, and filter; the obtained solid is redissolved in 5-20 mL of polymerization reaction solvent, and added dropwise into 100-400 mL of ether again, and filter; the obtained solid is vacuum dried overnight to obtain a photoinitiator monomer polymer P1.

3. The method for preparing rare earth metal coordinated photografted polymer metal labels for mass spectrometry flow cytometry according to claim 1, characterized in that: Step S2 specifically includes: S2-1, adding 0.48-1.92 g of N,N-dimethylacrylamide, 0.5-2 g of ethylene glycol methyl ether acrylate, 0.05-0.2 g of photoinitiator monomer polymer P1, and 1.23-4.92 g of N-hydroxysuccinimide acrylate to 22.5-90 mL of polymerization reaction solvent, and freezing with liquid nitrogen to deoxygenate; S2-2, after deoxygenation is completed, nitrogen protection is used, and a 200nm-400nm ultraviolet light source is used for irradiation and stirring for 15-60 minutes at room temperature; S2-3. After the reaction is completed, the reaction solution is added dropwise to 200-800 mL of ether and filtered. The obtained solid is redissolved in 5-20 mL of polymerization reaction solvent and added dropwise to 100-400 mL of ether again and filtered. The obtained solid is vacuum dried overnight to obtain the photografted polymer P2.

4. The method for preparing rare earth metal coordinated photografted polymer metal labels for mass spectrometry flow cytometry according to claim 1, characterized in that: Step S3 specifically includes: S3, preparation of rare earth metal coordinated photografted polymer P3: S3-1, dissolving 50-200 mg of the photografted polymer P2 in 1.5-6 mL of a polymerization reaction solvent, adding 0.5-2 mL of triethylamine under nitrogen protection, adding 0.5-2 mL of a polymerization reaction solvent containing 100-400 mg of DOTA under stirring, and stirring under nitrogen protection for 6-24 hours; S3-2, remove N,N-dimethylformamide by distillation under reduced pressure, add 1.5-6 mL of trifluoroacetic acid to the resulting oil, and stir under nitrogen for 6-24 hours; S3-3, removing trifluoroacetic acid by distillation under reduced pressure, and extracting the product with deionized water; S3-4. Wash the product with water using an ultrafiltration tube, concentrate it and then drain it to obtain the rare earth metal coordinated photografted polymer P3.

5. The method for preparing rare earth metal coordinated photografted polymer metal labels for mass spectrometry flow cytometry according to claim 1, characterized in that: Step S4 specifically includes: S4. Preparation of antibody-conjugated photografted polymer P4: S4-1, dissolving the rare earth metal coordinated photografted polymer P3 obtained in step S3 in a phosphate buffer solution containing 10-40 mM DL-dithiothreitol, heating to 40-60° C. and stirring for 0.5-2 hours; S4-2, after the reaction solution is cooled to room temperature, acetic acid is used to adjust the pH value of the solution to 3-5, and the product is washed with an ultrafiltration tube, and the washing liquid is acetic acid; S4-3, dissolving the obtained concentrated solution in 2.5-10 mL of phosphate buffer, adding 0.5-2 mL of N,N-dimethylformamide solution containing 75-300 mg of 1,8-di(maleimide)-3,6-dioxaoctane, and stirring at room temperature for 0.5-2 hours; S4-14. Filter and remove the precipitate produced by the reaction. Wash the filtrate with water using an ultrafiltration tube. Drain the obtained concentrated solution and freeze-dry it to obtain the antibody-conjugated photografted polymer P4.

6. The method for preparing rare earth metal coordinated photografted polymer metal tags for mass spectrometry flow cytometry according to claim 1, characterized in that: The initiator is azobisisobutyronitrile or azobisisovaleronitrile.

7. The method for preparing rare earth metal coordinated photografted polymer metal tags for mass spectrometry flow cytometry according to claim 6, characterized in that: The polymerization solvent is anhydrous N,N-dimethylformamide or 1,4-dioxane.

8. The method for preparing rare earth metal coordinated photografted polymer metal tags for mass spectrometry flow cytometry according to claim 7, characterized in that: The initiator is azobisisovaleronitrile, and the polymerization solvent is anhydrous N,N-dimethylformamide.

9. The method for preparing rare earth metal coordinated photografted polymer metal tags for mass spectrometry flow cytometry according to claim 1, characterized in that: The following steps are involved: S1. Preparation of photoinitiator monomer polymer P1: S1-1, add 0.48 g of N,N-dimethylacrylamide, 0.25 g of 2-(4-(2-hydroxy-2-methylpropionyl)phenoxy)ethyl acrylate, 8 mg of azobisisovaleronitrile, and 27 mg of tert-butyl dithiobenzoate to 20 mL of dry anhydrous N,N-dimethylformamide, and perform liquid nitrogen freezing and deoxygenation cycles three times; S1-2, heating to 60°C under nitrogen protection and stirring for 16 hours; S1-3, after the reaction system is cooled to room temperature, the reaction solution is added dropwise to 200 mL of ether and filtered; the obtained solid is redissolved in 10 mL of N,N-dimethylformamide, and added dropwise to 200 mL of ether again and filtered; the obtained solid is vacuum dried overnight to obtain a photoinitiator monomer polymer P1; S2. Preparation of photografted polymer P2: S2-1, 0.96 g of N,N-dimethylacrylamide, 1.0 g of ethylene glycol methyl ether acrylate, 10.1 g of photoinitiator monomer polymer P, and 2.46 g of N-hydroxysuccinimide acrylate were added to 45 mL of dry anhydrous N,N-dimethylformamide, and the system was frozen and deoxygenated with liquid nitrogen for three cycles; S2-2, after deoxygenation, nitrogen protection was applied, and irradiation with a 400 nm ultraviolet light source and stirring were performed at room temperature for 1 hour; S2-3, after the reaction is completed, the reaction solution is added dropwise to 400 mL of ether, filtered, the obtained solid is redissolved in 10 mL of N,N-dimethylformamide, and added dropwise to 200 mL of ether again, filtered, and the obtained solid is vacuum dried overnight to obtain a photografted polymer P2; S3, preparation of rare earth metal coordinated photografted polymer P3: S3-1, dissolve 100 mg of the photografted polymer P2 in 3 mL of dry N,N-dimethylformamide, add 1 mL of triethylamine under nitrogen protection, add 1 mL of N,N-dimethylformamide solution containing 200 mg of DOTA under stirring, and stir for 12 hours under nitrogen protection; S3-2, remove N,N-dimethylformamide by distillation under reduced pressure, add 3 mL of trifluoroacetic acid to the resulting oil, and stir under nitrogen for 12 hours; S3-3, removing trifluoroacetic acid by distillation under reduced pressure, and extracting the product with deionized water; S3-4, washing the product three times with water using an ultrafiltration tube, concentrating and then draining to obtain a rare earth metal coordinated photografted polymer P3; S4. Preparation of antibody-conjugated photografted polymer P4: S4-1, dissolving the rare earth metal coordinated photografted polymer P3 obtained in step S3 in a phosphate buffer solution containing 20 mM DL-dithiothreitol, heating to 50° C. and stirring for 1 hour; S4-2, after the reaction solution is cooled to room temperature, the pH value of the solution is adjusted to 4.0 using acetic acid, and the product is washed three times using an ultrafiltration tube, with the washing solution being 5 mM acetic acid; S4-3, dissolving the obtained concentrated solution in 5 mL of phosphate buffer, adding 1 mL of N,N-dimethylformamide solution containing 150 mg of 1,8-di(maleimide)-3,6-dioxaoctane, and stirring at room temperature for 1 hour; S4-14. Filter and remove the precipitate produced by the reaction. Wash the filtrate with water for 5 times using an ultrafiltration tube. Drain the obtained concentrated solution, freeze-dry it, and obtain the antibody-conjugated photografted polymer P4, which is stored at -20°C.

10. A rare earth metal coordinated photografted polymer metal tag for mass spectrometry flow cytometry, characterized in that: It is prepared by the method according to any one of claims 1 to 9.

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