A mass spectrometry flow cytometry universal element probe and its preparation method

Abstract

The present invention discloses a mass spectrometry flow cytometry universal element probe, which includes metal-doped polystyrene nanospheres; antibodies are modified on the surface of the metal-doped polystyrene nanospheres, and the metal includes at least one of rare earth metals, zirconium, and hafnium. The present invention also discloses a preparation method of the above-mentioned mass spectrometry flow cytometry universal element probe. The mass spectrometry flow cytometry element probe using the metal-doped polystyrene nanosphere material of the present invention can be doped with other types of metals in addition to rare earth elements, expands the mass spectrometry flow cytometry detection channel to metal channels other than rare earth elements, has high sensitivity, and can be used as a universal mass spectrometry flow cytometry element probe. Each step of the preparation method of the present invention is mature, the synthesis process is simple to operate, the size is uniform, and it has good biocompatibility, greatly reducing the cost of mass spectrometry flow cytometry detection reagents.

Description

Technical Field

[0001] The present invention belongs to the technical field of nanoprobe, and particularly relates to a mass cytometry universal elemental probe and a preparation method thereof. Background Art

[0002] Mass cytometry for high-throughput quantitative analysis of genes and proteins at the single-cell level is of great significance for hematology, oncology and immunology research. Different from traditional flow cytometry, mass cytometry uses heavy metal-labeled antibodies to conjugate cells, and then detects metal signals by inductively coupled plasma mass spectrometry to achieve cell population separation. This technology overcomes the disadvantages of fluorescence signal overlap and few detection channels in traditional flow cytometry, and can detect up to hundreds of parameters simultaneously for the same cell, greatly improving the detection efficiency. However, although current time-of-flight mass spectrometers can easily resolve 135 channels with atomic mass ranges from 75 to 209, more than 60% of the isotope channels are not actually used due to the lack of a suitable carrier to load heavy metal isotopes. Currently, the detection kits used in mass cytometry are mainly based on commercial Maxpar metalchelating polymer (MCP) reagents polymer elemental probes. Only 37 rare earth metals can be loaded on this polymer (corresponding to 37 detection channels), and only 50-100 metal ions can be conjugated to each label, making it impossible to detect low-abundance cell markers. Currently, mass cytometry elemental probes based on materials such as inorganic nanoparticles, quantum dots and polymer dots have also been developed. For example, Chinese patent document CN112834411A uses metal-organic framework materials as mass cytometry metal tags, and Chinese patent document CN113834802A uses lanthanide metal-doped carbon quantum dots as mass cytometry metal tags. However, these materials are difficult to synthesize and require complex surface modification to solve the problem of non-specific cell adsorption. In addition, these nanoprobes will determine the metal type at the beginning of synthesis, and can only load one metal element. Changing the metal type requires re-optimizing the synthesis conditions to maintain nanoparticle size, morphology, antibody conjugation performance, etc., and these properties cannot be stably maintained. It cannot provide a general strategy for synthesizing mass cytometry elemental probes. Summary of the Invention

[0003] Therefore, the technical problem to be solved by the present invention lies in the defects of the probes in the prior art, so as to provide a mass cytometry universal elemental probe and a preparation method thereof.

[0004] For this purpose, the present invention adopts the following technical solutions:

[0005] The present invention provides a mass cytometry universal elemental probe, including metal-doped polystyrene nanospheres;

[0006] The surface of the metal-doped polystyrene nanospheres is modified with an antibody, and the metal includes at least one of rare earth metals, zirconium, and hafnium.

[0007] Further, the antibody is modified on the surface of the metal-doped polystyrene nanospheres through an amino group.

[0008] The present invention also provides a preparation method of the above-mentioned mass spectrometry flow cytometry universal element probe, which includes the following steps:

[0009] S1: React a β-keto compound with a metal chloride to obtain a metal complex;

[0010] S2: Prepare carboxyl-modified polystyrene nanospheres by an emulsion polymerization method;

[0011] S3: React the metal complex obtained in step S1 with the carboxyl-modified polystyrene nanospheres obtained in step S2 to obtain metal-doped polystyrene nanospheres;

[0012] S4: Modify an antibody on the surface of the metal-doped polystyrene nanospheres to obtain the mass spectrometry flow cytometry universal element probe.

[0013] Further, in step S1, the molar ratio of the β-keto compound to the metal chloride is 4-10:1. The reaction is to dissolve the β-keto compound in ethanol or methanol, drop it into an aqueous solution of the corresponding metal chloride, add an alkali solution to adjust the pH value to 8-10, and stir for 4-5 hours until the reaction is completed;

[0014] The β-keto compound includes one of acetylacetone or β-diketone compounds, and the β-diketone compounds include 4,4,4-trifluoro-1-(2-furyl)-1,3-butanedione or 3-methyl-2,4-pentanedione;

[0015] The metal chloride includes at least one of rare earth metal, zirconium, and hafnium chlorides.

[0016] The emulsion polymerization method in step S2 is specifically as follows: Mix 15-30 mL of deionized water, 1-2 mL of styrene, 0.7-1.4 mL of methacrylic acid, and 150-300 mg of sodium bicarbonate and stir. During the stirring process, continuously introduce nitrogen. After heating to 70-90 °C, add 5-10 mg of potassium persulfate. After reacting for 30-60 min, add 0.1-0.2 mL of methacrylic acid and 5-10 mg of potassium persulfate, and react overnight.

[0017] In step S3, the mass ratio of the metal complex to the carboxyl-modified polystyrene nanospheres is 1:50. The specific preparation method is to dissolve the metal complex in dichloromethane, add an emulsifier and emulsify for 30 seconds, and then drop it into a 0.25 wt% aqueous solution of sodium dodecyl sulfate in which the carboxyl-modified polystyrene nanospheres are dispersed, and react overnight;

[0018] The emulsifier is at least one of sodium dodecyl sulfate, sodium dodecyl sulfonate, sodium dodecyl benzene sulfonate, sorbitan oleate, and sorbitan monostearate.

[0019] In step S4, the metal-doped polystyrene nanospheres are dispersed in a buffer solution, an aqueous solution of 1-ethyl-(3-dimethylaminopropyl)carbodiimide and an aqueous solution of N-hydroxysuccinimide are added, and the mixture is incubated in the dark for 30 minutes. After adding the antibody, the mixture is incubated in the dark for 3 hours to obtain the mass spectrometry flow cytometry universal element probe.

[0020] The mass ratio of the metal-doped polystyrene nanospheres to the antibody is 500:1 - 10.

[0021] Preferably, after preparing the metal complex in step S1, it further includes the steps of washing with ethanol and drying in a vacuum oven.

[0022] After preparing the carboxyl-modified polystyrene nanospheres in step S2, it further includes the steps of washing with ethanol and drying in a vacuum oven.

[0023] After preparing the metal-doped polystyrene nanospheres in step S3, it further includes the step of washing with ethanol.

[0024] After preparing the mass spectrometry flow cytometry universal element probe in step S4, it further includes the steps of washing with a buffer solution and storing in a buffer solution containing a preservative at 2 - 8°C.

[0025] The present invention also provides a mass spectrometry flow cytometry universal element probe prepared by the above preparation method.

[0026] The technical solution of the present invention has the following advantages:

[0027] (1) The mass spectrometry flow cytometry element probe of the present invention using metal-doped polystyrene nanosphere materials can be doped with other types of metals in addition to rare earth elements, expanding the mass spectrometry flow cytometry detection channels to metal channels other than rare earth elements; the number of metals on a single microsphere is on the order of 10 5 , which can increase the number of metal particles on a single label and significantly improve the detection sensitivity of mass spectrometry flow cytometry. The probe is prepared by doping metals into blank polystyrene microspheres, and changing the metal type will not affect the microsphere morphology, size, antibody conjugation performance, etc., and can be used as a universal mass spectrometry flow cytometry element probe.

[0028] (2) Different from the existing mass spectrometry flow cytometry polymer metal probes, the present invention ingeniously dopes metals into stable polystyrene nanospheres. The preparation method of each step is mature, the synthesis process is simple to operate, the size is uniform, and it has good biocompatibility, greatly reducing the cost of mass spectrometry flow cytometry detection reagents.

[0029] (3) When preparing polystyrene nanospheres in the present invention, polystyrene microspheres at the nanoscale are prepared by regulating the ratio of deionized water, monomers (styrene, methacrylic acid), and initiator (potassium persulfate), which expands the application of polystyrene microspheres. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following will briefly introduce the drawings required for the description of the specific embodiments or the prior art. Obviously, the drawings in the following description are some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0031] Figure 1 It is the synthesis route diagram of the mass spectrometry flow cytometry universal element probe in the embodiment of the present invention;

[0032] Figure 2 It is the scanning electron microscope photograph of the carboxyl-modified polystyrene nanospheres obtained in Example 1;

[0033] Figure 3 It is the mapping diagram of the metal-doped polystyrene nanospheres obtained in the examples, where (a) is the europium-doped polystyrene nanospheres in Example 1, (b) is the zirconium-doped polystyrene nanospheres in Example 2, (c) is the hafnium-doped polystyrene nanospheres in Example 3, and (d) is the lanthanum-doped polystyrene nanospheres in Example 4;

[0034] Figure 4 It is the particle size of the europium-doped polystyrene nanospheres obtained in Example 1 before and after antibody labeling, where A is before labeling and B is after labeling;

[0035] Figure 5 It is the surface potential diagram of the europium-doped polystyrene nanospheres obtained in Example 1 before and after antibody labeling, where A is before labeling and B is after labeling;

[0036] Figure 6 It is the sensitivity experiment result in the test example, where a is the mass spectrometry flow cytometry immune subpopulation result of human peripheral blood mononuclear cells by the FluoProbe polymer probe (1 μL 141-CD45, 1 μL 152-CD3, 1 μL 159-CD4) of Fluorochrome and 1 μL of the FluoProbe polymer probe 151-CD8 of Fluorochrome, and b is the mass spectrometry flow cytometry immune subpopulation result of human peripheral blood mononuclear cells by the FluoProbe polymer probe (1 μL 141-CD45, 1 μL 152-CD3, 1 μL 159-CD4) of Fluorochrome and 1 μL of the mass spectrometry flow cytometry 151Eu-CD8 probe obtained in Example 1 of the present application;

[0037] Figure 7These are the results of the detection channel expansion experiment in the experimental examples. Among them, a is the mass spectrometry flow cytometry immune cell sorting result of human peripheral blood mononuclear cells using the FluoProbes polymer probe (1 μL 151-CD45) from the United States; b is the mass spectrometry flow cytometry immune cell sorting result of human peripheral blood mononuclear cells using the mass spectrometry flow cytometry 90Zr-CD45 probe; c is the mass spectrometry flow cytometry immune cell sorting result of human peripheral blood mononuclear cells using the mass spectrometry flow cytometry 180Hf-CD45 probe; d is the mass spectrometry flow cytometry immune cell sorting result of human peripheral blood mononuclear cells using the mass spectrometry flow cytometry 139La-CD45 probe; e is the mass spectrometry flow cytometry immune cell sorting result of human peripheral blood mononuclear cells using the FluoProbes polymer probe (1 μL 152-CD3) from the United States in combination with the polymer probe (1 μL 151-CD45); f is the mass spectrometry flow cytometry immune cell sorting result of human peripheral blood mononuclear cells using the polymer probe (1 μL 152-CD3) in combination with the mass spectrometry flow cytometry 90Zr-CD45 probe; g is the mass spectrometry flow cytometry immune cell sorting result of human peripheral blood mononuclear cells using the polymer probe (1 μL 152-CD3) in combination with the mass spectrometry flow cytometry 180Hf-CD45 probe; h is the mass spectrometry flow cytometry immune cell sorting result of human peripheral blood mononuclear cells using the polymer probe (1 μL 152-CD3) in combination with the mass spectrometry flow cytometry 139La-CD45 probe. Detailed implementation manners

[0038] The following embodiments are provided to better further understand the present invention. They are not limited to the best implementation manner, and do not constitute a limitation to the content and protection scope of the present invention. Any product that is the same as or similar to the present invention obtained by anyone under the inspiration of the present invention or by combining the features of the present invention with those of other prior arts falls within the protection scope of the present invention.

[0039] For those experimental steps or conditions not specified in the embodiments, the operations or conditions of the conventional experimental steps described in the literature in this field can be followed.

[0040] The antibodies used in the detailed implementation manners are from biolegend.

[0041] The following specific embodiments are further descriptions of the present invention. The examples given cannot list all the implementation manners of the present invention. Only some of the implementation manners are taken as examples for illustration. The specific embodiments are as follows:

[0042] Example 1

[0043] This example provides a mass spectrometry flow cytometry universal element probe, and its synthesis route is as Figure 1 shown. The specific preparation method is as follows:

[0044] 1. Preparation of europium metal complex:

[0045] (1) Dissolve 440 mg of acetylacetone in 15 mL of ethanol and stir rapidly;

[0046] (2) Add 15 mL of 0.05 M EuCl3 aqueous solution dropwise. After the addition is complete, add sodium hydroxide solution to adjust the pH value to 9;

[0047] (3) Add 50 mL of water and stir for another 5 hours;

[0048] (4) Rotavap off most of the ethanol and then filter and dry;

[0049] (5) Wash the obtained solid with 20 mL of cold ethanol and dry it in a vacuum oven to obtain europium acetylacetonate complex.

[0050] 2. Preparation of carboxyl-modified polystyrene nanospheres

[0051] (1) Add 15 mL of deionized water, 1 mL of styrene, 0.7 mL of methacrylic acid, and 150 mg of sodium bicarbonate to a three-necked flask equipped with a condenser, mix, and stir rapidly;

[0052] (2) Continuously pass high-purity nitrogen into the solution to remove oxygen for 30 minutes;

[0053] (3) Heat to 80 °C and add an aqueous solution containing 5 mg of KPS initiator;

[0054] (4) After reacting for 30 min, add 0.1 mL of methacrylic acid and 5 mg of KPS initiator, and react overnight;

[0055] (5) Centrifuge at high speed and wash with ethanol. The structure is as shown in Figure 2 shown, spherical with a size of about 200 nm, and dry it in a vacuum oven for standby.

[0056] 3. Europium-doped polystyrene nanospheres:

[0057] (1) Take 50 mg of polystyrene nanospheres and disperse them in 50 mL of 0.25% sodium dodecyl sulfate aqueous solution, and stir rapidly;

[0058] (2) Dissolve 1 mg of europium complex in 1 mL of dichloromethane;

[0059] (3) Add 10 mL of 0.25% sodium dodecyl sulfate aqueous solution and emulsify at high speed for 30 seconds;

[0060] (4) Drop the emulsion into the polystyrene nanosphere solution in (1) and stir overnight;

[0061] (5) Rotavap off dichloromethane and centrifuge;

[0062] (6) Centrifuge and wash with ethanol at 8000 rpm for 15 min, repeat three times to obtain europium-doped polystyrene nanospheres, as shown in Figure 3As shown in Figure (a) of the mapping diagram, the metal was uniformly doped into the interior of the nanospheres.

[0063] 4. Preparation of the mass cytometry 151Eu-CD8 probe:

[0064] (1) Disperse 0.5 mg of europium-doped polystyrene nanospheres in 1 mL of 4-morpholineethanesulfonic acid buffer solution with pH = 6.2, and stir vigorously.

[0065] (2) Add 10 μL of aqueous solutions of 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide (10 mg / mL) respectively, and activate in the dark for 30 minutes.

[0066] (3) Centrifuge, wash three times with 4-morpholineethanesulfonic acid buffer solution with pH = 6.2, and disperse with 0.1 mL of 4-morpholineethanesulfonic acid buffer solution with pH = 6.2.

[0067] (4) Add 10 μg of CD8 antibody, incubate in the dark for 1 hour to obtain the mass cytometry 151Eu-CD8 probe. From Figure 4 the particle size before and after the labeled antibody and Figure 5 the surface potential diagram before and after the labeled antibody, it can be seen that the particle size of the nanospheres increased from 200 nm to 236 nm after antibody labeling, and the potential increased from -29.2 mV to -20.8 mV, proving that the antibody was successfully labeled on the surface of the nanospheres.

[0068] (5) Wash 3 times with 4-morpholineethanesulfonic acid buffer solution with pH = 6.2 and store at 4°C.

[0069] Example 2

[0070] This example provides a mass cytometry universal element probe, and its synthesis route is as Figure 1 shown. The specific preparation method is as follows:

[0071] 1. Preparation of hafnium metal complex

[0072] (1) Dissolve 440 mg of acetylacetone in 15 mL of ethanol and stir rapidly.

[0073] (2) Dropwise add 15 mL of 0.05 M aqueous solution of HfCl4, and after the addition is completed, add sodium hydroxide solution to adjust the pH value to 9.

[0074] (3) Add 50 mL of water and stir for another 5 hours.

[0075] (4) Rotate off most of the ethanol and then filter and dry.

[0076] (5) Wash the obtained solid with 20 mL of cold ethanol and dry in a vacuum oven to obtain hafnium acetylacetonate complex.

[0077] 2. Preparation of carboxyl - modified polystyrene nanospheres:

[0078] (1) Add 15 mL of deionized water, 1 mL of styrene, 0.7 mL of methacrylic acid, and 150 mg of sodium bicarbonate into a three - necked flask equipped with a condenser, and mix with rapid stirring;

[0079] (2) Continuously introduce high - purity nitrogen into the solution to remove oxygen for 30 minutes;

[0080] (3) Heat to 80 °C and add an aqueous solution containing 5 mg of KPS initiator;

[0081] (4) After reacting for 30 min, add 0.1 mL of methacrylic acid and 5 mg of KPS initiator, and react overnight;

[0082] (5) Centrifuge at high speed, wash with ethanol, and dry in a vacuum oven for standby.

[0083] 3. Hafnium - doped polystyrene nanospheres

[0084] (1) Take 50 mg of polystyrene nanospheres and disperse them in 50 mL of 0.25% sodium dodecyl sulfate aqueous solution, and stir rapidly

[0085] (2) Dissolve 1 mg of hafnium complex in 1 mL of dichloromethane;

[0086] (3) Add 10 mL of 0.25% sodium dodecyl sulfate aqueous solution and emulsify at high speed for 30 seconds;

[0087] (4) Drop this emulsion into the polystyrene nanosphere solution in (1) and stir overnight;

[0088] (5) Rotavap to remove dichloromethane and centrifuge;

[0089] (6) Centrifuge and wash with ethanol at 8000 rpm for 15 min, repeat three times to obtain hafnium - doped polystyrene nanospheres, as shown in Figure (b) of the mapping diagram in Figure 3 The metal is uniformly doped into the interior of the nanospheres;

[0090] 4. Preparation of mass cytometry 180Hf - CD45 probe:

[0091] (1) Disperse 0.5 mg of hafnium - doped polystyrene nanospheres in 1 mL of 4 - morpholineethanesulfonic acid buffer solution with pH = 6.2, and stir vigorously;

[0092] (2) Add 10 μL of aqueous solutions of 1 - ethyl - (3 - dimethylaminopropyl) carbodiimide and N - hydroxysuccinimide (10 mg / mL) respectively, and activate in the dark for 30 minutes;

[0093] (3) Centrifuge, wash three times with 4-morpholineethanesulfonic acid buffer solution with pH = 6.2, and disperse with 0.1 mL of 4-morpholineethanesulfonic acid buffer solution with pH = 6.2;

[0094] (4) Add 10 μg of CD45 antibody and incubate in the dark for 1 hour to obtain a mass spectrometry flow cytometry 180Hf-CD45 probe;

[0095] (5) Wash 3 times with 4-morpholineethanesulfonic acid buffer solution with pH = 6.2 and store at 4 °C.

[0096] Example 3

[0097] This example provides a mass spectrometry flow cytometry universal element probe, and its synthesis route is as Figure 1 shown, and the specific preparation method is as follows:

[0098] 1. Preparation of zirconium metal complex:

[0099] (1) Dissolve 440 mg of acetylacetone in 15 mL of ethanol and stir rapidly;

[0100] (2) Dropwise add 15 mL of 0.05 M ZrCl4 aqueous solution. After the addition is completed, add sodium hydroxide solution to adjust the pH value to 9;

[0101] (3) Add 50 mL of water and stir for another 5 hours;

[0102] (4) Spin off most of the ethanol and then filter and dry;

[0103] (5) Wash the obtained solid with 20 mL of cold ethanol and dry in a vacuum oven to obtain zirconium acetylacetonate complex.

[0104] 2. Preparation of carboxyl-modified polystyrene nanospheres

[0105] (1) Add 15 mL of deionized water, 1 mL of styrene, 0.7 mL of methacrylic acid, and 150 mg of sodium bicarbonate to a three-necked flask equipped with a condenser and mix and stir rapidly;

[0106] (2) Continuously pass high-purity nitrogen into the solution to remove oxygen for 30 minutes;

[0107] (3) Heat to 80 °C and add an aqueous solution containing 5 mg of KPS initiator;

[0108] (4) After reacting for 30 min, add 0.1 mL of methacrylic acid and 5 mg of KPS initiator, and react overnight;

[0109] (5) Centrifuge at high speed, wash with ethanol, and dry in a vacuum oven for standby.

[0110] 3. Zirconium metal-doped polystyrene nanospheres:

[0111] (1) Disperse 50 mg of polystyrene nanospheres into 50 mL of an aqueous solution of 0.25% sodium dodecyl sulfate, and stir rapidly.

[0112] (2) Dissolve 1 mg of zirconium complex in 1 mL of dichloromethane.

[0113] (3) Add 10 mL of an aqueous solution of 0.25% sodium dodecyl sulfate, and emulsify at high speed for 30 seconds.

[0114] (4) Drop the emulsion into the polystyrene nanosphere solution in (1), and stir overnight.

[0115] (5) Evaporate dichloromethane and centrifuge.

[0116] (6) Wash by centrifugation with ethanol at 8000 rpm for 15 min, and repeat three times to obtain zirconium-doped polystyrene nanospheres, as shown in Figure (c) of the mapping diagram, and the metal is uniformly doped into the interior of the nanospheres. Figure 3 as shown in Figure (c) of the mapping diagram, and the metal is uniformly doped into the interior of the nanospheres.

[0117] 4. Preparation of mass cytometry 90Zr-CD45 probe:

[0118] (1) Disperse 0.5 mg of zirconium-doped polystyrene nanospheres in 1 mL of 4-morpholineethanesulfonic acid buffer solution with pH = 6.2, and stir vigorously.

[0119] (2) Add 10 μL of aqueous solutions of 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide (10 mg / mL) respectively, and activate in the dark for 30 minutes.

[0120] (3) Centrifuge, wash three times with 4-morpholineethanesulfonic acid buffer solution with pH = 6.2, and disperse with 0.1 mL of 4-morpholineethanesulfonic acid buffer solution with pH = 6.2.

[0121] (4) Add 10 μg of CD45 antibody, and incubate in the dark for 1 hour to obtain the mass cytometry 90Zr-CD45 probe.

[0122] (5) Wash 3 times with 4-morpholineethanesulfonic acid buffer solution with pH = 6.2, and store at 4°C.

[0123] Example 4

[0124] This example provides a mass cytometry universal element probe, and its synthesis route is as Figure 1 shown, and the specific preparation method is as follows:

[0125] 1. Preparation of lanthanum metal complex:

[0126] (1) Dissolve 440 mg of acetylacetone in 15 mL of ethanol and stir rapidly;

[0127] (2) Add dropwise 15 mL of 0.05 M aqueous LaCl3 solution. After the addition is complete, add sodium hydroxide solution to adjust the pH value to 9;

[0128] (3) Add 50 mL of water and stir for another 5 hours;

[0129] (4) Rotavap off most of the ethanol and then filter and dry;

[0130] (5) Wash the obtained solid with 20 mL of cold ethanol and dry it in a vacuum oven to obtain lanthanum acetylacetonate complex.

[0131] 2. Preparation of carboxyl - modified polystyrene nanospheres

[0132] (1) Add 15 mL of deionized water, 1 mL of styrene, 0.7 mL of methacrylic acid, and 150 mg of sodium bicarbonate into a three - necked flask equipped with a condenser, and mix and stir rapidly;

[0133] (2) Continuously pass high - purity nitrogen into the solution to remove oxygen for 30 minutes;

[0134] (3) Heat to 80 °C and add an aqueous solution containing 5 mg of KPS initiator;

[0135] (4) After reacting for 30 min, add 0.1 mL of methacrylic acid and 5 mg of KPS initiator, and react overnight;

[0136] (5) Centrifuge at high speed, wash with ethanol, and dry in a vacuum oven for standby.

[0137] 3. Lanthanum - doped polystyrene nanospheres:

[0138] (1) Take 50 mg of polystyrene nanospheres and disperse them in 50 mL of 0.25% aqueous sodium dodecyl sulfate solution, and stir rapidly;

[0139] (2) Dissolve 1 mg of lanthanum complex in 1 mL of dichloromethane;

[0140] (3) Add 10 mL of 0.25% aqueous sodium dodecyl sulfate solution and emulsify at high speed for 30 seconds;

[0141] (4) Drop the emulsion into the polystyrene nanosphere solution in (1) and stir overnight;

[0142] (5) Rotavap off dichloromethane and centrifuge;

[0143] (6) Centrifuge and wash with ethanol at 8000 rpm for 15 min, repeat three times to obtain lanthanum - doped polystyrene nanospheres, as Figure 3As shown in Figure (d) of the mapping diagram, the metal was uniformly doped into the interior of the nanospheres.

[0144] 4. Preparation of the mass cytometry 139La-CD45 probe:

[0145] (1) Disperse 0.5 mg of lanthanum-doped polystyrene nanospheres in 1 mL of 4-morpholineethanesulfonic acid buffer solution with pH = 6.2, and stir vigorously.

[0146] (2) Add 10 μL of aqueous solution of 1-ethyl-(3-dimethylaminopropyl)carbodiimide and aqueous solution of N-hydroxysuccinimide (10 mg / mL) respectively, and activate for 30 minutes in the dark.

[0147] (3) Centrifuge, wash three times with 4-morpholineethanesulfonic acid buffer solution with pH = 6.2, and disperse with 0.1 mL of 4-morpholineethanesulfonic acid buffer solution with pH = 6.2.

[0148] (4) Add 10 μg of CD45 antibody, and incubate for 1 hour in the dark to obtain the mass cytometry 139La-CD45 probe.

[0149] (5) Wash 3 times with 4-morpholineethanesulfonic acid buffer solution with pH = 6.2, and store at 4°C.

[0150] Comparative example

[0151] Use the Fluidigm polymer probes (141-CD45, 152-CD3, 159-CD45, 151-CD8) of Fluidigm Corporation as the comparative example in the following test examples.

[0152] Test example

[0153] I. Sensitivity experiment:

[0154] 1. (1) Take 10^6 human peripheral blood mononuclear cells donated by healthy volunteers, and wash them with Fluidigm cell staining buffer solution.

[0155] (2) Add 5 μL of Fluidigm blocking solution and block for 10 minutes.

[0156] (4) Add Fluidigm polymer probes (1 μL of 141-CD45, 1 μL of 152-CD3, 1 μL of 159-CD45) and 1 μL of 151Eu-doped polystyrene nanosphere-CD8 to the cell suspension, and incubate for 45 minutes.

[0157] (5) Wash 2 times with Fluidigm cell staining buffer solution.

[0158] (6) Stain the cell nuclei with the Fluidigm cell Ir-DNA intercalator for 1 hour to obtain cell nuclei-labeled cells;

[0159] (7) Wash twice with the Fluidigm cell staining buffer and once with deionized water for standby;

[0160] 2. (1) Disperse the stained cells in the previous step in 1 mL of an aqueous solution containing 10% Fluidigm EQ Beads;

[0161] (2) Test the cell sample using a mass cytometry instrument;

[0162] (3) Analyze the test results using Cytobank software, and gate the CD45-positive, CD3-positive, CD4-positive, and CD8-positive cells. The results are as Figure 6 shown in b;

[0163] (4) Stain the same batch of cells and perform on-machine testing using the Fluidigm polymer probes (1 μL of 141-CD45, 1 μL of 152-CD3, 1 μL of 159-CD4, and 1 μL of 151-CD8) according to the above procedure. The results are as Figure 6 shown in a.

[0164] The test results are as Figure 6 shown. Both the Fluidigm polymer probe from the United States and the mass cytometry 151Eu-CD8 probe obtained in Example 1 of this application can be used in combination with the Fluidigm polymer CD4 probe to subgroup T cells (i.e., CD3-positive cells), and the subgrouping ratios are basically the same. From the Figure 6 comparison between a and b, it can be seen that by doping metal ions into the nanospheres in this application, the number of metal ions on each nanosphere label can reach 105, which is 2 - 3 orders of magnitude higher than that of the Fluidigm polymer label (50 - 100). The intensity of the mass cytometry 151Eu-CD8 probe in this application is 510, and the signal intensity of the Fluidigm polymer probe 151-CD8 is 200, with a significant difference in sensitivity.

[0165] II. Detection channel expansion experiment:

[0166] (1) Take four tubes of human peripheral blood mononuclear cells, with 10^6 cells in each tube, and wash them with the Fluidigm cell staining buffer;

[0167] (2) Add 5 μL of the Fluidigm blocking solution to each tube and block for 10 minutes;

[0168] (4) After adding 1 μL of Fluidigm polymer probe 152-CD3 to each tube of cell suspension, 1 μL of Fluidigm polymer probe 151-CD45 and 90Zr, 180Hf, and 139La-doped polystyrene nanospheres-CD45 were added to each tube, and incubated for 45 minutes;

[0169] (5) Wash twice with Fluidigm cell staining buffer;

[0170] (6) Stain the cell nuclei with Fluidigm cell Ir-DNA intercalator for 1 hour;

[0171] (7) Wash twice with Fluidigm cell staining buffer and once with deionized water for standby;

[0172] (8) Disperse the cells in 1 mL of aqueous solution containing 10% Fluidigm EQ Beads;

[0173] (9) Test the cell samples using a mass cytometry;

[0174] (10) Analyze the test results using Cytobank software, and gate the CD45-positive and CD3-positive cells.

[0175] The specific results are as Figure 7 shown. Both the Fluidigm polymer and metal-doped polystyrene nanospheres-CD45 probes can be used in combination with the Fluidigm polymer CD3 probe to cluster human peripheral blood mononuclear cells. The signal intensities of 90Zr, 180Hf, and 139La-doped polystyrene nanospheres-CD45 are 91, 1718, and 489 respectively, and the signal intensity of the Fluidigm polymer probe 151-CD45 is 120. The sensitivities of 90Zr, 180Hf, and 139La-doped polystyrene nanospheres-CD45 are approximately 0.76, 14, and 4.1 times that of the polymer probe.

[0176] Obviously, the above embodiments are merely examples for clear illustration and not limitations on the implementation manners. For those of ordinary skill in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to enumerate all the implementation manners here. And the obvious changes or modifications derived therefrom are still within the protection scope of the present invention.

Claims

1. A mass spectrometry flow cytometry universal element probe, characterized in that, Comprising metal-doped polystyrene nanospheres; The surface of the metal-doped polystyrene nanospheres is modified with an antibody, and the metal includes at least one of rare earth metals, zirconium, and hafnium; The antibody is coupled to the surface of the metal-doped polystyrene nanospheres through an amino group; The preparation method of the mass spectrometry flow cytometry universal element probe includes the following steps: S1: React a β-keto compound with a metal chloride to obtain a metal complex; S2: Prepare carboxyl-modified polystyrene nanospheres by emulsion polymerization; S3: React the metal complex obtained in step S1 with the carboxyl-modified polystyrene nanospheres obtained in step S2 to obtain metal-doped polystyrene nanospheres; S4: Modify an antibody on the surface of the metal-doped polystyrene nanospheres to obtain the mass spectrometry flow cytometry universal element probe; In step S1, the molar ratio of the β-keto compound to the metal chloride is 4 to 10:

1. The reaction is to dissolve the β-keto compound in ethanol or methanol, drop it into an aqueous solution of the corresponding metal chloride, add an alkali solution to adjust the pH value to 8-10, and stir for 4-5 hours until the reaction is completed; The β-keto compound includes one of acetylacetone or β-diketone compounds, and the β-diketone compounds include 4,4,4-trifluoro-1-(2-furyl)-1,3-butanedione or 3-methyl-2,4-pentanedione; The metal chloride includes at least one of chlorides of rare earth metals, zirconium, and hafnium.

2. A preparation method of the mass spectrometry flow cytometry universal element probe according to claim 1, characterized in that, Including the following steps: S1: React a β-keto compound with a metal chloride to obtain a metal complex; S2: Prepare carboxyl-modified polystyrene nanospheres by emulsion polymerization; S3: React the metal complex obtained in step S1 with the carboxyl-modified polystyrene nanospheres obtained in step S2 to obtain metal-doped polystyrene nanospheres; S4: Modify an antibody on the surface of the metal-doped polystyrene nanospheres to obtain the mass spectrometry flow cytometry universal element probe; In step S1, the molar ratio of the β-keto compound to the metal chloride is 4 to 10:

1. The reaction is to dissolve the β-keto compound in ethanol or methanol, drop it into an aqueous solution of the corresponding metal chloride, add an alkali solution to adjust the pH value to 8-10, and stir for 4-5 hours until the reaction is completed; The β-keto compound includes one of acetylacetone or β-diketone compounds, and the β-diketone compounds include 4,4,4-trifluoro-1-(2-furyl)-1,3-butanedione or 3-methyl-2,4-pentanedione; The metal chloride includes at least one of chlorides of rare earth metals, zirconium, and hafnium.

3. According to the preparation method described in claim 2, characterized in that, In step S2, the emulsion polymerization method is specifically as follows: Mix 15-30 mL of deionized water, 1-2 mL of styrene, 0.7-1.4 mL of methacrylic acid, and 150-300 mg of sodium bicarbonate and stir. Continuously introduce nitrogen during the stirring process. After heating to 70-90 °C, add 5-10 mg of potassium persulfate. After reacting for 30-60 min, add 0.1-0.2 mL of methacrylic acid and 5-10 mg of potassium persulfate, and react overnight.

4. According to the preparation method described in claim 3, characterized in that, In step S3, the mass ratio of the metal complex to the carboxyl-modified polystyrene nanospheres is 1:

50. The specific preparation method is as follows: dissolve the metal complex in dichloromethane, add an emulsifier and emulsify for 30 seconds, then drop it into a 0.25 wt% sodium dodecyl sulfate aqueous solution in which the carboxyl-modified polystyrene nanospheres are dispersed, and react overnight; The emulsifier is at least one of sodium dodecyl sulfate, sodium dodecyl sulfonate, sodium dodecylbenzenesulfonate, sorbitan oleate, and sorbitan monostearate.

5. According to the preparation method described in claim 4, characterized in that, In step S4, the metal-doped polystyrene nanospheres are dispersed in a buffer solution, an aqueous solution of 1-ethyl-(3-dimethylaminopropyl)carbodiimide and an aqueous solution of N-hydroxysuccinimide are added, and incubated in the dark for 30 minutes. After adding the antibody, incubate in the dark for 3 hours to obtain the mass spectrometry flow cytometry universal element probe; The mass ratio of the metal-doped polystyrene nanospheres to the antibody is 500:1 - 10.

6. According to the preparation method described in any one of claims 2-5, characterized in that, After the metal complex is prepared in step S1, it further includes the steps of washing with ethanol and drying in a vacuum oven; After the carboxyl-modified polystyrene nanospheres are prepared in step S2, it further includes the steps of washing with ethanol and drying in a vacuum oven; After the metal-doped polystyrene nanospheres are prepared in step S3, it further includes the step of washing with ethanol; After the mass spectrometry flow cytometry universal element probe is prepared in step S4, it further includes the steps of washing with a buffer solution and storing in a buffer solution containing a preservative at 2 - 8°C.

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