Biotinylated transition metal synchrotron radiation nanoprobe as well as preparation method and application thereof

Abstract

The invention provides a biotinylated transition metal synchrotron radiation nanoprobe as well as a preparation method and application thereof. The nanoprobe comprises transition metal-based nanoparticles and a coating substance on the outer layer of the nanoparticles, the transition metal comprises any one or a combination of at least two of iron, cobalt or nickel; the coating substance comprises biotinylated protein and a fluorescent dye. The surface of the probe is coated with biotin and fluorescent dye, and the probe can be combined with a commercial probe with biotin recognition capability to realize recognition of biomolecules such as protein, polysaccharide and the like. The method can be applied to a synchrotron radiation X-ray microscope, and three-dimensional high-resolution space imaging of a target biomolecule or subcellular structure is realized. And the probe is a multi-signal probe capable of being used for synchrotron radiation X-ray biological imaging, can assist in carrying out synchrotron radiation pre-experiment, fully utilizes the scarce synchrotron radiation resource, and saves the experiment time cost and economic cost.

Description

Technical Field

[0001] The present invention belongs to the field of biological imaging, and specifically relates to a biotinylated transition metal synchrotron radiation nanoprobe and a preparation method and application thereof, and in particular to a transition metal-containing nanoprobe suitable for synchrotron radiation X-ray biological imaging and a method for preparing the probe, as well as the use of the transition metal-containing nanoprobe for synchrotron radiation cell in situ detection. Background Art

[0002] Cells are the basic form of life, and proteins are important components of building cells. The spatial localization and distribution of proteins directly affect their biological functions, reflecting the biological state and biological processes of cells. From the cellular level, spatial and temporal imaging studies of proteins are important research methods in life sciences and medicine, which plays an important role in the study of cell pathway mechanisms, disease pathology, etc.

[0003] Synchrotron X-ray microscopy is a technique that uses X-rays generated by synchrotron radiation sources for microscopic imaging. It has the advantages of high resolution, high sensitivity, and non-destructive testing, and has broad application prospects in fields such as materials and biomedicine.

[0004] Patent CN116429544A discloses a synchrotron radiation X-ray imaging staining method for human brain tissue imaging, which combines a low-temperature freezing preservation method with an optimized Golgi staining method to achieve effective staining of human brain tissue, improve the efficiency of neuron labeling, and achieve synchrotron radiation X-ray imaging of the morphology and connections of neurons in human brain tissue.

[0005] Synchrotron radiation X-ray imaging technology is widely used in the field of biological imaging due to its three-dimensional super-resolution imaging capability. However, as a label-free imaging technology, synchrotron radiation X-ray imaging technology is difficult to distinguish different target proteins with small differences in X-ray contrast. Therefore, the development of a universal synchrotron radiation protein probe with a strong X-ray absorption signal is of great significance for expanding the application scope of synchrotron radiation imaging technology and conducting a deeper study of cell physiological processes from an imaging perspective. Summary of the invention

[0006] In view of the shortcomings of the prior art, the purpose of the present invention is to provide a biotinylated transition metal synchrotron radiation nanoprobe and its preparation method and application. The nanoprobe is a multi-signal nanoprobe that can assist in synchrotron radiation pre-experiments, make full use of scarce synchrotron radiation resources, and save experimental time and economic costs. The nanoprobe can also be combined with a commercial streptavidin-avidin system and has a wide range of applicability. The present invention also provides a method for synchrotron radiation X-ray imaging using the nanoprobe to provide three-dimensional high-resolution positioning information of biological macromolecules.

[0007] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions:

[0008] In the first aspect, the present invention provides a biotinylated transition metal synchrotron radiation nanoprobe, the nanoprobe comprising: a transition metal-based nanoparticle and a coating material on the outer layer of the nanoparticle; the transition metal comprises: any one of iron, cobalt or nickel or a combination of at least two of them; the coating material comprises: a biotinylated protein and a fluorescent dye.

[0009] In the present invention, the nanoprobe includes transition metal-based nanoparticles and a coating material compounded on the outer layer of the nanoparticles, wherein the transition metal includes iron, cobalt or nickel, etc. The coating material of the outer layer includes biotinylated protein and fluorescent dye. Among them, the transition metal nanoparticles provide X-ray contrast, and the coating material provides targeting, so that specific labeling of biological macromolecules can be achieved in an X-ray microscope. The fluorescent dye in the coating material provides a fluorescent signal, which can assist in pre-experiments before synchrotron radiation experiments, ensure the accuracy of experimental results, and make full use of synchrotron radiation machine time resources. The biotin in the coating material can be combined with a commercial streptavidin-biotin labeling system to expand the probe labeling capability.

[0010] Preferably, the biotinylated protein is a protein molecule modified with a biotin molecule, and the molar ratio of the protein molecule to the biotin molecule is 1:(1-20), for example, it can be 1:1, 1:2, 1:4, 1:5, 1:6, 1:8, 1:10, 1:12, 1:14, 1:15, 1:16, 1:18 or 1:20, etc.

[0011] In the present invention, when the molar ratio of protein molecules to biotin molecules is lower than 1:1, the probe affinity will decrease. Biotin molecules have a certain hydrophobicity and can affect the surface properties of proteins. When the molar ratio of protein molecules to biotin molecules is higher than 1:20, it is difficult to synthesize, and during the purification process, the protein is easily aggregated. In summary, the molar ratio of protein molecules to biotin molecules is 1:(1-20), which is easy to synthesize, has good affinity, and can achieve a good labeling effect.

[0012] Preferably, the molar ratio of the protein molecule to the transition metal is 1:(10-200), for example, it can be 1:10, 1:20, 1:40, 1:60, 1:80, 1:100, 1:120, 1:140, 1:160, 1:180 or 1:200, etc.

[0013] In the present invention, the transition metal has strong X-ray absorption and mainly provides X-ray absorption signals. When the molar ratio of the protein molecule to the transition metal is less than 1:10, the X-ray absorption signal is too low and difficult to identify. When the molar ratio of the protein molecule to the transition metal is greater than 1:200, the transition metal will affect the surface biotin sites and affect the targeting specificity of the nanoparticles. In summary, considering the specificity of the nanoparticles and the X-ray absorption signal, we select the molar ratio of the protein molecule to the transition metal to be 1:(10-200).

[0014] Preferably, the protein molecule is selected from any one of bovine serum albumin BSA, human serum albumin HSA or chicken ovalbumin OVA, or a combination of at least two of them.

[0015] Preferably, the fluorescent dye is selected from: any one or a combination of at least two of FITC (fluorescein isothiocyanate), RBITC (Rose Bengal isothiocyanate B), Cyanine3, Cyanine5, Cyanine7 or Alexa Fluor series fluorescent dyes.

[0016] Preferably, the fluorescent dye is a fluorescent dye modified with succinimidyl ester, and the fluorescent dye is coupled to the protein molecule via the succinimidyl ester.

[0017] Preferably, the particle size of the nanoprobe is 1-50 nm, for example, it can be 1 nm, 10 nm, 20 nm, 30 nm, 40 nm or 50 nm.

[0018] In the present invention, nanoprobes smaller than 1 nm are difficult to synthesize, and those larger than 50 nm are non-specifically adsorbed in cells and have poor specificity.

[0019] In a second aspect, the present invention provides a method for preparing the biotinylated transition metal synchrotron radiation nanoprobe according to the first invention, the method comprising:

[0020] (S1) mixing the biotinylated protein with an aqueous solution of a transition metal salt, adjusting the pH, performing a first stirring and mixing, and obtaining a transition metal nanoprobe precursor after purification;

[0021] (S2) mixing the fluorescent dye with the transition metal nanoprobe precursor, adjusting the pH, performing a second stirring and mixing, and obtaining a biotinylated transition metal synchrotron radiation nanoprobe after purification.

[0022] Preferably, in step (S1), the biotinylated protein is prepared by a method comprising the following steps: mixing and stirring a dimethyl sulfoxide solution of biotin succinimidyl ester with an alkaline solution of protein; and obtaining the biotinylated protein after purification.

[0023] Preferably, the concentration of the dimethyl sulfoxide solution of biotin succinimide ester is 1-20 g / L, for example, it can be 1 g / L, 2 g / L, 4 g / L, 6 g / L, 8 g / L, 10 g / L, 12 g / L, 14 g / L, 16 g / L, 18 g / L or 20 g / L.

[0024] Preferably, the concentration of the alkaline solution of protein is 10-50 g / L, for example, it can be 10 g / L, 15 g / L, 20 g / L, 25 g / L, 30 g / L, 35 g / L, 40 g / L, 45 g / L or 50 g / L.

[0025] Preferably, the pH of the alkaline solution of the protein is in the range of 8-10, for example, 8, 9 or 10.

[0026] Preferably, the mixing and stirring temperature is 20-30°C, for example, 20°C, 25°C or 30°C.

[0027] In the present invention, the rotation speed of the mixing and stirring is 200-1000 rpm, for example, it can be 200 rpm, 400 rpm, 600 rpm, 800 rpm or 1000 rpm.

[0028] Preferably, the purification method is dialysis, and the dialysis time is 24-48 hours, for example, it can be 24-30 hours, 36 hours, 42 hours or 48 hours.

[0029] Preferably, in step (S1), the transition metal salt is selected from any one or a combination of at least two of inorganic salts of divalent iron, trivalent iron, divalent cobalt or divalent nickel, and the inorganic salt is selected from chloride, nitrate, acetate or sulfate.

[0030] Preferably, in step (S1), in the mixed reaction system, the final concentration of the transition metal salt is 10-100 mM, for example, 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM or 100 mM, etc.

[0031] Preferably, in step (S1), in the mixed reaction system, the final concentration of the biotinylated protein is 10-50 g / L, for example, 10 g / L, 20 g / L, 30 g / L, 40 g / L or 50 g / L.

[0032] Preferably, in step (S1), the pH is adjusted to 10-14 with alkali, for example, 10, 11, 12, 13 or 14.

[0033] Preferably, the base is selected from any one of sodium bicarbonate, sodium carbonate or sodium hydroxide, or a combination of at least two of them.

[0034] Preferably, in step (S1), the temperature of the first stirring and mixing is 20-37°C, for example, 20°C, 25°C, 30°C, 35°C or 37°C, etc.; the time is 4-8h, for example, 4h, 5h, 6h, 7h or 8h, etc.

[0035] In the present invention, the rotation speed of the mixing and stirring is 400-800 rpm, for example, it can be 400 rpm, 600 rpm or 800 rpm.

[0036] Preferably, in step (S1), the purification method is dialysis, and the dialysis time is 12-24 hours, for example, it can be 12 hours, 14 hours, 16 hours, 18 hours, 20 hours, 22 hours or 24 hours.

[0037] Preferably, the fluorescent dye is dissolved in DMSO and then mixed with the transition metal nanoprobe precursor solution.

[0038] Preferably, the concentration of the fluorescent dye dissolved in DMSO is 1-20 g / L, for example, 1 g / L, 5 g / L, 15 g / L or 20 g / L.

[0039] Preferably, the concentration of the transition metal nanoprobe precursor solution is 5-20 g / L, for example, 5 g / L, 7 g / L, 10 g / L, 13 g / L, 15 g / L, 17 g / L or 20 g / L.

[0040] Preferably, in the mixed system, the final concentration of the transition metal nanoprobe precursor is 5-20 g / L, for example, 5 g / L, 7 g / L, 10 g / L, 13 g / L, 15 g / L, 17 g / L or 20 g / L.

[0041] Preferably, in the mixed system, the final concentration of the fluorescent dye is 0.05-1 g / L, for example, it can be 0.05 g / L, 0.1 g / L, 0.2 g / L, 0.3 g / L, 0.4 g / L, 0.5 g / L, 0.6 g / L, 0.7 g / L, 0.8 g / L, 0.9 g / L or 1 g / L, etc.

[0042] Preferably, in step (S2), the pH is adjusted to 10-14 with alkali, for example, 10, 11, 12, 13 or 14.

[0043] Preferably, the base is selected from any one of sodium bicarbonate, sodium carbonate or sodium hydroxide, or a combination of at least two of them.

[0044] Preferably, in step (S2), the temperature of the second stirring and mixing is 20-30°C, for example, 20°C, 25°C or 30°C, etc.; the time is 4-8h, for example, 4h, 5h, 6h, 7h or 8h, etc.

[0045] In the present invention, the rotation speed of the mixing and stirring is 400-800 rpm, for example, it can be 400 rpm, 600 rpm or 800 rpm.

[0046] Preferably, in step (S2), the purification method is dialysis, and the dialysis time is 6-12 hours, for example, it can be 6, 7, 8, 9, 10, 11 or 12 hours.

[0047] In a third aspect, the present invention provides a method for intracellular protein imaging, the method comprising: labeling the cells to be tested with a biotin-modified labeling molecule, adding fluorescent dye-modified streptavidin for incubation, and then incubating the cells with the biotinylated transition metal synchrotron radiation nanoprobe described in the first aspect, and performing in situ fluorescence imaging and / or synchrotron radiation X-ray imaging after incubation.

[0048] Preferably, the labeling molecule specifically binds to a biological macromolecule in a cell, and the labeling molecule is an antibody, an antigen recognition fragment, a ligand or a receptor that binds to a biological macromolecule in a cell.

[0049] Preferably, the intracellular biomacromolecule is selected from any one of: human epidermal growth factor receptor, interferon-stimulated molecule STING, cyclic GMP-AMP synthase cGAS, TOMM20 protein, calcium-binding protein Calnexin, microtubule protein or microfilament protein.

[0050] Preferably, the fluorescent dye is selected from: any one or a combination of at least two of FITC (fluorescein isothiocyanate), RBITC (Rose Bengal isothiocyanate B), Cyanine3, Cyanine5, Cyanine7 or Alexa Fluor series fluorescent dyes.

[0051] In the present invention, the nanoprobe is a multi-signal probe that can be used for synchrotron radiation X-ray bioimaging, which can assist in synchrotron radiation pre-experiments, make full use of scarce synchrotron radiation resources, and save experimental time and economic costs.

[0052] In a fourth aspect, the present invention provides application of the biotinylated transition metal synchrotron radiation nanoprobe described in the first aspect or the intracellular protein imaging method described in the third aspect in biomolecule imaging.

[0053] In the present invention, the application fields of the above-mentioned biotinylated transition metal synchrotron radiation nanoprobe include labeling target biological molecules or subcellular structures in synchrotron radiation X-ray imaging, and verifying the labeling effect by fluorescence imaging in synchrotron radiation pre-experiments.

[0054] The numerical range described in the present invention not only includes the point values ​​listed above, but also includes any point values ​​between the above numerical ranges that are not listed. Due to space limitations and for the sake of simplicity, the present invention no longer exhaustively lists the specific point values ​​included in the range.

[0055] Compared with the prior art, the present invention has the following beneficial effects:

[0056] The nanoprobe of the present invention comprises transition metal-based nanoparticles and a coating material compounded on the outer layer of the nanoparticles, wherein the transition metal comprises iron, cobalt, and nickel. The outer coating material comprises protein, biotin, and fluorescent dye. The transition metal nanoparticles provide X-ray contrast, and the coating material provides targeting, so that specific labeling of biological macromolecules can be achieved in an X-ray microscope. The fluorescent dye in the coating material provides a fluorescent signal, which can assist in pre-experiments before synchrotron radiation experiments, ensure the accuracy of experimental results, and make full use of synchrotron radiation machine time resources. The biotin in the coating material can be combined with a commercial streptavidin-biotin labeling system to expand the probe labeling capability. BRIEF DESCRIPTION OF THE DRAWINGS

[0057] Figure 1 This is a TEM image of the nanoparticle probe prepared in Example 1 of the present invention.

[0058] Figure 2 This is the TEM spectrum of the nanoparticle probe prepared in Example 2 of the present invention.

[0059] Figure 3 This is the TEM spectrum of the nanoparticle probe prepared in Example 3 of the present invention.

[0060] Figure 4 This is an image of antibody co-localization fluorescence staining of cell Ki67 protein by the nanoparticle probe obtained in Example 1 of the present invention.

[0061] Figure 5 This is a fluorescent confocal image of the nanoparticle probe obtained in Example 2 of the present invention labeling the cell HER2 protein.

[0062] Figure 6 This is a synchrotron radiation X-ray two-dimensional imaging pattern of the nanoparticle probe obtained in Example 1 of the present invention on the cell Ki67 protein.

[0063] Figure 7The nanoparticle probe obtained in Example 1 of the present invention performs synchrotron radiation X-ray three-dimensional imaging on the cell Ki67 protein, including cell membrane structure, cell nucleus, nucleolus and probe-labeled Ki67 protein.

[0064] Figure 8 This is a synchrotron radiation X-ray three-dimensional imaging pattern of the nanoparticle probe obtained in Example 3 of the present invention on the cell HER2 protein. DETAILED DESCRIPTION

[0065] The technical solution of the present invention is further described below by specific implementation methods. It should be understood by those skilled in the art that the embodiments are only to help understand the present invention and should not be regarded as specific limitations of the present invention.

[0066] If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the field or the product instructions are used. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0067] The sources of the experimental materials used in the following examples are as follows:

[0068] Biotin-NHS biotin active ester: Tianjin Xiensiopude Technology Co., Ltd., B-26000.

[0069] BSA bovine serum albumin: Amresco, 0332.

[0070] OVA chicken ovalbumin: Bioss, bs-0283P.

[0071] Ki67-biotin antibody: Novus, NB500-170B.

[0072] HER2-biotin antibody: Thermo Fisher, MA5-12995.

[0073] Example 1

[0074] Preparation of transition metal nanoparticle probes

[0075] A) 5 mg of biotin-NHS was dissolved in 1 mL of DMSO, and then added to 10 mL of 0.1 M sodium bicarbonate solution containing 100 mg of BSA, the pH of the alkaline solution of BSA was 9. Stirred at room temperature for 12 hours. After that, dialyzed for 48 hours using a dialysis bag with a molecular weight cutoff of 100,000, freeze-dried and stored to obtain a protein molecule modified with biotin molecules, called biotinylated protein, and detected by matrix-assisted laser desorption ionization time-of-flight mass spectrometry. The molar ratio of protein molecules to biotin molecules in the protein molecule modified with biotin molecules was 1:10.

[0076] B) To 5 mL of biotin-modified BSA molecules with a concentration of 20 g / L calculated based on the BSA content, 1.6 mL of 100 mM ferrous chloride solution and 0.4 mL of 200 mM ferric chloride solution were added; then 0.4 mL of 2 M sodium hydroxide aqueous solution was added, the pH was adjusted to 10, and the mixture was vigorously stirred for 6 h at 37° C., and dialyzed for 24 h using a dialysis bag with a molecular weight cutoff of 100,000. The mixture was recorded as a nanoparticle probe precursor, and after ICP-MS detection, the molar ratio of protein molecules to transition metals in the nanoparticle probe precursor was 1:50.

[0077] C) Add 10 μL of 10 g / L DMSO solution of succinimidyl ester of fluorescein isothiocyanate to 1 mL of aqueous solution of nanoparticle probe precursor with a BSA content of 10 g / L. Then add 0.1 mL of 1M sodium bicarbonate solution and adjust the pH to 12. Stir at room temperature for 6 h, dialyze for 24 h using a dialysis bag with a molecular weight cutoff of 100,000, and record it as a nanoparticle probe.

[0078] See also Figure 1 , Figure 1 TEM image of the nanoprobe of the present invention. Figure 1 It can be seen that the average particle size of the nanoprobes prepared in the example of the present invention is 4.7 nm, and the nanoprobes have good dispersibility and uniform size.

[0079] Example 2

[0080] Preparation of transition metal nanoparticle probes

[0081] A) 3 mg of biotin-NHS was dissolved in 1 mL of DMSO, and then added to 10 mL of 0.1 M sodium bicarbonate solution containing 100 mg of HSA, the pH of the alkaline solution of HSA was 9; stirred at room temperature for 12 hours. Then, dialyzed for 48 hours using a dialysis bag with a molecular weight cutoff of 100,000, freeze-dried and stored to obtain a protein molecule modified with biotin molecules, called biotinylated protein, and detected by matrix-assisted laser desorption ionization time-of-flight mass spectrometry. The molar ratio of protein molecules to biotin molecules in the protein molecule modified with biotin molecules was 1:6.

[0082] B) 1.8 mL of 400 mM nickel chloride solution was added to 5 mL of biotin-modified HSA molecules with a concentration of 50 g / L calculated by HSA content; then 0.4 mL of 2 M sodium hydroxide aqueous solution was added, the pH was adjusted to 12, and the mixture was vigorously stirred for 6 h at 37° C., and dialyzed for 24 h using a dialysis bag with a molecular weight cutoff of 100,000. The mixture was recorded as a nanoparticle probe precursor. After ICP-MS detection, the molar ratio of protein molecules to transition metals in the nanoparticle probe precursor was 1:65.

[0083] C) Add 100 μL of DMSO solution of succinimidyl ester of rhodamine B isothiocyanate with a concentration of 10 g / L to 1 mL of aqueous solution of nanoparticle probe precursor with a HSA content of 20 g / L. Then add 0.1 mL of 1M sodium bicarbonate solution and adjust the pH to 12. Stir at room temperature for 6 h, dialyze for 24 h using a dialysis bag with a molecular weight cutoff of 100,000, and record it as a nanoparticle probe.

[0084] See also Figure 2 , Figure 2 TEM image of the nanoprobe of the present invention. Figure 2 It can be seen that the average particle size of the nanoprobes prepared in the example of the present invention is 8.6 nm, and the nanoprobes have good dispersibility and uniform size.

[0085] Example 3

[0086] Preparation of transition metal nanoparticle probes

[0087] A) 10 mg of biotin-NHS was dissolved in 1 mL of DMSO, and then added to 10 mL of 0.1 M sodium bicarbonate solution containing 100 mg of chicken ovalbumin OVA, the pH range of the alkaline solution of OVA was 9, and stirred at room temperature for 12 hours. After that, it was dialyzed for 48 hours using a dialysis bag with a molecular weight cutoff of 100,000, and freeze-dried for storage to obtain a protein molecule modified with biotin molecules, called biotinylated protein, and detected by matrix-assisted laser desorption ionization time-of-flight mass spectrometry. The molar ratio of protein molecules to biotin molecules in the protein molecule modified with biotin molecules was 1:15.

[0088] B) To 5 mL of biotin-modified OVA molecules (hereinafter referred to as OVA-biotin) with a concentration of 20 g / L calculated based on the OVA content, 0.6 mL of 200 mM cobalt trichloride solution was added; then 0.4 mL of 2 M sodium hydroxide aqueous solution was added, the pH was adjusted to 9, and the mixture was vigorously stirred for 6 h at 37° C., and dialyzed for 24 h using a dialysis bag with a molecular weight cutoff of 100,000, which was referred to as a nanoparticle probe precursor. After matrix-assisted laser desorption ionization time-of-flight mass spectrometry detection, the molar ratio of protein molecules to transition metals in the nanoparticle probe precursor was 1:20.

[0089] C) Add 5 μL of DMSO solution of succinimidyl ester of Cyanine 5 with a concentration of 10 g / L to 1 mL of aqueous solution of nanoparticle probe precursor with an OVA content of 10 g / L. Then add 0.1 mL of 1 M sodium bicarbonate solution and adjust the pH to 10. Stir at room temperature for 8 h, dialyze for 24 h using a dialysis bag with a molecular weight cutoff of 100,000, and record it as a nanoparticle probe.

[0090] See also Figure 3 , Figure 3 TEM image of the nanoprobe of the present invention. Figure 3 It can be seen that the average particle size of the nanoprobes prepared in the example of the present invention is 5 nm, and the nanoprobes have good dispersibility and uniform size.

[0091] Example 4

[0092] This embodiment provides a method for preparing a transition metal nanoparticle probe, the method comprising:

[0093] A) 1 mg of biotin-NHS was dissolved in 1 mL of DMSO, and then added to 10 mL of 0.1 M sodium bicarbonate solution containing 500 mg of BSA, the pH of the alkaline solution of BSA was 9. Stirred at room temperature for 12 hours. After that, dialyzed for 48 hours using a dialysis bag with a molecular weight cutoff of 100,000, freeze-dried and stored to obtain a protein molecule modified with biotin molecules, called biotinylated protein, and detected by matrix-assisted laser desorption ionization time-of-flight mass spectrometry. The molar ratio of protein molecules to biotin molecules in the protein molecule modified with biotin molecules was 1:1.

[0094] B) To 5 mL of biotin-modified BSA molecules with a concentration of 20 g / L calculated based on the BSA content, 1.6 mL of 100 mM ferrous chloride solution and 0.4 mL of 200 mM ferric chloride solution were added; then 0.4 mL of 2 M sodium hydroxide aqueous solution was added, the pH was adjusted to 10, and the mixture was vigorously stirred for 6 h at 37° C., and dialyzed for 24 h using a dialysis bag with a molecular weight cutoff of 100,000. The mixture was recorded as a nanoparticle probe precursor, and after ICP-MS detection, the molar ratio of protein molecules to transition metals in the nanoparticle probe precursor was 1:40.

[0095] C) Add 100 μL of 10 g / L DMSO solution of succinimidyl ester of fluorescein isothiocyanate to 1 mL of aqueous solution of nanoparticle probe precursor with a BSA content of 20 g / L. Then add 0.1 mL of 1M sodium bicarbonate solution and adjust the pH to 12. Stir at room temperature for 6 h, dialyze for 24 h using a dialysis bag with a molecular weight cutoff of 100,000, and record it as a nanoparticle probe.

[0096] The probe has good targeting and fluorescence intensity, and meets the use requirements.

[0097] Example 5

[0098] This embodiment provides a method for preparing a transition metal nanoparticle probe, the method comprising:

[0099] A) 15 mg of biotin-NHS was dissolved in 1 mL of DMSO, and then added to 10 mL of 0.1 M sodium bicarbonate solution containing 100 mg of BSA, the pH of the alkaline solution of BSA was 9. Stirred at room temperature for 12 hours. After that, dialyzed for 48 hours using a dialysis bag with a molecular weight cutoff of 100,000, freeze-dried and stored to obtain a protein molecule modified with biotin molecules, called biotinylated protein, and detected by matrix-assisted laser desorption ionization time-of-flight mass spectrometry. The molar ratio of protein molecules to biotin molecules in the protein molecule modified with biotin molecules was 1:13.

[0100] B) To 5 mL of biotin-modified BSA molecules with a concentration of 20 g / L calculated based on the BSA content, 1.6 mL of 100 mM ferrous chloride solution and 0.4 mL of 200 mM ferric chloride solution were added; then 0.4 mL of 2 M sodium hydroxide aqueous solution was added, the pH was adjusted to 10, and the mixture was vigorously stirred for 6 h at 37° C., and dialyzed for 24 h using a dialysis bag with a molecular weight cutoff of 100,000. The mixture was recorded as a nanoparticle probe precursor, and after ICP-MS detection, the molar ratio of protein molecules to transition metals in the nanoparticle probe precursor was 1:65.

[0101] C) Add 10 μL of 10 g / L DMSO solution of succinimidyl ester of fluorescein isothiocyanate to 1 mL of aqueous solution of nanoparticle probe precursor with a BSA content of 10 g / L. Then add 0.1 mL of 1M sodium bicarbonate solution and adjust the pH to 12. Stir at room temperature for 6 h, dialyze for 24 h using a dialysis bag with a molecular weight cutoff of 100,000, and record it as a nanoparticle probe.

[0102] The probe has good targeting and fluorescence intensity, and meets the use requirements.

[0103] Example 6

[0104] This embodiment provides a method for preparing a transition metal nanoparticle probe, which differs from the method in Embodiment 1 only in that the molar ratio of the protein molecule to the transition metal is 1: 5. The obtained transition metal nanoparticle probe has a weak X-ray absorption signal, making it difficult to achieve labeling.

[0105] Example 7

[0106] This embodiment provides a method for preparing a transition metal nanoparticle probe, which differs from the method in Embodiment 1 only in that the molar ratio of the protein molecule to the transition metal is 1: 220. The obtained transition metal nanoparticle probe has an uneven particle size distribution and high labeling nonspecificity, which is difficult to meet labeling requirements.

[0107] Example 8

[0108] This embodiment provides a method for preparing a transition metal nanoparticle probe, which differs from the method in Embodiment 1 only in that, when preparing the nanoparticle probe precursor, a base is used to adjust the pH to 9. The nanoparticle probe precursor obtained during preparation has an uneven particle size distribution, and the labeling is highly nonspecific, which is difficult to meet the labeling requirements.

[0109] Example 9

[0110] This example provides a method for preparing a transition metal nanoparticle probe, which differs from Example 1 only in that the stirring time is 2 hours when preparing the nanoparticle probe precursor. The obtained nanoparticle probe precursor is clear during the reaction, but precipitates during the dialysis purification process, and subsequent experiments cannot be performed.

[0111] Example 10

[0112] This embodiment provides a method for preparing a transition metal nanoparticle probe, which differs from the method in Example 1 only in that the stirring time is 10 hours when preparing the nanoparticle probe precursor. The obtained nanoparticle probe precursor precipitates during the reaction, and subsequent experiments cannot be performed.

[0113] The following examples use the proliferation marker Ki67 protein as a targeted model protein to implement in situ cell detection using nanoprobes.

[0114] Embodiment 11

[0115] Cellular fluorescence imaging using transition metal nanoparticle probes

[0116] A) Human breast cancer cells (hereinafter referred to as MDA-MB-231) were fixed with 4% paraformaldehyde for 15 minutes. Then, the cells were incubated with 3% BSA solution for 40 minutes to avoid nonspecific adsorption. Endogenous biotin and avidin were then blocked using an endogenous biotin-avidin blocking kit.

[0117] B) Add 50-fold diluted 3% BSA solution of biotinylated Ki67-biotin antibody to the cells treated in step A, incubate at room temperature for 1 hour, and then wash with PBS for 3 times.

[0118] C) Then, under light-proof conditions, add 2 μg / mL red fluorescent dye-modified streptavidin solution to the cells treated in step B, incubate at room temperature for 30 minutes, and wash with PBS three times.

[0119] D) Add the green fluorescent dye-modified nanoparticle probe obtained in Example 1 to the cells treated in step C, and continue incubating for 30 minutes, add a nuclear dye (DAPI) to stain the cells for 15 minutes, and observe the cells using a laser confocal microscope.

[0120] See also Figure 4 . Figure 4 This is an image of antibody co-localization fluorescence staining of cell Ki67 protein by the nanoparticle probe obtained in Example 1 of the present invention. Figure 4 It can be seen that the nanoparticle probe obtained in Example 1 of the present invention has green fluorescence and can achieve co-localization with streptavidin modified with red fluorescent dye, and the red fluorescent dye streptavidin targets the biotinylated Ki67 antibody, and the two are mainly distributed in the cell nucleus and aggregated in the nucleolus. This shows that the nanoprobe can effectively and specifically target the Ki67 protein of the cell.

[0121] Example 12

[0122] Cellular fluorescence imaging using transition metal nanoparticle probes

[0123] A) Human breast cancer cells (hereinafter referred to as SK-BR-3) were fixed with 4% paraformaldehyde for 15 minutes. Then, the cells were incubated with 3% BSA solution for 40 minutes to avoid nonspecific adsorption. Endogenous biotin and avidin were then blocked using an endogenous biotin-avidin blocking kit.

[0124] B) Add 200-fold diluted 3% BSA solution of biotinylated HER2 antibody to the cells treated in step A, incubate at room temperature for 1 hour, and then wash with PBS for 3 times.

[0125] C) Then, under light-protected conditions, add 2 μg / mL streptavidin solution to the cells treated in step B and incubate at room temperature for 30 minutes. Wash with PBS three times.

[0126] D) Add the red fluorescent dye-modified nanoparticle probe obtained in Example 2 to the cells treated in step C, and continue incubating for 30 minutes, add a nuclear dye (DAPI) to stain the cells for 15 minutes, and observe the cells using a laser confocal microscope.

[0127] See also Figure 5 . Figure 5 This is an image of antibody fluorescence staining of HER2 protein in cells by the nanoparticle probe obtained in Example 2 of the present invention. Figure 5 It can be seen that the nanoparticle probe obtained in Example 2 of the present invention has red fluorescence, can achieve the labeling of HER2, and is mainly distributed on the surface of the cell membrane, indicating that the nanoprobe can effectively and specifically target the HER2 protein of the cell.

[0128] Example 13

[0129] Synchrotron X-ray Imaging of Cells Using Transition Metal Nanoparticle Probes

[0130] A) MCF-7 cells were seeded on silicon nitride windows and fixed with 4% paraformaldehyde in PBS for 30 minutes. The cells were then incubated with 3% BSA solution for 40 minutes to avoid nonspecific adsorption. Endogenous biotin and avidin were then blocked using an endogenous biotin-avidin blocking kit.

[0131] B) Add 50-fold diluted 3% BSA solution of biotinylated Ki67 antibody to the cells treated in step A, incubate at room temperature for 1 hour, and then wash with PBS 3 times.

[0132] C) Add 2 μg / mL streptavidin solution to the cells treated in step B and incubate at room temperature for 30 minutes. Wash with PBS three times.

[0133] D) The nanoparticle probes were incubated with the cells immobilized on the silicon nitride window obtained in step A for 30 minutes and then washed twice with PBS.

[0134] E) The labeled cell sample obtained in step D is subjected to gradient dehydration and stored in a dry place at room temperature.

[0135] F) Performing synchrotron X-ray cell imaging experiments on the dehydrated sample obtained in step E. An alternative method is to use the absorption edge of metal based on nanoparticle probes.

[0136] See also Figure 6 , Figure 7 . Figure 6 This is a synchrotron radiation X-ray two-dimensional imaging pattern of the nanoparticle probe obtained in Example 1 of the present invention on the cell Ki67 protein. Figure 7 This is a synchrotron radiation X-ray three-dimensional imaging pattern of the nanoparticle probe obtained in Example 1 of the present invention on the cell Ki67 protein.

[0137] Depend on Figure 6 It can be seen that the nanoparticle probe obtained in Example 1 of the present invention can be used for cell synchrotron radiation X-ray Ki67 protein imaging, and its X-ray absorption signal intensity is positively correlated with the nanoprobe labeling density, which can be used for qualitative comparative analysis of the distribution of Ki67 protein in cells. Figure 6It can be seen that Ki67 protein is mainly irregularly distributed in the cells and mainly accumulates in the nucleolus region.

[0138] Depend on Figure 6 It can be seen that the two-dimensional data of the distribution of the nanoprobe-labeled Ki67 protein obtained in Example 1 of the present invention can be obtained through data processing. Figure 7 Three-dimensional distribution data of Ki67 protein in cells.

[0139] Embodiment 14

[0140] Synchrotron X-ray Imaging of Cells Using Transition Metal Nanoparticle Probes

[0141] A) SK-BR-3 cells were seeded on silicon nitride windows and fixed with 4% paraformaldehyde in PBS for 30 minutes. The cells were then incubated with 3% BSA solution for 40 minutes to avoid nonspecific adsorption. Endogenous biotin and avidin were then blocked using an endogenous biotin-avidin blocking kit.

[0142] B) Add 200-fold diluted 3% BSA solution of biotinylated HER2 antibody to the cells treated in step A, incubate at room temperature for 1 hour, and then wash with PBS for 3 times.

[0143] C) Add 2 μg / mL streptavidin solution to the cells treated in step B and incubate at room temperature for 30 minutes. Wash with PBS three times.

[0144] D) The nanoparticle probes were incubated with the cells immobilized on the silicon nitride window obtained in step A for 30 minutes and then washed twice with PBS.

[0145] E) The labeled cell sample obtained in step D is subjected to gradient dehydration and stored in a dry place at room temperature.

[0146] F) Performing synchrotron X-ray cell imaging experiments on the dehydrated sample obtained in step E. An alternative method is to use the absorption edge of metal based on nanoparticle probes.

[0147] See also Figure 8 . Figure 8 This is a synchrotron radiation X-ray three-dimensional imaging pattern of the nanoparticle probe obtained in Example 3 of the present invention on the cell HER2 protein.

[0148] Depend on Figure 8 It can be seen that the nanoparticle probe obtained in Example 3 of the present invention can be used for cell synchrotron radiation X-ray HER2 protein imaging, and its X-ray absorption signal intensity is positively correlated with the nanoprobe labeling density, which can be used for qualitative comparative analysis of the distribution of HER2 protein in cells. Figure 8 It can be seen that Ki67 protein is mainly irregularly distributed on the cell membrane, showing regional cluster aggregation.

[0149] In summary, the present invention provides a biotinylated transition metal synchrotron radiation nanoprobe, which is composed of transition metal nanoparticles and nanoparticle outer layer coating material, and the probe surface is coated with biotin and fluorescent dye, which can be used in conjunction with commercial probes with biotin recognition ability to achieve recognition of biological molecules such as proteins and polysaccharides. It can be applied to synchrotron radiation X-ray microscopes to achieve three-dimensional high-resolution spatial imaging of target biological molecules or subcellular structures.

[0150] The applicant declares that the above is only a specific implementation mode of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily thought of by those skilled in the art within the technical scope disclosed by the present invention shall fall within the protection scope and disclosure scope of the present invention.

Claims

1. A biotinylated transition metal synchrotron radiation nanoprobe, characterized in that: The nanoprobe comprises: transition metal-based nanoparticles and coating materials on the outer layer of the nanoparticles; the transition metal comprises: any one of iron, cobalt or nickel or a combination of at least two; the coating materials comprises: biotinylated protein and fluorescent dye.

2. The biotinylated transition metal synchrotron radiation nanoprobe according to claim 1, characterized in that: The biotinylated protein is a protein molecule modified with a biotin molecule, and the molar ratio of the protein molecule to the biotin molecule is 1:(1-20); Preferably, the molar ratio of the protein molecule to the fluorescent dye is 1:(1-20); Preferably, the molar ratio of the protein molecule to the transition metal is 1:(10-200); Preferably, the protein molecule is selected from any one of bovine serum albumin BSA, human serum albumin HSA or chicken ovalbumin OVA, or a combination of at least two of them.

3. The biotinylated transition metal synchrotron radiation nanoprobe according to claim 1 or 2, characterized in that: The fluorescent dye is selected from: any one or a combination of at least two of FITC, RBITC, Cyanine3, Cyanine5, Cyanine7 or Alexa Fluor series fluorescent dyes; Preferably, the fluorescent dye is a fluorescent dye modified with succinimide ester, and the fluorescent dye is coupled to the protein molecule via the succinimide ester; Preferably, the particle size of the nanoprobe is 1-50 nm.

4. A method for preparing a biotinylated transition metal synchrotron radiation nanoprobe according to any one of claims 1 to 3, characterized in that: The method comprises: (S1) mixing the biotinylated protein with an aqueous solution of a transition metal salt, adjusting the pH, performing a first stirring and mixing, and obtaining a transition metal nanoprobe precursor after purification; (S2) mixing the fluorescent dye with the transition metal nanoprobe precursor, adjusting the pH, performing a second stirring and mixing, and obtaining a biotinylated transition metal synchrotron radiation nanoprobe after purification.

5. The method for preparing the biotinylated transition metal synchrotron radiation nanoprobe according to claim 4, characterized in that: In step (S1), the biotinylated protein is prepared by a method comprising the following steps: mixing and stirring a dimethyl sulfoxide solution of biotin succinimidyl ester with an alkaline solution of protein; and obtaining the biotinylated protein after purification; Preferably, the concentration of the dimethyl sulfoxide solution of biotin succinimide ester is 1-20 g / L; Preferably, the concentration of the alkaline solution of the protein is 10-50 g / L; Preferably, the pH range of the alkaline solution of the protein is 8-10; Preferably, the mixing and stirring temperature is 20-30°C.

6. The method for preparing the biotinylated transition metal synchrotron radiation nanoprobe according to claim 4 or 5, characterized in that: In step (S1), the transition metal salt is selected from any one or a combination of at least two of inorganic salts of divalent iron, trivalent iron, divalent cobalt or divalent nickel, and the inorganic salt is selected from chloride, nitrate, acetate or sulfate; Preferably, in step (S1), in the mixed reaction system, the final concentration of the transition metal salt is 10-100 mM; Preferably, in step (S1), in the mixed reaction system, the final concentration of the biotinylated protein is 10-50 g / L; Preferably, in step (S1), a base is used to adjust the pH to 10-14; Preferably, the base is selected from any one or a combination of at least two of sodium bicarbonate, sodium carbonate or sodium hydroxide; Preferably, in step (S1), the temperature of the first stirring and mixing is 20-37°C and the time is 4-8h.

7. The method for preparing the biotinylated transition metal synchrotron radiation nanoprobe according to any one of claims 4 to 6, characterized in that: In step (S2), the fluorescent dye is dissolved in DMSO and then mixed with the transition metal nanoprobe precursor solution; Preferably, the concentration of the fluorescent dye dissolved in DMSO is 1-20 g / L; Preferably, the concentration of the transition metal nanoprobe precursor solution is 5-20 g / L; Preferably, in the mixed system, the final concentration of the transition metal nanoprobe precursor is 5-20 g / L; Preferably, in the mixed system, the final concentration of the fluorescent dye is 0.05-1 g / L; Preferably, in step (S2), the pH is adjusted to 10-14 using a base; Preferably, the base is selected from any one or a combination of at least two of sodium bicarbonate, sodium carbonate or sodium hydroxide; Preferably, in step (S2), the temperature of the second stirring and mixing is 20-30°C and the time is 4-8h.

8. A method for intracellular protein imaging, characterized in that: The method comprises: labeling the cells to be tested with a biotin-modified labeling molecule, adding fluorescent dye-modified streptavidin for incubation, and then incubating the cells with the biotinylated transition metal synchrotron radiation nanoprobe described in any one of claims 1 to 3, and performing in situ fluorescence imaging and / or synchrotron radiation X-ray imaging after incubation.

9. The method for intracellular protein imaging according to claim 8, characterized in that: The labeling molecule specifically binds to the biological macromolecule in the cell, and the labeling molecule is an antibody, antigen recognition fragment, ligand or receptor that binds to the biological macromolecule in the cell; Preferably, the intracellular biomacromolecule is selected from any one of: human epidermal growth factor receptor, interferon-stimulated molecule STING, cyclic GMP-AMP synthase cGAS, TOMM20 protein, calnexin, tubulin or microfilament protein; Preferably, the fluorescent dye is selected from: any one or a combination of at least two of FITC, RBITC, Cyanine3, Cyanine5, Cyanine7 or AlexaFluor series fluorescent dyes.

10. Use of the biotinylated transition metal synchrotron radiation nanoprobe according to any one of claims 1 to 3 or the intracellular protein imaging method according to claim 8 or 9 in biomolecule imaging.

Images