X-ray click chemistry labeling probe based on synchrotron light source and preparation method and application thereof

Abstract

The application provides an X-ray click chemistry probe based on a synchronous light source and a preparation method and application thereof, and comprises the following steps: 1) synthesizing a polydopamine-metal with a group required for a click reaction on a polydopamine surface; 2) adding a modification reagent to a culture medium with cells to modify biomolecules which can be targeted to various subcellular organelles; 3) adding the polydopamine-metal synthesized in step 1) to the culture medium after modification in step 2) until a click reaction is completed; and 4) adding the cells obtained in step 3) to a substrate suitable for synchronous radiation X-ray imaging, and observing imaging under synchronous radiation X-ray, so that biomolecules in the cells have specific absorption peaks under X-ray, and the method is obtained. According to the application, a method for high-specificity recognition and high-resolution imaging of various biomolecules in cells based on click chemistry and synchronous X-ray is provided, and has a good biomedical application prospect.

Description

Technical Field

[0001] This invention relates to the field of biochemistry, and more specifically to a synchrotron X-ray click chemical labeling probe, its preparation method, and its applications. Background Technology

[0002] One of the fundamental goals of cell biology is to understand the complex interactions of biomolecules in cellular life processes. Synchrotron radiation X-ray microscopy, with its wavelengths ranging from 0.1 to 10 nm, is naturally a super-resolution microscopy technique, theoretically capable of achieving resolutions of several nanometers. Compared to electron beams, X-rays have greater penetrating power into biological samples, allowing for imaging of intact cells without the need for sectioning. The soft X-ray "water window" band can provide natural-contrast imaging of cells. In particular, X-rays possess excellent energy (elemental) resolution; by utilizing the differences in the X-ray fluorescence emission spectra of different elements, multicolor tags can be developed for use in the preparation of biological probes, enabling the identification and imaging of various biomolecules within cells.

[0003] Existing molecular probes suitable for X-ray microscopy include immune probes and genetic marker probes, enabling fine imaging of multiple intracellular biological targets. However, immune labeling techniques are prone to crosstalk between antigens and antibodies, and genetic marker techniques have few systems capable of independent stepwise expression, making it difficult to simultaneously label and image multiple intracellular biological targets.

[0004] Chinese patent application (CN201911094934.5) discloses an X-ray multicolor genetic marker probe based on a synchronous light source. However, because the imaging tag does not have a fixed morphology, it will diffuse in the cell, which will reduce the positioning accuracy. Furthermore, the probe is difficult to image three or more biological targets at the same time and can only image fixed dead cells, which has certain defects. Summary of the Invention

[0005] The purpose of this invention is to provide an X-ray click chemical labeling probe based on a synchronous light source, its preparation method, and its application, thereby solving the problem that existing genetic labeling probes and immune probes suitable for X-rays are difficult to recognize, accurately locate, and perform multicolor imaging of various biomolecules in living cells.

[0006] To solve the above-mentioned technical problems, the present invention adopts the following technical solution:

[0007] According to a first aspect of the present invention, a method for preparing an X-ray click chemical probe based on a synchrotron light source is provided. The preparation method includes the following steps: 1) synthesizing a polydopamine-metal with a polydopamine surface modified with groups required for the click reaction; 2) adding a modifying agent to a culture medium containing cells to modify biomolecules that can target various subcellular organelles; 3) adding the polydopamine-metal obtained in step 1) to the culture medium after modification in step 2) until the click reaction is completed; 4) dropping the cells obtained in step 3) onto a substrate suitable for synchrotron X-ray imaging, and observing the cells under synchrotron X-ray imaging. The biomolecules in the cells have specific absorption peaks under X-rays, thus obtaining an X-ray click chemical labeling probe based on a synchrotron light source.

[0008] The preparation method provided by this invention operates on the principle of modifying polydopamine particles and biomolecules to enable a click reaction between them. Simultaneously, the polydopamine particles can chelate Cu. 2+ Metal ions, when observed using synchronous X-ray imaging, exhibit specific absorption peaks under X-rays, thus enabling the characteristic identification and localization of various biomolecules within cells.

[0009] The polydopamine-metal used in step 1) is polydopamine particles that chelate metal ions. The metals are non-selective and include Fe, Cu, Zn, Mn, Ti, Ni, Ca, Cr, Cd, Pb and Hg, etc.

[0010] The click reaction groups used in step 2) include azide, alkynyl, mercapto, and dibenzocyclooctyne. Azide-polyethylene glycol-amine is preferred for modifying azide groups, alkynyl-polyethylene glycol-amine is preferred for modifying alkynyl groups, mercapto-polyethylene glycol-amine is preferred for modifying mercapto groups, and dibenzocyclooctyne-polyethylene glycol-amine is preferred for modifying dibenzocyclooctyne.

[0011] The modifying reagent used in step 2) is used to modify the groups required for the click reaction on biomolecules. Different biomolecules require different reagents to obtain better modification effects. For example, EdU reagent can be used to modify alkyne groups on DNA and target the cell nucleus, which is rich in DNA.

[0012] It should be understood that different modifying reagents are required for different groups of different biomolecules. For example, Ac4ManNAz is needed to modify the azide group of mannose on the cell membrane, while Ac4ManNAl is needed to modify the alkyne group of mannose on the cell membrane. For DNA, Ac4ManNAz cannot effectively modify the azide group, and AmdU is required.

[0013] The concentration of polydopamine-metal used in step 3) is 5 to 40 μg / mL, with 10 μg / mL being preferred.

[0014] In step 3), for different biological targets, polydopamine particles of different sizes can be selected as the core to obtain better positioning accuracy. When the biological target is at the micrometer scale, it is better to select polydopamine particles with a particle size of more than 100 nm. When the biological target is at the submicrometer scale, it is better to select polydopamine particles with a particle size of less than 100 nm.

[0015] In step 4), different incident energies are selected according to different metal ions. Taking Fe, Cu, Zn, Mn, Ti, Ni, and Ca as examples, the appropriate incident energies for Fe are 280-1500 eV and 7112-15000 eV, for Cu are 280-2000 eV and 8979-18000 eV, and for Zn are 280-2100 eV and 9659-2000 eV. 0000eV, the incident energies applicable to Mn are 280-1400eV and 6514-14000eV, the incident energies applicable to Ti are 280-1300eV and 4952-13000eV, the incident energies applicable to Ni are 280-1900eV and 8332-17000eV, and the incident energies applicable to Ca are 280-1200eV and 4059-12000eV.

[0016] The corresponding metal elements can be imaged within the selected X-ray incident energy range, but there is also an optimal incident energy selection. Taking scanning transmission X-ray microscopy for Fe imaging as an example, if dual-energy X-ray absorption analysis is to be performed, the incident energy is selected at 704 eV and 709.2 eV.

[0017] In step 4), the optimal X-ray incident energy range is different for polydopamine cores with different particle sizes. For example, for polydopamine with a particle size of less than 100 nm, high-energy X-rays, i.e., hard X-rays, are preferred. For polydopamine with a particle size of more than 100 nm, low-energy X-rays, i.e., soft X-rays, are preferred.

[0018] According to a second aspect of the present invention, an X-ray click chemical labeling probe based on a synchrotron light source is provided, prepared according to the above-described preparation method.

[0019] The types of click reactions that the probe can undergo include click reactions between azide and alkynyl groups, click reactions between mercapto and alkynyl groups, and click reactions between dibenzocyclooctyne (DBCO) and azide.

[0020] According to a third aspect of the present invention, an application of a synchronous X-ray click chemical labeling probe in intracellular biomolecule recognition and imaging is provided.

[0021] According to a preferred embodiment of the present invention, imaging of the cell nucleus is achieved by selecting abundant DNA in the cell nucleus for modification to induce a click reaction. For example, EdU, a nucleoside, can be used as a label for nuclear DNA. It can be incorporated into replicating DNA via cellular enzymes, incorporating an alkyne group into the DNA structure. By modifying polydopamine particles with an azide-polyethylene glycol-amine mixture, and then adding the particles to the cell culture medium after chelating metal ions, a click reaction occurs between the azide and alkyne groups, thereby localizing the DNA to the cell nucleus. Similarly, by modifying the biomolecule to be labeled with the groups required for the click reaction, labeling of other intracellular biomolecules can also be achieved. For example, mannose on the cell membrane can be modified with Ac4ManNA1 or Ac4ManNAz to modify alkyne or azide groups, respectively.

[0022] It should be noted that Chinese patent application (CN201911094934.5) discloses a synchrotron X-ray multicolor genetic marker probe, its preparation method, and its application. This method uses synchrotron X-ray imaging and utilizes the specific fluorescence peaks of each DAB-metal polymer under X-rays to achieve the characteristic recognition and localization of multiple biomolecules within cells. However, compared with this prior patent, the present invention has more significant advantages, which are detailed below:

[0023] 1) The prior patent relates to a synchrotron X-ray imaging probe. Due to the inherent defects of the imaging tag, it will spread in the cell, resulting in a decrease in positioning accuracy. However, the click chemical imaging probe prepared in this invention overcomes the above defects. The click chemical imaging tag has a fixed morphology and controllable particle size, so a suitable imaging energy range can be selected (different energies have different sensitivities to metals), and the imaging signal-to-noise ratio is better. Therefore, the positioning accuracy for different biological targets in the cell is higher.

[0024] 2) The prior patent discloses a limited number of systems in which the genetic marker probes can be expressed independently and stepwise (only photocatalytic mini singlet oxygen-producing protein (MiniSOG) and APEX2 catalyze the generation of DAB and metal polymers), making it difficult to simultaneously image three or more biological targets. However, the X-ray click chemistry probe provided by this invention is based on click chemistry reactions. Since there are many types of click chemistry and different catalytic types, it is very promising to break through the limitations of existing technologies on the number of biomolecules that can be imaged simultaneously and can be better applied to cell biology research.

[0025] 3) The prior patent discloses an X-ray genetic marker probe based on a synchronous light source that can only image fixed dead cells. However, the X-ray click chemistry probe provided by this invention is based on click chemistry reactions, in which strain-promoted azide alkyne cycloaddition and other reactions are bioorthogonal reactions. The principle is that biochemical reactions that do not interfere with the biochemical reactions of the organism occur in the organism, i.e., bioorthogonal reactions. Therefore, it can image living cells without producing toxicity to the cells, thereby better tracking the life process of cells.

[0026] Therefore, the X-ray click chemical probe based on a synchronous light source and its preparation method provided by the present invention not only has higher positioning accuracy for different biological targets within cells, but also can simultaneously image more types of biological targets. More importantly, it can also image living cells without causing toxicity to cells.

[0027] The main inventive point of this invention lies in optimizing polydopamine particles of different sizes as the core for biological targets of different scales to obtain good biological target localization accuracy, and optimizing different X-ray incident energy ranges for polydopamine cores of different sizes to obtain good imaging signals. Specifically, for large-diameter polydopamine particle cores, low-energy X-rays, i.e., soft X-rays, are selected. For small-diameter polydopamine particle cores, low-energy X-rays are not effective for imaging, but when high-energy X-rays are used, the labeled biomolecules have better imaging effects. See Examples 4 and 5 for details. The metal selected is Fe. The cell nucleus is relatively large, so large-diameter polydopamine particles (120 nm) are selected as the core and low-energy X-rays are used to obtain the distribution of DNA within it. However, for the cell membrane, because the cell membrane is thin, small-diameter polydopamine particles (50 nm) are selected as the core. In this case, low-energy X-rays cannot clearly show the outline of the cell membrane, but when high-energy X-rays (9000 eV) are selected, the outline of the cell membrane is clearly shown.

[0028] The significant advancements of this invention lie in the fact that synchrotron X-ray microscopy and its suitable probe labeling techniques are crucial for understanding the complex spatiotemporal interactions of biomolecules in the natural cellular environment. However, currently developed immunostaining probes and genetic marker probes suitable for synchrotron X-ray microscopy struggle to achieve multicolor imaging of intracellular biomolecules. This invention leverages the high elemental resolution and non-interference characteristics of X-ray microscopy, combined with the numerous, highly specific, rapid, and reliable properties of click chemistry reaction systems, to construct a synchrotron X-ray click chemistry labeling probe. Through the click reaction of polydopamine particles chelating metal ions with biomolecules, biomolecules are labeled in situ within the cell. Observation using synchrotron X-ray imaging reveals specific absorption peaks for each metal under X-rays, enabling the characteristic identification and localization of multiple intracellular biomolecules.

[0029] Secondly, the X-ray click chemistry probe prepared according to this invention consists of a polydopamine core and metal ions bonded to it, achieving efficient recognition of intracellular / extracellular biological targets through click reactions between functional groups. The metal ions loaded on the probe provide a good signal under synchrotron radiation microscopy. Since click chemistry reactions include cycloaddition reactions, nucleophilic ring-opening reactions, non-alcoholic carbonyl chemistry, and addition reactions of carbon-carbon double and triple bonds, there is still room for further development, and crosstalk between reaction systems can be well avoided. This invention, by developing a third type of imaging probe suitable for X-ray microscopy, achieves precise recognition and localization of various intracellular biomolecules, which is of great significance in the field of cell imaging.

[0030] In summary, this invention provides a method for highly specific recognition and high-resolution imaging of a variety of biomolecules within living cells, which has promising prospects for biomedical applications. Attached Figure Description

[0031] Figure 1 This is an SEM image of small-diameter polydopamine particles;

[0032] Figure 2 This study investigates the HeLa cytotoxicity of polydopamines with different particle sizes.

[0033] Figure 3 The study focused on the cytotoxicity of polydopamine-Cu and polydopamine-Fe modified with azide groups.

[0034] Figure 4 This is a synchrotron X-ray image of polydopamine-Fe labeled nuclear DNA with azide groups, where white indicates the distribution of polydopamine-Fe labeled intracellular DNA;

[0035] Figure 5AThis is a scanning transmission X-ray image of cell membrane mannose labeled with polydopamine-Fe modified with azide groups;

[0036] Figure 5B This is a transmission X-ray image of cell membrane mannose labeled with polydopamine-Fe modified with azide groups. Detailed Implementation

[0037] The present invention will be further described below with reference to specific embodiments. It should be understood that the following embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments that do not specify specific conditions were performed according to conventional methods and conditions, or according to the product instructions. All reagents and raw materials used in this invention are commercially available.

[0038] The inventors selected human HeLa cells, modified polydopamine with azide groups, chose a click reaction type of azide-alkynyl click reaction, selected DNA in the cell nucleus and mannose on the cell membrane as biological targets, and selected Fe as the metal ion. 3+ and Cu 2+ The invention constructs an X-ray click chemical labeling probe and applies it to imaging studies of this cell. The following examples illustrate the implementation effects of the invention.

[0039] Example 1: Synthesis of small-diameter polydopamine particles.

[0040] Synthesis of polydopamine. Mix 20 mL of anhydrous ethanol and 45 mL of ddH₂O, add 5 mL of 28% ammonia solution, and stir at 30 °C for 30 minutes. Dissolve 250 mg of dopamine hydrochloride (purchased from Sigma) in 5 mL of ddH₂O and add to the reaction mixture; react for 24 hours. Adjust the pH to neutral and sonicate the solution at 100 W output power for 3 minutes. Dialyze (30 kDa) three to four times for purification.

[0041] The amount of ammonia used affects the particle size of polydopamine. Using 5 mL of 28% ammonia, polydopamine with an average particle size of 50 nm can be synthesized. The azide-modified polydopamine was observed under SEM. Figure 1 ).

[0042] Conclusion: SEM results show that polydopamine particles were successfully synthesized. The polydopamine particles exhibited intact morphology and good monodispersity. The small-diameter polydopamine particle cores can improve imaging accuracy for labeling submicron-scale biological targets, thus enabling applications in various scenarios.

[0043] Example 2: Cytotoxicity of polydopamine particles of different sizes.

[0044] The synthesis of polydopamine was the same as in Example 1, except that different amounts of ammonia were used: 0.75 mL and 1 mL.

[0045] The amount of ammonia used affects the particle size of polydopamine. Using 1 mL of 28% ammonia can synthesize polydopamine with an average particle size of 120 nm; using 0.75 mL of 28% ammonia can synthesize polydopamine with an average particle size of 180 nm.

[0046] Assay for HeLa cell toxicity. HeLa cells were purchased from the Cell Bank of the Chinese Academy of Sciences Type Culture Collection Committee. HeLa cells were cultured at 7 × 10⁶ cells per well. 4 Cells were seeded in 96-well plates and cultured to a normal growth state. The cells were then cultured for 24 hours in medium supplemented with polydopamine of varying particle sizes. The final concentration of polydopamine used in the experiment was 10 μg / mL. Cytotoxicity was determined using the MTT assay. 10 μL of MTT (0.5 mg / mL) solution was added to each well, and the cells were incubated at 37°C for 3 hours to obtain crystals. The supernatant was collected, and the product was lysed with 200 μL of DMSO. The absorbance was recorded at 590 nm using a microplate reader. The absorbance of untreated cells was used as a control, and its absorbance was used as a reference value for calculating 100% cell viability.

[0047] Conclusion: The results of HeLa cytotoxicity assays showed that ( Figure 2 HeLa cells treated with polydopamine of different particle sizes all exhibited high viability. This example demonstrates the excellent biocompatibility of polydopamine particles.

[0048] Example 3: Cytotoxicity of azido-modified polydopamine-Cu and azido-modified polydopamine-Fe.

[0049] The synthesis of polydopamine is the same as in Example 1, except that the amount of ammonia used is 1 mL.

[0050] Polydopamine was surface-modified with azido groups. 1 mL of 1 mg / mL polydopamine was mixed with 5 mL of 28% ammonia solution, the pH was adjusted to 9, and 20 mg of azido-polyethylene glycol-amine (purchased from VanSho Biotechnology) was added. The reaction was allowed to proceed for 12 hours. The mixture was then dialyzed (30 kDa) three to four times for purification.

[0051] Preparation of azido-modified polydopamine-Cu. 1 mL of 1 mg / mL azido-modified polydopamine was mixed with 100 μL of 10 mg / mL Cu. 2+ The solutions (provided in CuCl2 form) were mixed and dissolved in PB buffer solution at pH 5.5. The mixture was reacted at 40°C for 1 hour, and unreacted metal ions were separated to obtain polydopamine-Cu modified with azide groups.

[0052] Preparation of azido-modified polydopamine-Fe. 1 mL of 1 mg / mL azido-modified polydopamine was mixed with 100 μL of 10 mg / mL Fe. 3+The solutions (provided in FeCl3 form) were mixed and dissolved in PBS buffer at pH 7. The mixture was reacted at 40°C for 1 hour, and unreacted metal ions were separated to obtain polydopamine-Fe modified with azide groups.

[0053] The culture and cytotoxicity determination of HeLa cells were the same as in Example 1. The final concentrations of both the azide-modified polydopamine-Cu and polydopamine-Fe were 10 μg / mL.

[0054] Conclusion: The results of HeLa cytotoxicity assays showed that ( Figure 3 At a concentration of 10 μg / mL, HeLa cells exhibited high viability. The viability of HeLa cells with azido-modified polydopamine-Cu was slightly lower than that with azido-modified polydopamine-Fe, but still above 90%, indicating excellent biocompatibility.

[0055] Example 4: Application of polydopamine-Fe modified with azide groups on labeled DNA.

[0056] The preparation of polydopamine-Fe modified with azide groups is the same as in Example 3.

[0057] HeLa cells were cultured. The HeLa cells were purchased from the Cell Bank of the Chinese Academy of Sciences Type Culture Collection Committee. Logarithmically growing cells were harvested, and silicon nitride windows were placed in cell culture plates. The plates were sterilized under UV light, with 7 × 10⁶ cells per well. 4 Cells were seeded in 96-well plates and cultured until normal growth was achieved. EdU (Invitrogen) solution was diluted 1000:1 with cell culture medium to prepare 50 μM EdU medium. 100 μL of 50 μM EdU medium was added to each well and incubated for two hours. At this point, alkyne groups were modified on the DNA of HeLa cells.

[0058] Polydopamine-Fe modified with azide groups was added to the culture medium to a final concentration of 10 μg / mL. After 2 hours of click reaction, the culture medium was discarded. Cells were washed with PBS 1-2 times, 5 minutes each time. The solution in the wells was removed, and X-ray imaging was performed simultaneously.

[0059] At the BL08U1-A soft X-ray absorption spectroscopy beamline of the Shanghai Synchrotron Radiation Facility, incident light energies of 704 eV and 709.2 eV were selected to perform scanning transmission X-ray dual-energy imaging on cells labeled with synchrotron X-ray click chemical labeling probes.

[0060] Conclusion: After labeling, the localization and distribution of DNA in the cell nucleus can be clearly observed under a synchrotron X-ray microscope. Figure 4 The results of dual-energy imaging showed a significant Fe signal in the nucleus region (the region with the lowest gray value in the nucleus), which is consistent with the common knowledge that DNA is mainly distributed in the nucleus.

[0061] Example 5: Application of azide-modified polydopamine-Fe in labeling mannose.

[0062] The preparation of azide-modified polydopamine-Fe was the same as in Example 3. Using 5 mL of ammonia, polydopamine particles with a particle size of 50 nm were synthesized as the core.

[0063] HeLa cells were cultured. The HeLa cells were purchased from the Cell Bank of the Chinese Academy of Sciences Type Culture Collection Committee. Silicon nitride windows were placed in cell culture plates and sterilized under UV light, with a density of 7 × 10⁶ cells per well. 4 Cells were seeded in 96-well plates and cultured until normal growth was achieved. 100 μL of 100 μM Ac4 ManNAl (purchased from Qiyue Biotechnology) medium was prepared. 100 μL of 100 μM Ac4 ManNAl medium was added to each well and the cells were incubated until confluence. At this point, the mannose groups on the HeLa cell membrane were modified with alkyne groups.

[0064] Polydopamine-Fe modified with azide groups was added to the culture medium to a final concentration of 10 μg / mL. After a 2-hour click reaction, the culture medium was discarded. Cells were washed with PBS 1-2 times, 5 minutes each time. The solution in the wells was removed, and X-ray imaging was performed simultaneously.

[0065] At the BL08U1-A soft X-ray absorption spectroscopy beamline of the Shanghai Synchrotron Radiation Facility in China, with an incident light energy of 709.2 eV, scanning transmission X-ray imaging was performed on cells labeled with synchrotron X-ray click chemical labeling probes. Figure 5A At the 01B1 X-ray Microscopy beamline of the Taiwan Light Source, with an incident light energy of 9000 eV, transmission X-ray imaging was performed on synchrotron X-ray click-labeled chemical cells. Figure 5B ).

[0066] Conclusion: The results of transmission X-ray imaging show that the cell membrane outline can be clearly observed and marked under synchrotron radiation X-ray microscopy, which is consistent with the fact that mannose is mainly distributed on the cell membrane. However, the cell membrane outline is not obvious under scanning transmission X-ray imaging, indicating that high-energy X-rays, i.e., hard X-rays, are preferable when using small-particle-size polydopamine cores to improve positioning accuracy.

[0067] The above description is merely a preferred embodiment of the present invention and is not intended to limit the scope of the invention. Various variations can be made to the above embodiments of the present invention. All simple and equivalent changes and modifications made in accordance with the claims and description of this application fall within the protection scope of the claims of this patent. Any aspects not described in detail in this invention are within the scope of conventional technology.

Claims

1. A method for preparing an X-ray click chemical probe based on a synchrotron light source, characterized in that, The preparation method includes the following steps: 1) Synthesize a polydopamine-metal with a surface modified with groups required for the click reaction, wherein the polydopamine-metal is a polydopamine particle that chelates metal ions and is non-selective for metals, including: Fe, Cu, Zn, Mn, Ti, Ni, Ca, Cr, Cd, Pb and Hg, and the groups required for the click reaction include azide, alkynyl, mercapto and dibenzocyclooctyne; 2) Add modification reagents to the culture medium containing cells to modify biomolecules that can target various subcellular organelles; 3) Add the polydopamine-metal synthesized in step 1) to the culture medium modified in step 2) until the click reaction is complete. The concentration of the polydopamine-metal is 5~40 μg / mL. For different biological targets, polydopamine particles of different sizes can be selected as the core to obtain better positioning accuracy. When the biological target is at the micron scale, polydopamine particles with a particle size of more than 100 nm are selected. When the biological target is at the submicron scale, polydopamine particles with a particle size of less than 100 nm are selected. 4) The cells obtained in step 3) are dropped onto a substrate suitable for synchrotron X-ray imaging. Through biochemical reactions that do not interfere with the biochemical reactions of the organism, live cells can be imaged and observed under synchrotron X-rays without causing toxicity to the cells. The biomolecules in the cells have specific absorption peaks under X-rays, thus obtaining an X-ray click chemical labeling probe based on a synchrotron light source.

2. The preparation method according to claim 1, characterized in that, In step 2), the modifying agent is used to modify the groups required for the click reaction on the biomolecule. Different biomolecules require different modifying agents to obtain better modification effects.

3. The preparation method according to claim 1, characterized in that, In step 3), the click reaction types include click reaction between azide and alkynyl, click reaction between mercapto and alkynyl, and click reaction between dibenzocyclooctyne and azide.

4. The preparation method according to claim 1, characterized in that, In step 4), the applicable X-ray incident energy range is different for polydopamine cores with different particle sizes; for polydopamine cores with a particle size of less than 100 nm, high-energy X-rays are selected, and for polydopamine with a particle size of more than 100 nm, low-energy X-rays are selected.

5. The preparation method according to claim 1, characterized in that, In step 4), different incident energies are selected according to different metal ions. The incident energies suitable for Fe are 280-1500 eV and 7112-15000 eV, suitable for Cu are 280-2000 eV and 8979-18000 eV, suitable for Zn are 280-2100 eV and 9659-20000 eV, suitable for Mn are 280-1400 eV and 6514-14000 eV, suitable for Ti are 280-1300 eV and 4952-13000 eV, suitable for Ni are 280-1900 eV and 8332-17000 eV, and suitable for Ca are 280-1200 eV and 4059-12000 eV.

6. A synchrotron light source-based X-ray click chemical labeling probe prepared by the preparation method according to any one of claims 1 to 5.

7. The application of the synchronous X-ray click chemical labeling probe according to claim 6 in the recognition and imaging of biomolecules in living cells.

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