A synchrotron X-ray visible click chemistry imaging label and its preparation method

Abstract

The present invention provides a click chemistry imaging label visible to synchrotron X-rays and a preparation method thereof. The preparation method comprises the following steps: 1) adding ammonia water to an ethanol solution, adding a dopamine hydrochloride solution to react to obtain polydopamine; 2) neutralizing the solution, purifying the polydopamine by dialysis after ultrasound; 3) adding polydopamine to ammonia water, adjusting the pH to weak alkalinity, adding a click reaction group modification reagent, and separating and purifying after the reaction; 4) dissolving the metal ions and the modified polydopamine in a buffer system; 5) reacting for a period of time until a compound is generated; and 6) dripping the solution of the obtained compound onto a substrate, and observing the image under synchrotron X-rays. The present invention successfully prepares a click chemistry imaging label visible to synchrotron X-rays, laying a good foundation for further preparing a variety of synchrotron X-ray click chemistry imaging probes and achieving high-specificity recognition and high-resolution imaging of a variety of biological molecules in cells.

Description

Technical Field

[0001] The present invention relates to the field of biochemical technology, and more particularly to a click chemistry imaging label visible to synchrotron X-rays and a preparation method thereof. Background Art

[0002] One of the basic goals of cell biology is to understand the complex interactions of biological molecules in cellular life activities. Synchrotron radiation X-ray microscopy, because the wavelength of X-rays is in the range of 0.1 to 10 nm, it is naturally a super-resolution microscopy technology, and the resolution can theoretically reach several nanometers. Compared with electron beams, X-rays have stronger penetration into biological samples and can image intact cells without sectioning. The soft X-ray "water window" band can image cells with natural contrast. In particular, X-rays have excellent energy (element) resolution, and the differences in X-ray fluorescence emission spectra of different elements can be used to develop multi-color labels, which can be used to prepare biological probes to achieve the identification and imaging of multiple biological molecules in cells.

[0003] Existing molecular probes compatible with X-ray microscopy include immune probes and genetic labeling probes, enabling detailed imaging of multiple biological targets within cells. However, immune labeling techniques are prone to crosstalk between antigens and antibodies, and genetic labeling techniques offer limited systems capable of independent, step-by-step expression. Both techniques struggle to simultaneously label and image multiple biological targets within cells.

[0004] A Chinese patent application (CN201710228701.4) discloses a synchrotron X-ray visible imaging tag and its preparation method. However, this type of synchrotron radiation X-ray imaging tag does not have a fixed morphology. When subsequently applied to cell imaging, the positioning accuracy will be reduced due to the diffusion of the tag within the cell. This is a major inherent defect of this type of imaging tag. Summary of the Invention

[0005] The purpose of the present invention is to provide a click chemistry imaging label visible to synchronized X-rays and a preparation method thereof, so as to solve the problem that existing genetic labeling probes and immune probes suitable for X-rays do not have a fixed morphology and, when applied to cell imaging, the positioning accuracy is reduced due to the diffusion of the label within the cell.

[0006] In order to solve the above technical problems, the present invention adopts the following technical solutions:

[0007] According to a first aspect of the present invention, a method for preparing a click chemistry imaging label visible to synchrotron X-rays is provided, the preparation method comprising the following steps: 1) adding ammonia water to an ethanol solution and stirring the solution for a period of time, then adding a dopamine hydrochloride solution to react and obtain polydopamine; 2) neutralizing the solution to neutrality, ultrasonicating the solution for a period of time, and dialysis to purify the polydopamine; 3) adding the polydopamine to ammonia water, adjusting the pH to weak alkalinity, adding a click reaction group modification reagent, reacting for a period of time, and separating and purifying the polydopamine; 4) dissolving metal ions and the modified polydopamine obtained in step 3) in a buffer system, wherein the metal is non-selective and includes: Fe, Cu, Zn, Mn, Ti, Ni, Ca, Cr, Cd, Pb and Hg; 5) reacting for a period of time until a compound is generated; and 6) dropping the solution of the compound obtained in step 5) onto a substrate suitable for synchrotron X-ray imaging, and imaging and observing under synchrotron X-rays to obtain a click chemistry imaging label visible to synchrotron X-rays.

[0008] According to the preparation method provided by the present invention, its working principle is: the surface of polydopamine can be modified with groups such as azide to provide the necessary groups for the click reaction, and polydopamine can chelate a large number of Cu 2+ Using synchrotron X-ray imaging, we observed that metal ions have specific absorption peaks under X-rays, thus preparing a click chemistry imaging label visible to synchrotron X-rays.

[0009] The ratio of ammonia used in step 1) is adjustable, and polydopamine of varying particle sizes can be synthesized by adjusting the ratio of ammonia. For example, polydopamine can be synthesized by adding 28% ammonia to a mixed solution of 20 mL of 100% ethanol, 45 mL of ddH2O, and 250 mg of dopamine hydrochloride dissolved in 5 mL of ddH2O. The volume of 28% ammonia ranges from 0.2 mL to 5 mL, and preferably 1 mL is used to synthesize polydopamine with an average particle size of 120 nm.

[0010] The ultrasound used in step 2) has an ultrasound power of 80 to 200 W, preferably 100 W, and an ultrasound time of 1 to 20 min, preferably 3 min.

[0011] In step 3), the pH value is 7.5 to 11, preferably 9.

[0012] The click reaction groups used in step 3) include azide, alkynyl, sulfhydryl and dibenzocyclooctyne (DBCO). The azide modification reagent is preferably azide-polyethylene glycol-amine, the alkyne modification reagent is preferably alkynyl-polyethylene glycol-amine, the sulfhydryl modification reagent is preferably sulfhydryl-polyethylene glycol-amine, and the DBCO modification reagent is preferably DBCO-polyethylene glycol-amine.

[0013] The metal ions used in step 4) are provided by any one of hydrochloride, sulfate, nitrate and phosphate, such as Cu 2+ The hydrochloride salt is most effective.

[0014] The buffer system used in step 4) includes phosphate buffer, phosphate buffered saline or tris-hydrochloric acid buffer, the pH of the buffer is 5 to 8, such as Cu 2+ Phosphate buffer at pH 5.5 is preferred to obtain better solubility.

[0015] In step 5), the reaction temperature is 4-60° C., and the reaction time is 30 min-4 h. The preferred reaction temperature is 40° C., and the reaction time is 1 h.

[0016] The imaging substrate used in step 6) is a silicon nitride window, a copper-mesh carbon support film, or a Mylar film. The optimal imaging substrate is selected based on the substrate molecule. The silicon nitride window thickness is 50-150 nm, with an optimal value of 100 nm. The copper-mesh carbon film thickness is 5-100 nm, with an optimal value of 20 nm. The Mylar film thickness is 2-25 μm, with an optimal value of 10 μm.

[0017] The energy of the synchronous X-ray imaging used in step 6) is 280-20000eV. Different incident energies are selected according to different metal ions. Taking Fe, Cu, Zn, Mn, Ti, Ni and Ca as examples, the incident energies suitable for Fe are 280-1500eV and 7112-15000eV, the incident energies suitable for Cu are 280-2000eV and 8979-18000eV, and the incident energy suitable for Zn is 280- 2100eV and 9659-20000eV, the incident energy suitable for Mn is 280-1400eV and 6514-14000eV, the incident energy suitable for Ti is 280-1300eV and 4952-13000eV, the incident energy suitable for Ni is 280-1900eV and 8332-17000eV, and the incident energy suitable for Ca is 280-1200eV and 4059-12000eV.

[0018] 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 Cu as an example, for dual-energy X-ray absorption analysis, the incident energies are selected between 938.8eV and 942.8eV.

[0019] This click chemistry tag enables click reactions between azide and alkyne groups, sulfhydryl and alkyne groups, and DBCO and azide. The diverse catalytic systems provide a promising foundation for its application in multicolor imaging of diverse biomolecules.

[0020] According to a second aspect of the present invention, a synchrotron X-ray visible click chemistry imaging label prepared according to the above preparation method is provided.

[0021] It should be noted that a Chinese patent application (CN201710228701.4) discloses a synchrotron X-ray visible imaging label and a preparation method thereof. This solution utilizes the characteristics of X-ray microscopy imaging technology that has unique X-ray fluorescence characteristic spectra for different elements and does not interfere with each other. The substrate molecules and different metal ions are mixed, and a variety of synchrotron X-ray imaging labels are prepared through in vitro chemical catalytic reactions and complex reactions between DAB and metal ions. However, this type of synchrotron radiation X-ray imaging label does not have a fixed morphology. When it is subsequently applied to cell imaging, the positioning accuracy will be reduced due to the diffusion of the label in the cell. This is a major inherent defect of this type of imaging label. The click chemistry imaging label prepared by the present invention successfully overcomes the above-mentioned difficulties. Since the click chemistry imaging label has a fixed morphology and controllable particle size, it has higher positioning accuracy for different biological targets in the cell when it is subsequently applied to cell imaging.

[0022] The main point of the invention is to optimize the concentration of metal in the imaging label (taking copper as an example), and also optimize the amount of Cu ion solution used in the preparation of polydopamine-Cu modified with azide groups, and analyze the amount of Cu ions chelated by polydopamine particles under different usage amounts. 2+ When the mass ratio of Cu ions to polydopamine reaches 0.5 or above, the maximum chelation quantity can be achieved. 2+ The dosage was optimized to add 100 μL of 10 mg / mL Cu per 1 mL of 1 mg / mL polydopamine. 2+ ion solution, thereby ensuring sufficient Cu on polydopamine 2+ ions, providing X-ray imaging signals. Although this label and the catalytic polymerization labels based on DAB and the like in previous patents both utilize the X-ray absorption characteristics of metal ions, the labels in the previous patents are actually applied by in situ polymerization at the biological target in the cell to achieve specific labeling. They have no fixed morphology and are very easy to diffuse. The click chemistry imaging label prepared according to the present invention adds metal ions during synthesis to produce a chelation effect with the polydopamine core. The label itself contains metal ions and has a fixed morphology. In actual application, a click reaction occurs in the cell to achieve specific labeling. The reaction principles of the two are completely different. In addition, the present invention also stipulates the imaging energy of imaging labels containing different metals.

[0023] The positive progress of the present invention is that synchrotron X-ray microscopy technology and probe labeling technology suitable for it are of great significance for understanding the complex spatiotemporal interactions of biomolecules in natural cellular environments. However, there is still a lack of probe technology that is compatible with synchrotron radiation X-ray microscopy technology for simultaneous identification and imaging of multiple biotargets in cells. The present invention utilizes the high elemental resolution of X-ray microscopy technology and the characteristics of non-interference between elemental spectra, combined with the strong binding ability of polydopamine to metal ions, to successfully prepare a variety of synchrotron X-ray visible click chemistry imaging labels, laying a good foundation for further preparation of a variety of X-ray click chemistry imaging probes and the realization of specific identification and imaging of multiple biomolecules in cells at the same time.

[0024] Secondly, the click chemistry-based imaging label provided by the present invention comprises a polydopamine core and a metal ion liganded thereto. Since click chemistry reactions include cycloadditions, nucleophilic ring-opening reactions, non-aldol carbonyl chemistry, and addition reactions to carbon-carbon double and triple bonds, there is room for further expansion and crosstalk between reaction systems can be effectively avoided. Therefore, by developing a synchrotron X-ray-visible click chemistry imaging label, the present invention paves the way for the future development of a third type of imaging probe compatible with X-ray microscopy, enabling the precise identification and localization of multiple biomolecules within cells, which is of great significance in the field of cell imaging.

[0025] In summary, the present invention provides a click chemistry imaging label with a fixed morphology and controllable particle size, which can locate different biological targets in cells with higher precision and has good biomedical application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 TEM images of polydopamine particles, A shows the result of adding 0.75 mL of ammonia water, and B shows the result of adding 1 mL of ammonia water;

[0027] Figure 2A is a TEM image of polydopamine-Cu modified with an azide group;

[0028] Figure 2B This is the energy spectrum of polydopamine-Cu modified with azide group;

[0029] Figure 3 is the amount of copper ions chelated on polydopamine particles at different mass ratios;

[0030] Figure 4 is a synchrotron radiation X-ray imaging image of azide-modified polydopamine-Cu, where the white color indicates the distribution of Cu on polydopamine;

[0031] Figure 5This is a synchrotron radiation X-ray imaging image of DBCO-modified polydopamine-Cu, where the white color indicates the distribution of Cu on polydopamine. DETAILED DESCRIPTION

[0032] The present invention will be further described below with reference to specific examples. It should be understood that the following examples are intended only to illustrate the present invention and are not intended to limit the scope of the present invention. Experimental methods in the following examples where specific conditions are not specified were performed according to conventional methods and conditions, or according to commercial specifications. Reagents and raw materials used in the present invention are all commercially available.

[0033] The present invention mainly selects Cu 2+ As a representative of metal ions, polydopamine was modified with azide and DBCO as representatives to prepare synchrotron X-ray click chemistry imaging labels, and applied to synchrotron X-ray imaging research. The following examples specifically illustrate the implementation effects of the present invention.

[0034] Example 1 Preparation of polydopamine particles with different particle sizes.

[0035] Synthesis of polydopamine. Mix 20 mL of anhydrous ethanol and 45 mL of ddH2O, add 1 mL and 0.75 mL of 28% aqueous ammonia, and stir at 30°C for 30 minutes. Dissolve 250 mg of dopamine hydrochloride (purchased from Sigma) in 5 mL of ddH2O and inject into the reaction mixture for 24 hours. Adjust the pH to neutral and sonicate the solution at 100 W output power for 3 minutes. Dialyze (30 kD) three to four times for purification.

[0036] The amount of ammonia used will affect 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 can synthesize polydopamine with an average particle size of 180 nm. Observation of polydopamine modified with azide groups under TEM ( Figure 1 ).

[0037] Conclusion: TEM results show that polydopamine particles were successfully synthesized. The polydopamine particles are complete in morphology and well monodispersed. Polydopamine particles of different sizes lay a good foundation for their application in different scenarios. For example, when observing single polydopamine particles chelated with metal ions under synchrotron radiation X-rays to verify that they have synchrotron radiation X-ray signals, polydopamine particles with larger particle sizes are preferred. However, if this label is further applied to synchrotron radiation X-ray click chemistry probes, for intracellular biological targets of different sizes, in order to improve the positioning accuracy, polydopamine particles of different particle sizes need to be selected as the core of the imaging label.

[0038] Example 2 Preparation of azide-modified polydopamine-Cu.

[0039] The synthesis of polydopamine was the same as in Example 1, except that 1 mL of ammonia was used.

[0040] To modify the surface of polydopamine with azide groups, mix 1 mL of 1 mg / mL polydopamine with 5 mL of 28% aqueous ammonia, adjust the pH to 9, add 20 mg of azide-polyethylene glycol-amine (purchased from Fanshuo Biotechnology), and allow to react for 12 hours. Purify by dialysis (30 kD) three to four times.

[0041] 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 solution (provided in the form of CuCl2) was mixed and dissolved in PB buffer at pH = 5.5. The mixture was reacted at 40°C for 1 hour. The unreacted metal ions were separated and removed to obtain polydopamine-Cu modified with an azide group. The polydopamine-Cu modified with an azide group was observed under TEM ( Figure 2A ) and perform spectrum analysis ( Figure 2B ).

[0042] Conclusion: TEM results show that the azido-modified polydopamine-Cu is morphologically intact and monodisperse. Energy dispersive spectrometry analysis shows a copper signal peak.

[0043] Example 3Cu 2+ Optimization of solution usage.

[0044] The preparation of polydopamine-Cu modified with azide group was the same as in Example 2, with 1 mL of ammonia and 1 mL of Cu 2+ The amounts of the solution used were 1 μL, 5 μL, 10 μL, 20 μL, 50 μL and 100 μL, respectively, such that the mass ratios of metal ions to azide-modified polydopamine were 0.01, 0.05, 0.1, 0.2, 0.5 and 1, respectively.

[0045] The unreacted metal ions were removed using a PD-10 desalting column (purchased from Cytiva) and the complex was purified. 2+ The concentration was determined by inductively coupled plasma mass spectrometry (ICP-MS). For ICP-MS analysis, 1 mL of concentrated nitric acid (68% w / w) was first added to 100 μL of the test sample and heated for digestion. The residue was dissolved in ddH2O, the volume was fixed to 10 mL, and then diluted 100 times with dilute nitric acid (2% w / w) for the final ICP-MS analysis. The measurement results are shown in Figure 2. Figure 3 shown.

[0046] Conclusion: ICP-MS results show that polydopamine modified with azide group chelates Cu 2+There was no significant difference in the amount of Cu when the mass ratio reached 0.5 and when it reached 1, and the chelated amount was 99 μg / mg, indicating that the solution provided Cu when the mass ratio was 0.5. 2+ The amount of copper ions chelated is 89 μg / mg when the mass ratio is 0.2. Based on this, it can be determined that Cu 2+ The amount of solution used.

[0047] Example 4 Synchrotron X-ray Imaging of Azide-Modified Polydopamine-Cu and DBCO-Modified Polydopamine-Cu

[0048] The preparation of polydopamine-Cu modified with an azide group was the same as in Example 2, except that the amount of ammonia water used was 0.75 mL.

[0049] The synthesis of polydopamine was the same as in Example 1, with 0.75 mL of ammonia water used.

[0050] To modify the surface of polydopamine with DBCO, mix 1 mL of 1 mg / mL polydopamine with 5 mL of 28% ammonia solution, adjust the pH to 9, and add 20 mg of DBCO-polyethylene glycol-amine (purchased from Qiyue Biotechnology). Allow to react for 12 hours. Purify by dialysis (30 kD) three to four times.

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

[0052] The above solutions were dropped onto the silicon nitride window, dried, and placed in a vacuum sample chamber. The X-ray imaging experiment was carried out at the BL08U1 soft X-ray spectroscopy microscopy line of the Shanghai Synchrotron Radiation Facility, and the experimental method was soft X-ray transmission imaging. The incident energy was selected as 938.8eV and 942.8eV, corresponding to polydopamine-Cu modified with azide and polydopamine-Cu modified with DBCO, respectively. The moving motor was used to complete the search and focusing of polydopamine-Cu modified with azide and polydopamine-Cu modified with DBCO, and then scanning transmission X-ray dual-energy imaging was performed with an imaging resolution of 30nm. The imaging results are shown in Figure 2. Figure 4 and Figure 5 As shown, the pixels with lower gray values ​​on the polydopamine particles are Cu signal areas.

[0053] Conclusion: Both azide-modified polydopamine-Cu and DBCO-modified polydopamine-Cu exhibit excellent X-ray absorption properties and can be used as synchronized X-ray imaging labels for further application research. The above descriptions are merely preferred embodiments of the present invention and are not intended to limit its scope. Various variations are possible. Any simple, equivalent changes and modifications made in accordance with the claims and description of this application fall within the scope of protection of the patent claims. Any information not fully described herein is conventional technology.

Claims

1. A method for preparing a click chemistry imaging label visible to synchrotron X-rays, characterized in that: The preparation method comprises the following steps: 1) Ammonia water is added to an ethanol solution and stirred for a period of time, and then a dopamine hydrochloride solution is added to react to obtain polydopamine; the ratio of the ammonia water is adjustable, and polydopamine of different particle sizes can be synthesized by adjusting the ratio of the ammonia water; 2) Neutralize the solution to neutrality, sonicate for a period of time, and then dialysis to purify polydopamine; 3) adding polydopamine to ammonia water, adjusting the pH to weak alkalinity, adding a click reaction group modification reagent, reacting for a period of time, and separating and purifying the reaction product, wherein the click reaction group includes an azide group, an alkyne group, a thiol group, or a dibenzocyclooctyne group; 4) dissolving the metal ions and the modified polydopamine obtained in step 3) in a buffer system, wherein the metal ions are non-selective and include: Fe, Cu, Zn, Mn, Ti, Ni, Ca, Cr, Cd, Pb, and Hg; 5) reacting for a period of time until a compound is formed; and 6) adding a solution of the compound obtained in step 5) dropwise onto a substrate suitable for synchrotron X-ray imaging, and observing the image under synchrotron X-rays to obtain a click chemistry imaging label visible to synchrotron X-rays. The prepared click chemistry imaging label can provide click reactions between azide and alkyne groups, click reactions between sulfhydryl and alkyne groups, and click reactions between DBCO and azide. The click chemistry imaging label has a fixed morphology and controllable particle size, and can achieve specific recognition of multiple biomolecules in cells through click reactions.

2. The preparation method according to claim 1, characterized in that In step 2), the ultrasonic power is 80-200 W, and the ultrasonic time is 1-20 min.

3. The preparation method according to claim 1, characterized in that In step 3), the pH range is 7.5-11.

4. The preparation method according to claim 1, characterized in that In step 4), the metal ions are provided by any one of hydrochloride, sulfate, nitrate and phosphate; the buffer system used includes phosphate buffer, phosphate buffered saline or Tris-hydrochloric acid buffer, with a pH of 5-8.

5. The preparation method according to claim 1, wherein In step 5), the reaction temperature is 4-60°C, and the reaction time is 30 min-4 h; in step 6), the substrate is a silicon nitride window, a copper mesh carbon support film, or a Mylar film.

6. The preparation method according to claim 1, characterized in that In step 6), the energy of the synchrotron radiation X-ray imaging is 280-20000 eV, and different incident energies are selected according to different metal ions. The incident energy suitable for Fe is 280-1500 eV and 7112-15000 eV, the incident energy suitable for Cu is 280-2000 eV and 8979-18000 eV, the incident energy suitable for Zn is 280-2100 eV and 9659-20000 eV, the incident energy suitable for Mn is 280-1400 eV and 6514-14000 eV, the incident energy suitable for Ti is 280-1300 eV and 4952-13000 eV, and the incident energy suitable for Ni is 280-1900 eV and 8332-17000 eV. eV, and the incident energies applicable to Ca are 280-1200 eV and 4059-12000 eV.

7. A synchrotron X-ray visible click chemistry imaging label prepared according to the preparation method according to any one of claims 1 to 6.

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