A method for imaging primary neuronal cells using dual-modal bioorthogonal spherical nucleic acid probes
By designing a dual-modal bio-orthogonal spherical nucleic acid probe and co-incubating it with neuronal cells using click reaction functional groups, the problem of insufficient accuracy of X-ray imaging probes in existing technologies was solved, and highly specific and sensitive neuronal cell imaging was achieved.
Patent Information
- Application Number
- CN202411344701.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2024-09-25
- Publication Date
- 2025-10-03
- Estimated Expiration
- 2044-09-25
AI Technical Summary
Existing X-ray imaging probes are few and difficult to achieve high-precision positioning. Traditional methods are multi-step and error-prone, and cannot effectively label neuronal cell structures.
DNA sequences modified with click reaction functional groups and fluorescent groups are designed and modified, and dual-modal bio-orthogonal spherical nucleic acid probes are prepared by salt aging or microwave method. The click reaction functional groups are used to co-incubate with neuronal cells, and imaging is performed using X-ray imaging technology.
It achieved high-specificity and high-sensitivity neuronal cell imaging, overcame the problem of false-positive signals, and expanded the application of synchrotron radiation X-ray imaging.
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Figure CN119198800B_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the field of biochemical technology, and more particularly to a method for imaging primary neuronal cells using a dual-modal bio-orthogonal spherical nucleic acid probe. Background Art
[0002] The large penetration depth of X-rays makes X-ray imaging technology promising for in situ imaging of cells. Synchrotron radiation offers high brightness and high collimation. Synchrotron-based X-ray microscopy (XRM), with its inherent nanoscale resolution and excellent penetration, can provide the sensitivity and spatial resolution required to analyze ion distribution at the subcellular level, enabling in situ analysis of cells under near-physiological conditions. The contrast of X-ray imaging stems from the fact that different biochemical components at different locations in the sample absorb varying amounts of X-rays as X-rays pass through it. Biological samples composed of light elements absorb X-rays weakly, resulting in poor imaging quality. However, high-atomic-number elements absorb X-rays strongly, and their use as stains or contrast agents to label samples can enhance contrast and generate sufficient signal for specific imaging of cellular biomolecules or structures. Current conventional methods primarily modify imaging probes with various biological ligands (including antibodies, oligonucleotide aptamers, and peptides). However, these methods involve multiple steps and are prone to errors, resulting in limitations.
[0003] Bioorthogonal click reactions are rapid, specific, and high-yield covalent reactions between two molecules that occur under physiological conditions. Click reactions offer advantages such as high selectivity, mild conditions, and rapid kinetics. By effectively combining bioorthogonal click reactions with metabolic engineering, highly specific and sensitive molecular labeling strategies have been developed. These strategies are widely used in many biomedical fields, particularly in the selective labeling of biomolecules in vivo, targeted imaging, and imaging-based drug delivery.
[0004] Spherical nucleic acids (SNAs), as a novel nucleic acid delivery system, have been used for immunomodulation, gene regulation, drug delivery, biosensing, and bioimaging. Furthermore, high-atomic-number elements like Au have a certain degree of absorption in the X-ray band. Au-labeled neurons can be used to observe neuronal morphology and structure using STXM. Summary of the Invention
[0005] The purpose of the present invention is to provide a method for imaging primary neuronal cells using a dual-modal bio-orthogonal spherical nucleic acid probe, thereby solving the problem that there are few existing cell imaging probes suitable for X-rays and that existing imaging probes are difficult to achieve high-precision positioning.
[0006] In order to solve the above technical problems, the present invention adopts the following technical solutions:
[0007] According to the present invention, a method for imaging primary neuronal cells using a dual-modal bio-orthogonal spherical nucleic acid probe is provided, comprising the following steps: 1) designing a DNA sequence modified with a click reaction functional group, a fluorescent group, and labeled with a thiol or polyadenine; 2) using a salt aging method or a microwave method to prepare a dual-modal bio-orthogonal spherical nucleic acid probe from the DNA sequence designed in step 1) and a probe core material; 3) extracting and culturing primary neuronal cells; 4) co-incubating the primary neuronal cells with a click reaction functional group modification reagent to allow the primary neuronal cells to metabolically integrate the click reaction functional group; 5) using the dual-modal bio-orthogonal spherical nucleic acid probe prepared in step 2) to label the primary neuronal cells modified with the click reaction functional group in step 4) with BO-SNAs probes based on a click reaction; and 6) imaging the primary neuronal cells labeled with BO-SNAs probes in step 5).
[0008] The method provided by the present invention works by designing a DNA sequence with click-reactive functional groups and coupling it to a gold nanosphere core to create a BO-SNAs probe, which can specifically label cellular biomolecules through a click reaction. Because the DNA sequence carries fluorescent groups, fluorescence imaging of neurons is possible. Furthermore, because the metal core exhibits highly specific X-ray absorption peaks, it enables the characteristic recognition and localization of different biomolecules within neurons.
[0009] The click reaction functional groups that can be modified in the DNA sequence in step 1) include Alkyne, DBCO, Azide, etc.
[0010] The fluorescent group modified with the DNA sequence in step 1) is non-selective and includes Alexa Fluor 488, Atto590, Cy3, Cy5, Cy7, etc.
[0011] In step 1), the modified fluorescent group must be kept at a certain distance from the probe core material to prevent the probe core material from absorbing fluorescence through FRET, resulting in fluorescence quenching.
[0012] The DNA sequence in step 1) can be specifically designed to have properties such as synthesizing metal clusters.
[0013] The BO-SNAs probe core in step 2) has multiple selectivities and may include inorganic material cores such as AuNPs, AgNPs, FeNPs and their composite materials, as well as organic material cores such as proteins, for example, bovine catalase, etc.
[0014] Methods for preparing inorganic core BO-SNAs probes in step 2) include salt aging and microwave-assisted heating. The salt aging method involves gradually adding salt to a mixture of thiol (SH) or polyadenine (A)-labeled DNA and an inorganic core to reduce charge repulsion and prepare spherical nucleic acids. The microwave-assisted heating method is a further improvement on the salt aging method for preparing spherical nucleic acids. Spherical nucleic acids are directly prepared by microwave-assisted drying the preparation system, significantly reducing the preparation time of the salt aging method and increasing the yield of spherical nucleic acids.
[0015] According to a preferred embodiment of the present invention, the process for extracting primary neuronal cells in step 3) is as follows: after the 18-day gestational age SD pregnant mouse dies by dislocation, the fetal mouse brain is quickly removed, the fetal mouse cerebral cortical neuronal cells are isolated and cultured, and subsequent operations are performed after culturing on a confocal dish or silicon nitride window for 7-14 days.
[0016] The functional group modification reagents for the click reaction in step 4) include cell membrane modification reagents such as Ac4ManNAz and AHA. Other modification reagents can also be used to achieve labeling of other biological molecules in the cell, such as the nuclear DNA marker EdU.
[0017] In step 4), the primary neurons were incubated with the click reaction functional group modification reagent. The original culture medium was aspirated, the cell culture system was rinsed with sterile water, and fresh culture medium was added for incubation to allow the cells to metabolically incorporate the click reaction functional groups. For cell membrane glycan modification, the primary neurons were incubated with 250 μM Ac4ManNAz for 12 hours; for cell membrane protein modification, the primary neurons were incubated with 500 μM AHA for 1 hour.
[0018] In step 5), the BO-SNAs probe labeling of primary neuronal cells is mainly based on copper ion-catalyzed click chemistry reaction (CuAAC) and ring strain-driven alkyne-azide coupling reaction (SPAAC), thereby achieving bioorthogonal spherical nucleic acid (BO-SNAs) probe labeling of cell membranes.
[0019] It should be understood that in the presence of a reducing agent and / or a stabilizing ligand, a cycloaddition reaction occurs between azide and alkyne under copper catalysis to form a stable triazole moiety [1,4-disubstituted (trans)-1,2,3-triazole] similar to an amide bond, which is called a CuAAC reaction. According to a preferred embodiment of the present invention, the BO-SNAs probe is labeled based on the CuAAC click reaction in step 5), specifically comprising: removing the click reaction functional group modification reagent, treating the cells with 4% paraformaldehyde for 15 minutes to fix the cells; BO-Au-SNAs probe (Alkyne-Au-SNAs), BTTAA-CuSO4 complex and sodium ascorbate are shaken and mixed in a PBS system, added to the cell surface and incubated for 1.5 hours, and the BO-SNAs probe is labeled on the cell surface through a click reaction.
[0020] It should also be understood that SPAAC (Strain-promoted azide-alkyne cycloaddition) is a bioorthogonal reaction and a variant of the azide-alkyne Huisgen cycloaddition, which is carried out in the absence of a cytotoxic copper catalyst. The molecules coupled are usually azide and cyclooctyne, and this reaction is also known as copper-free click chemistry. According to a preferred embodiment of the present invention, the BO-SNAs probe labeling based on the SPAAC click reaction in step 5) specifically includes: after removing the click reaction functional group modification reagent, directly adding the BO-SNAs probe (DBCO-AuSNAs) to the culture medium and incubating with the cells for 20 minutes to label the BO-SNAs probe on the cell surface. After removing the BO-SNAs probe, the cells are treated with 4% paraformaldehyde for 15 minutes to fix the cells.
[0021] The primary neuronal cell imaging in step 6) includes confocal imaging and STXM imaging. Confocal imaging is performed by setting the emission and absorption spectra of corresponding fluorescent groups to perform fluorescence imaging of cells labeled with BO-SNAs probes. STXM imaging is performed by performing synchrotron X-ray two-dimensional imaging of neurons using the 08U beamline of the Shanghai Synchrotron Radiation Facility and the 07W beamline of the Hefei Synchrotron Radiation Facility.
[0022] X-rays have the characteristics of short wavelength and strong penetrating power. X-ray-based imaging technology can achieve non-destructive three-dimensional high-resolution imaging studies of cells. Current traditional cell labeling methods mainly label cells by modifying imaging probes with different biological ligands (including antibodies, oligonucleotide aptamers and peptides, etc.). However, these methods use multiple steps and are prone to errors, which have certain limitations. Click reactions have the advantages of high selectivity, mild conditions and fast kinetics. The effective combination of bioorthogonal click reactions and metabolic engineering can construct a highly specific and sensitive molecular labeling strategy. Bioorthogonal click chemistry has been widely used in the in vivo labeling and imaging of various biomolecules, such as antibodies, proteins, DNA, RNA and lipids.
[0023] The key invention of the present invention is that by designing a DNA sequence modified with a click reaction functional group, a fluorescent group, and labeled with a thiol or polyadenine, the DNA sequence is assembled with the probe core material by a salt aging method or a microwave method, and the spherical nucleic acid is modified with a bio-orthogonal click group, a dual-modal bio-orthogonal spherical nucleic acid probe is prepared for the first time, which is used as a new type of synchrotron radiation X-ray probe and applied to synchrotron radiation X-ray imaging of primary neurons. The core of high atomic number elements such as AuNPs has strong absorption of X-ray, which can generate sufficient signals to specifically image the biological molecules or structures of neuronal cells. The technical difficulties of the present invention mainly lie in the selection of the core material of the BO-SNAs probe, the design of the DNA sequence, and the selection of cell labeling sites.
[0024] Compared with the prior art, the present invention has the following positive effects:
[0025] Currently available exogenous X-ray-sensitive labeling probes for cell staining often result in cross-reactivity and poor specificity. Therefore, developing X-ray-sensitive probes with high labeling specificity is highly desirable for cell imaging. Applying bioorthogonal click reactions to cell labeling allows for the introduction of more imaging agents through covalent linkage, resulting in more efficient and reliable imaging applications, which is more effective and reliable than traditional active targeting strategies.
[0026] The traditional principle of applying SNA nanostructures to biological imaging is based on fluorescence resonance energy transfer (FRET). AuNPs can absorb fluorescence through FRET, thereby quenching the fluorophore. When the target molecule is nearby, the SNA will quickly capture it and cause a conformational change in the nucleic acid shell, pulling the fluorophore away from the core and restoring it with another energy source. However, in a biological environment, the presence of interfering molecules (such as nucleases) can destroy the probe structure and cause false positive signals. Synchrotron X-ray imaging can be performed by using the strong absorption of X-rays by the core of high atomic number elements of SNA. As long as the SNA structure is not completely destroyed, imaging can be performed, which can effectively overcome the problem of false positive signals.
[0027] In summary, the present invention provides an effective molecular probe for labeling primary neuronal cell structures and has successfully applied it to synchrotron radiation X-ray imaging. By introducing spherical nucleic acid probes into synchrotron radiation X-ray imaging, the application of SNAs in biological imaging has been further expanded, providing new ideas for the design of synchrotron radiation X-ray imaging probes. BRIEF DESCRIPTION OF THE DRAWINGS
[0028] Figure 1 Schematic diagram of the preparation of a bio-orthogonal click-reaction gold nanospherical nucleic acid (BO-Au-SNAs) probe provided by the present invention: a DNA sequence modified with a click-reaction functional group and a fluorescent group and labeled with thiol (SH) or polyadenine is designed, and an Au-SNAs probe (BO-Au-SNAs) with a click-reaction functional group is synthesized by a salt aging method;
[0029] Figure 2 Characterization diagram of Alkyne-Au-SNAs probe: TEM image of BO-Au-SNAs probe and rat adrenal pheochromocytoma cells (PC12) labeled with BO-Au-SNAs probe. The basic structural morphology of PC12 cells can be directly observed by fluorescence imaging and STXM imaging.
[0030] Figure 3 Schematic diagram of neuronal labeling using bio-orthogonal click reaction gold nanospheres (BO-Au-SNAs) probes: Primary neurons cultured for 7-14 days are co-incubated with bio-orthogonal click reaction modification reagents to express bio-orthogonal click reaction functional groups in the neurons, and then labeled with BO-Au-SNAs probes.
[0031] Figure 4 Confocal fluorescence imaging results and synchrotron radiation STXM imaging results of neuronal cell membrane glycans labeled with Alkyne-Au-SNAs probes. 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] Example 1 Synthesis of bio-orthogonal spherical nucleic acid (BO-SNAs) probes.
[0034] The inventors modified primary neuronal cell membranes with azide groups using bioorthogonal click reaction reagents. The bioorthogonal click reactions used copper-catalyzed click chemistry (CuAAC) and ring-strain-driven alkyne-azide coupling (SPAAC) were selected as bioorthogonal click reaction types. Bioorthogonal spherical nucleic acid (BO-SNAs) probes were constructed and applied to label neuronal cell membranes for imaging studies. The following examples illustrate the effectiveness of the present invention.
[0035] Synthesis of bio-orthogonal spherical nucleic acid (BO-SNAs) probes. First, a DNA sequence modified with a click reaction functional group, a fluorescent group, and labeled with thiol (SH) or polyadenine was designed, Alkyne-TTTTTTTT-iCy5-TTTTTTTT-SH, which was ordered from Sangon Biotech (Shanghai) Co., Ltd. It should be understood that this is only an example and not a limitation. The present invention is not limited to this DNA sequence. Thiol can also be replaced by polyA. The modified fluorescent group must ensure a certain distance from the organic core to prevent the organic core (AuNPS) from absorbing fluorescence through FRET, resulting in fluorescence quenching. 300μL 83nM 5nm AuNPs, 30μL 100μM DNA sequence, and 30μL 5×TBE solution were mixed, shaken at constant temperature for 4h, and then 10.5μL 3M NaCl was added every 30min to mix thoroughly and then allowed to stand. After adding 4 times, shake overnight. Centrifuge at 14000rpm for 30min, remove the supernatant, and wash 3 times with 0.5×TBE solution. Finally, resuspend in 0.5×TBE solution (see Figure 1 ).
[0036] TEM characterization of BO-Au-SNAs probe. 2 nM BO-Au-SNAs probe was dropped onto the surface of the carbon-supported copper mesh. After adsorption for 5 min, excess solution was removed from the edge of the copper mesh using filter paper. After the copper mesh surface was completely dry, TEM imaging was performed with an imaging voltage of 200 kV (e.g. Figure 2 (as shown in A in the figure).
[0037] Rat adrenal pheochromocytoma cells (PC12) were cultured. PC12 cells were purchased from the Cell Bank of the Committee for the Collection of Typical Cultures of the Chinese Academy of Sciences. Cells in the logarithmic growth phase were cultured at 1×10 5They were cultured in concocal dishes and waited for subsequent confocal fluorescence imaging. The silicon nitride window was placed in a 24-well cell culture plate and sterilized with UV light. 2×10 5 Cells were seeded in 24-well plates and prepared for subsequent STXM imaging. After the cells reached normal growth, the original culture medium was removed and fresh culture medium was added. Each dish or well was then incubated with 500 μM AHA modification reagent for 2 hours. This resulted in the modification of the PC12 cell membrane with azide groups.
[0038] PC12 cells were labeled with bioorthogonal gold nanoparticle spherical nucleic acid (Alkyne-Au-SNAs) probes using copper-catalyzed click chemistry (CuAAC). PC12 cells modified with azide functional groups were washed with 1% PBS for 2-5 minutes and fixed with 4% paraformaldehyde for 15 minutes. The fixative was removed and the cells were washed three times with 1% PBS for 5 minutes each. 20-50 nM Alkyne-Au-SNAs, BTTAA-CuSO4 complex (50 μM CuSO4, BTTAA / CuSO4 6:1, mol / mol), and 2.5 mM sodium ascorbate were mixed with oscillation in PBS and added dropwise to the cell surface. The cells were incubated for 1.5 hours, labeling the Alkyne-Au-SNAs probes on the cell membrane via a click reaction. The cells were then washed three times with 1% PBS for 5 minutes each to remove any unbound probes for imaging.
[0039] Confocal microscopy was used to observe the Alkyne-Au-SNAs probe-labeled PC12 cells (eg Figure 2 (shown in B).
[0040] At the BL08U 1-A soft X-ray absorption spectroscopy line station of Shanghai Synchrotron Radiation Facility in China, the incident light energy was selected as 520 eV, and scanning transmission X-ray imaging of PC12 cells labeled with Alkyne-Au-SNAs probes was performed (e.g. Figure 3 (as shown in C).
[0041] Conclusion: TEM results demonstrate the successful synthesis of bioorthogonal gold nanoparticle spherical nucleic acid (Alkyne-Au-SNAs) probes. The BO-Au-SNAs probes exhibited intact morphology and good monodispersity. PC12 cells labeled with the BO-Au-SNAs probes were directly visualized using both fluorescence imaging and STXM imaging.
[0042] Example 2 Bioorthogonal gold nanospherical nucleic acid (BO-Au-SNAs) probes were used to label neuronal cells and perform fluorescence and X-ray imaging.
[0043] Culture of primary neurons. The day before taking the brain, treat the culture dish or 24-well cell culture plate with silicon nitride window with polylysine to enhance the cell adhesion ability. Remove the polylysine before taking the brain and add fresh BME culture medium. The 18-day pregnant SD mouse was killed by dislocation and immersed in 75% alcohol for disinfection. Quickly remove the fetal brain, separate the fetal cerebral cortex tissue blocks, place them in HBSS culture medium and gently aspirate, and change the HBSS culture medium twice. Then resuspend the tissue in 4.5mL HBSS culture medium, add 0.5mL trypsin and enzymatically hydrolyze in a 37℃ water bath for 12-15min, then add 0.5mL DNase and enzymatically hydrolyze at room temperature for 2-5min. Clear the tissue once with HBSS culture medium. Add fresh BME culture medium to wash the tissue twice. Resuspend the cells, count them, and use 0.8×1 0 5 Culture in concocal dishes and wait for subsequent confocal fluorescence imaging or at 1.2×10 5 Cells were seeded in 24-well plates and prepared for STXM imaging. After 4 hours of culture, the BME medium was replaced with Neurobassal medium. The medium was changed every 3 days. Subsequent experiments were performed after the cells had been cultured for 7-14 days.
[0044] Azide modification of primary neuronal cell membranes. For azide modification of cell membrane glycans, cells were incubated in a medium containing 250 μM Ac4ManNAz for 12 hours. For modification of cell membrane proteins, cells were incubated in a medium containing 500 μM AHA for 1 hour. Thus, the cell membranes of primary neurons were modified with azide functional groups.
[0045] Based on the copper ion catalyzed click chemistry reaction (CuAAC), bio-orthogonal gold nanoparticle spherical nucleic acid (Alkyne-Au-SNAs) probes were used to label neuronal cells. Neuronal cells modified with azide functional groups were washed with 1% PBS for 2-5 minutes and fixed with 4% paraformaldehyde for 15 minutes. The fixative was removed and washed 3 times with 1% PBS for 5 minutes each time. 20-50nM Alkyne-Au-SNAs, BTTAA-CuSO4 complex (50μM CuSO4, BTTAA / CuSO4 6:1, mol / mol) and 2.5mM sodium ascorbate were oscillated in a PBS system and added dropwise to the cell surface for incubation for 1.5 hours to label the Alkyne-Au-SNAs probe on the cell membrane through a click reaction. It was then washed 3 times with 1% PBS for 5 minutes each time to wash away unbound probes for imaging (such as Figure 3 shown).
[0046] Neuronal cells were labeled with bioorthogonal gold nanoparticle spherical nucleic acid (DBCO-Au-SNAs) probes via ring strain-driven alkyne-azide coupling (SPAAC). Neuronal cells modified with azide functional groups were washed with sterile water for 2-5 minutes and incubated in a medium containing 5-20 nM DBCO-Au-SNAs for 20 minutes. The DBCO-Au-SNAs probes were then labeled on the cell membrane via a click reaction. Neuronal cells modified with azide functional groups were washed with 1% PBS for 2-5 minutes and fixed with 4% paraformaldehyde for 15 minutes. The fixative was removed and the cells were washed three times with 1% PBS for 5 minutes each. The fixative was then removed and the cells were washed with 1% PBS for 5 minutes each. The fixative was then removed and the cells were then left for imaging.
[0047] Dehydration of silicon nitride window samples of neuronal cells imaged with light sources. After labeling the cell samples on the silicon nitride window with BO-Au-SNAs probes, they were dehydrated in a series of 30%, 50%, 70%, 80%, 90%, and 100% alcohol gradients, with each gradient dehydration time of 5 minutes.
[0048] Confocal microscopy can be used to observe Alkyne-Au-SNAs probe-labeled neuronal cells, and two-dimensional and three-dimensional imaging (such as Figure 4 (as shown in A and B).
[0049] At the BL08U 1-A soft X-ray absorption spectroscopy line station of Shanghai Light Source in China, the incident light energy was selected as 520eV, and scanning transmission X-ray imaging of neuronal cells labeled with Alkyne-Au-SNAs probes was performed (such as Figure 4 At the BL07 W soft X-ray imaging beamline of the Hefei Light Source in China, the incident light energy was selected as 520 eV, and transmission X-ray imaging of neurons labeled with Alkyne-Au-SNAs probes was performed (as shown in Figure 5). Figure 4 (as shown in D in the figure).
[0050] Conclusion: After labeling, the basic structure of neuronal fibers (such as Figure 4 shown).
[0051] The above description is merely a preferred embodiment of the present invention and is not intended to limit the scope of the present invention. Various modifications are possible. Any simple, equivalent changes and modifications made in accordance with the claims and description of the present invention fall within the scope of protection of the patent claims. Anything not fully described in the present invention is within the conventional art.
Claims
1. A method for imaging primary neuronal cells using a dual-modal bio-orthogonal spherical nucleic acid probe, characterized in that: The following steps are involved: 1) Design a DNA sequence modified with a click-reactive functional group and a fluorescent group, and labeled with a thiol or polyadenine; 2) synthesizing the DNA sequence designed in step 1) and a probe core material into a dual-modal bio-orthogonal spherical nucleic acid probe using a salt aging method or a microwave method, wherein the probe core material is an inorganic material core, and the inorganic material core comprises: AuNPs, AgNPs, FeNPs, or a composite thereof; 3) Extract and culture primary neuronal cells; 4) co-incubating the primary neuronal cells with a click reaction functional group modification reagent to allow the primary neuronal cells to metabolically integrate the click reaction functional group; 5) using the dual-modal bio-orthogonal spherical nucleic acid probe prepared in step 2) to label the primary neuronal cells modified with click reaction functional groups in step 4) with BO-SNAs probes based on a click reaction; 6) Perform fluorescence imaging and X-ray imaging on the primary neuronal cells labeled with the BO-SNAs probe in step 5).
2. The method according to claim 1, characterized in that In step 1), the click reaction functional groups include: Alkyne, DBCO; the non-selective fluorescent groups include: Cy3, Cy5.
3. The method according to claim 1, characterized in that In step 1), the modified fluorescent group must be kept at a certain distance from the probe core material to prevent the probe core material from absorbing fluorescence through FRET, resulting in fluorescence quenching.
4. The method according to claim 1, wherein In step 2), a salt aging method is used to prepare a dual-modal bio-orthogonal spherical nucleic acid probe, including: mixing the probe core material, DNA sequence, and 5×TBE solution, oscillating at a constant temperature for 3 to 4 hours, adding NaCl every 30 minutes to mix, and then standing. After adding NaCl four times, oscillating overnight, centrifuging at 14,000 rpm for 30 minutes, removing the supernatant, washing three times with 0.5×TBE solution, and finally resuspending with 0.5×TBE solution.
5. The method according to claim 1, wherein In step 3), the primary neuronal cells extracted are cerebral cortical neuronal cells of 18-day-old SD rat fetuses.
6. The method according to claim 1, characterized in that In step 4), the click reaction functional group modification reagents include: Ac4ManNAz, AHA cell membrane modification reagent.
7. The method according to claim 1, characterized in that In step 5), primary neuronal cells were labeled with BO-SNAs probes based on a copper ion-catalyzed click chemistry reaction and a ring strain-driven alkyne-azide coupling reaction.
8. The method according to claim 1, characterized in that In step 5), The method for labeling primary neuronal cells with BO-SNAs probes based on copper ion-catalyzed click chemistry reaction includes: After removing the click reaction functional group modification reagent, the cells were treated with 4% paraformaldehyde for 15 minutes to fix the cells. The BO-Au-SNAs probe, BTTAA-CuSO4 complex, and sodium ascorbate were mixed by oscillation in a PBS system and added to the cell surface for incubation for 1.5 hours to label the BO-SNAs probe on the cell surface through the click reaction. Methods for labeling primary neuronal cells with BO-SNAs probes based on ring strain-driven alkyne-azide coupling reactions include: After removing the click reaction functional group modification reagent, the BO-SNAs probe was directly added to the culture medium and incubated with the cells for 20 minutes to label the BO-SNAs probe on the cell surface. After removing the BO-SNAs probe, the cells were treated with 4% paraformaldehyde for 15 minutes to fix the cells.
9. The method according to claim 1, characterized in that In step 6), imaging methods for primary neuronal cells include confocal imaging and STXM imaging.