Method for imaging primary neuronal cells by using bimodal biological orthogonal spherical nucleic acid probe

Abstract

The invention discloses a method for imaging primary neuronal cells by using a bimodal biological orthogonal spherical nucleic acid probe. The method comprises the following steps: 1) designing a DNA sequence modified with a click reaction functional group and a fluorophore and marked with mercaptan or polyadenine; (2) preparing a BO-SNAs probe from the DNA sequence and a probe core material; 3) extracting and culturing primary neuronal cells; 4) co-incubating the click reaction functional group modification reagent and the cells; (5) carrying out BO-SNAs probe labeling on the cells by using a BO-SNAs probe on the basis of a click reaction; and 6) imaging the primary neuronal cells. According to the method, the BO-SNAs probe labeled neuronal cells based on the biological orthogonal click reaction are subjected to synchrotron radiation X-ray imaging for the first time, a new thought is provided for synchrotron radiation X-ray imaging probe design, and a new tool is provided for synchrotron radiation X-ray brain imaging.

Description

Technical Field

[0001] The present invention relates to the field of biochemical technology, and more specifically to a method for imaging primary neuronal cells using a dual-modal bio-orthogonal spherical nucleic acid probe. Background Art

[0002] The greater penetration depth of X-rays makes X-ray imaging technology have the potential for in situ imaging of cells. Synchrotron radiation light has the characteristics of high brightness and high collimation. Due to its inherent nanoscale resolution and excellent penetration ability, synchrotron-based X-ray microscopy (XRM) can provide the sensitivity and spatial resolution required for ion distribution at the subcellular level, and can be used for in situ analysis of cells under near-physiological conditions. The contrast of TXM imaging comes from the fact that when X-rays pass through the sample, the biochemical composition of different locations in the sample is different, resulting in different absorption of X-rays. Biological samples composed of light elements absorb X-rays weakly, and the quality of imaging them is often poor. However, high atomic number elements absorb X-rays strongly, and their use as stains or contrast agents to mark samples can improve contrast and generate sufficient signals for specific imaging of cell biological molecules or structures. The current traditional methods mainly modify imaging probes through different biological ligands (including antibodies, oligonucleotide aptamers and peptides, etc.), but these methods use multiple steps and are prone to errors, and have certain limitations.

[0003] Bioorthogonal click reaction refers to a rapid, specific and high-yield covalent reaction between two molecules under physiological conditions. Click reaction has the advantages of high selectivity, mild conditions and fast kinetics. The effective combination of bioorthogonal click reaction and metabolic engineering has constructed a highly specific and sensitive molecular labeling strategy, which has been widely used in many biomedical fields, especially in the selective labeling of biomolecules in vivo, targeted imaging and imaging drug delivery.

[0004] As a new nucleic acid delivery system, spherical nucleic acids (SNAs) have been used in immunomodulation, gene regulation, drug delivery, biosensing and bioimaging. In addition, high atomic number elements such as Au have a certain absorption in the X-ray band. After Au labeling of neuronal cells, the morphological structure of neurons can be observed by STXM. Summary of the invention

[0005] The purpose of the present invention is to provide a method for primary neuronal cell imaging 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) using a click reaction functional group modification reagent to co-incubate the primary neuronal cells so that the primary neuronal cells 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] According to the method provided by the present invention, its working principle is: design a DNA sequence with a click reaction functional group, and prepare it into a BO-SNAs probe by coupling it with a nano-gold ball core, so that it can specifically label cell biological molecules through a click reaction. Since the DNA sequence carries a fluorescent group, fluorescent imaging of neuronal cells can be performed. At the same time, because its metal material core has a high specific absorption peak under X-rays, it can realize the characteristic recognition and positioning of different biological molecules in neuronal cells.

[0009] The click reaction functional groups that can be modified in the DNA sequence in step 1) include Alkyne, DBCO, Azide and the like.

[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 the properties of synthesizing metal clusters.

[0013] The BO-SNAs probe core in step 2) has multiple selectivity and may include inorganic material cores such as AuNPs, AgNPs, FeNPs and their composite materials, as well as organic material cores such as proteins, such as bovine catalase, etc.

[0014] The method for preparing the inorganic material core BO-SNAs probe in step 2) includes salt aging method, microwave assisted heating method, etc. The salt aging method is to prepare spherical nucleic acids by gradually adding salt to a mixture of thiol (SH) or polyadenine (A) labeled DNA and inorganic material core to reduce charge repulsion. The microwave assisted heating method is a further improvement on the salt aging method for preparing spherical nucleic acids. The spherical nucleic acids are directly prepared by microwave assisted heating and drying the preparation system, which greatly reduces the preparation time of the salt aging method and improves the yield of spherical nucleic acids.

[0015] According to a preferred embodiment of the present invention, the process of extracting primary neuronal cells in step 3) is as follows: a pregnant SD mouse at day 18 of gestation is subjected to dislocation and the fetal brain is quickly removed, and the fetal cerebral cortical neuronal cells are isolated and cultured, and subsequent operations are performed after culturing on a confocal dish or a silicon nitride window for 7-14 days.

[0016] In step 4), the click reaction functional group modification reagents 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 neuron cells were co-incubated with the click reaction functional group modification reagent. The original culture medium was first aspirated, the cell culture system was washed with sterile water, and new culture medium was added for co-incubation to allow the cell metabolism to integrate the click reaction functional group. For cell membrane polysaccharide modification, the primary neurons were co-incubated with 250 μM Ac4ManNAz for 12 h; for cell membrane protein modification, the primary neurons were co-incubated with 500 μM AHA for 1 h.

[0018] In the step 5), the BO-SNAs probe labeling of primary neuronal cells is mainly based on the copper ion catalyzed click chemistry reaction (CuAAC) and the ring strain driven alkyne-azide coupling reaction (SPAAC), so as to achieve the bioorthogonal spherical nucleic acid (BO-SNAs) probe labeling of the cell membrane.

[0019] It should be understood that in the presence of a reducing agent and / or a stabilizing ligand, a cycloaddition reaction between azide and alkyne occurs 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 labeling based on the CuAAC click reaction in step 5) specifically includes: after removing the click reaction functional group modification reagent, the cells are treated with 4% paraformaldehyde for 15 minutes to fix the cells; the 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 by a click reaction.

[0020] It should also be understood that SPAAC (Strain-promoted azide-alkyne cycloaddition) is a bioorthogonal reaction and a variant reaction of the azide-alkyne Huisgen cycloaddition, and the reaction is carried out in the absence of a live cytotoxic copper catalyst. The molecules of the two coupling parties are usually azide and cyclooctyne, and this reaction is also called 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, the BO-SNAs probe is labeled on the cell surface, and after removing the BO-SNAs probe, the cells are treated with 4% paraformaldehyde for 15 minutes to fix the cells.

[0021] The primary neuron cell imaging in step 6) includes confocal imaging and STXM imaging. Confocal imaging is to perform fluorescence imaging on BO-SNAs probe-labeled cells by setting the emission spectrum and absorption spectrum of the corresponding fluorescent group. STXM imaging is to perform synchrotron radiation X-ray two-dimensional imaging of neurons by using the 08U beamline of the Shanghai Light Source and the 07W beamline of the Hefei Light Source.

[0022] X-rays have the characteristics of short wavelength and strong penetrating power. X-ray-based imaging technology can realize non-destructive three-dimensional high-resolution imaging research of cells. The current traditional cell labeling method mainly labels cells by modifying imaging probes with different biological ligands (including antibodies, oligonucleotide aptamers and peptides, etc.), but these methods use multiple steps and are prone to errors, and 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 a variety of 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 a bimodal bio-orthogonal spherical nucleic acid probe is prepared for the first time by modifying the spherical nucleic acid with a bio-orthogonal click group, so that it can be 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 positive and progressive effects of the present invention are:

[0025] Currently, available exogenous X-ray sensitive labeled probes for staining cells often result in cross-reactions leading to poor specificity. Therefore, the development of X-ray sensitive probes with high labeling specificity is highly desirable for cell imaging. Applying bioorthogonal click reactions to cell labeling technology can achieve more introduction of imaging agents through covalent bonding, thereby achieving more effective imaging applications, which is more efficient and reliable than traditional active targeting strategies.

[0026] The traditional principle of applying SNA nanostructures to bioimaging is based on fluorescence resonance energy transfer (FRET). AuNP 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 radiation X-ray imaging can be performed through the strong absorption of X-rays by the core of SNA high atomic number elements. As long as the SNA structure is not completely destroyed, imaging can be performed, which can well 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 A schematic diagram of the preparation of a bio-orthogonal click reaction nano-gold spherical 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 through fluorescence imaging and STXM imaging.

[0030] Figure 3 Schematic diagram of labeling neurons with bio-orthogonal click reaction nano-gold spherical nucleic acid (BO-Au-SNAs) probes: primary neuronal cells cultured for 7-14 days are co-incubated with bio-orthogonal click reaction modification reagents to make the neuronal cells express bio-orthogonal click reaction functional groups, and then the neuronal cells are labeled with BO-Au-SNAs probes;

[0031] Figure 4 Confocal fluorescence imaging results and synchrotron radiation STXM imaging results for Alkyne-Au-SNAs probe-labeled neuronal cell membrane glycans. DETAILED DESCRIPTION

[0032] The present invention will be further described below in conjunction with specific examples. It should be understood that the following examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods for which specific conditions are not specified in the following examples are carried out according to conventional methods and conditions, or are selected according to the product specifications. The 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 selected a cell membrane bio-orthogonal click reaction modification reagent to modify the primary neuron cell membrane with an azido group, and the bio-orthogonal click reaction type selected copper ion catalyzed click chemistry reaction (CuAAC) and ring strain driven alkyne-azide coupling reaction (SPAAC), constructed bio-orthogonal spherical nucleic acid (BO-SNAs) probes and applied them to label neuron cell membranes for imaging research. The following examples specifically illustrate the implementation effect 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 be kept at a certain distance from the organic core to prevent the organic core (AuNPS) from absorbing fluorescence through FRET, resulting in fluorescence quenching. 300 μL 83nM 5nm AuNPs, 30 μL 100 μM DNA sequence, and 30 μL 5×TBE solution were mixed, and 10.5 μL 3M NaCl was added every 30 minutes after constant temperature oscillation for 4 hours, and then mixed and allowed to stand. After adding 4 times, oscillation was allowed to proceed overnight. After centrifugation at 14000rpm for 30 minutes, the supernatant was removed and washed 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 support film copper mesh. After adsorption for 5 min, excess solution was removed from the edge of the copper mesh using filter paper. After the surface of the copper mesh was completely dry, TEM imaging was performed. The imaging voltage was 200 KV (e.g. Figure 2 (shown in A).

[0037] Rat adrenal pheochromocytoma cells (PC12) were cultured. PC12 cells were purchased from the Cell Bank of the Chinese Academy of Sciences Typical Culture Collection Committee. Cells in the logarithmic growth phase were taken and 1×10 5The cells were cultured in concocal dishes and waited for confocal fluorescence imaging. The silicon nitride windows were placed in 24-well cell culture plates and sterilized with ultraviolet light. 5 The cells were seeded in a 24-well plate and waited for subsequent STXM imaging. After the cells were cultured to a normal growth state, the original culture medium was removed, and new culture medium was added. Each dish or each well was incubated with 500 μM AHA modification reagent and the cells for 2 hours. At this point, the cell membrane of PC12 cells was modified with azide groups.

[0038] Based on the copper-catalyzed click chemistry reaction (CuAAC), bio-orthogonal nanogold spherical nucleic acid (Alkyne-Au-SNAs) probes were labeled with PC12 cells. 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 washed with 1% PBS for 3 times, 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 mixed by oscillation in a PBS system, added dropwise on the cell surface and incubated for 1.5 hours to label the Alkyne-Au-SNAs probe on the cell membrane through a click reaction. Then wash with 1% PBS for 3 times, 5 minutes each time, and wash the unbound probes for imaging.

[0039] Confocal microscopy was used to observe the Alkyne-Au-SNAs probe labeled PC12 cells (such as Figure 2 (shown in B).

[0040] 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 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 show that the bio-orthogonal gold nanoparticle spherical nucleic acid (Alkyne-Au-SNAs) probes were successfully synthesized. The BO-Au-SNAs probes were complete in morphology and had good monodispersity. The basic structural morphology of PC12 cells was directly observed by fluorescence imaging and STXM imaging using BO-Au-SNAs probes to label PC12 cells.

[0042] Example 2 Bioorthogonal gold nanoparticle spherical 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 the brain was taken out, the culture dish or 24-well cell culture plate with silicon nitride window was treated with poly-lysine to enhance the cell adhesion ability. Before the brain was taken out, the poly-lysine was removed and fresh BME culture medium was added. The 18-day gestational SD pregnant mice were killed by dislocation and the pregnant mice were immersed in 75% alcohol for disinfection. The fetal brain was quickly taken out, and the fetal cerebral cortex tissue blocks were separated and placed in HBSS culture medium for gentle aspiration, and the HBSS culture medium was changed twice. After that, the tissue was resuspended in 4.5mL HBSS culture medium, 0.5mL trypsin was added and enzymatically hydrolyzed in a 37℃ water bath for 12-15min, and then 0.5mL DNase was added for enzymatic hydrolysis at room temperature for 2-5min. The tissue was cleared once with HBSS culture medium. Fresh BME culture medium was added to wash the tissue twice. The cells were resuspended and counted, and 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 waited for subsequent STXM imaging. After 4 hours of cell culture, the BME medium was replaced with Neurobassal medium. The medium was replaced every 3 days. Subsequent experiments could be performed after the cells were cultured for 7-14 days.

[0044] Azide modification of primary neuronal cell membranes. For azide modification of cell membrane polysaccharides, cells were incubated in a medium containing 250 μM Ac4ManNAz for 12 h. For modification of cell membrane proteins, cells were incubated in a medium containing 500 μM AHA for 1 h. Thus, the cell membranes of primary neurons were modified with azide functional groups.

[0045] Neuronal cells were labeled with bioorthogonal nanogold spherical nucleic acid (Alkyne-Au-SNAs) probes based on copper ion-catalyzed click chemistry (CuAAC). 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 and mixed in a PBS system, and then dropped on the cell surface and incubated for 1.5 hours to label the Alkyne-Au-SNAs probes on the cell membrane through a click reaction. Wash with 1% PBS 3 times for 5 minutes each time to wash out the unbound probes for imaging (such as Figure 3 shown).

[0046] Based on the ring strain driven alkyne-azide coupling reaction (SPAAC), bioorthogonal nanogold spherical nucleic acid (DBCO-Au-SNAs) probes were used to label neuronal cells. The neuronal cells modified with azide functional groups were washed with sterile water for 2-5 minutes, and the cells were incubated in a 5-20nM DBCO-Au-SNAs culture medium system for 20 minutes to label the DBCO-Au-SNAs probes on the cell membrane through a click reaction. The 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 with 1% PBS 3 times for 5 minutes each time, and the fixative was washed and left for imaging.

[0047] Dehydration of the silicon nitride window samples of neuronal cells imaged by light sources. After labeling the cell samples on the silicon nitride window with BO-Au-SNAs probes, they were dehydrated with 30%, 50%, 70%, 80%, 90% and 100% series of gradient alcohols, with a dehydration time of 5 min for each gradient.

[0048] Using confocal microscopy to observe Alkyne-Au-SNAs probe-labeled neurons, 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 520 eV, and scanning transmission X-ray imaging of Alkyne-Au-SNAs probe-labeled neuronal cells 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 to be 520 eV, and transmission X-ray imaging of neurons labeled with Alkyne-Au-SNAs probes was performed (as shown in Figure 2). Figure 4 (shown in D).

[0050] Conclusion: After labeling, the basic structure of neuronal fibers (such as Figure 4 shown).

[0051] The above is only a preferred embodiment of the present invention, and is not intended to limit the scope of the present invention. The above embodiments of the present invention can also be modified in various ways. All simple, equivalent changes and modifications made according to the claims and the contents of the specification of the present invention fall within the scope of protection of the claims of the present invention. Anything not described in detail in the present invention is within the conventional technology.

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) Designing a DNA sequence modified with a click reaction functional group and a fluorescent group and labeled with a thiol or polyadenine; 2) using a salt aging method or a microwave method to synthesize the DNA sequence designed in step 1) and the probe core material into a dual-modal bio-orthogonal spherical nucleic acid probe; 3) Extract and culture primary neuronal cells; 4) co-incubating the click reaction functional group modification reagent with the primary neuron cells to allow the primary neuron 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) Imaging the primary neuronal cells labeled with BO-SNAs probes 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 fluorescent groups are non-selective and 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, characterized in that: In step 2), the probe core material includes: an inorganic material core or an organic material core, the inorganic material core includes: AuNPs, AgNPs, FeNPs or a composite material thereof, and the organic material core includes bovine catalase.

5. The method according to claim 1, characterized in that 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, adding 4 times and then oscillating overnight, centrifuging at 14000 rpm for 30 minutes, removing the supernatant, adding 0.5×TBE solution to wash 3 times, and finally resuspending with 0.5×TBE solution.

6. The method according to claim 1, characterized in that In step 3), the primary neuronal cells extracted are cerebral cortical neuronal cells of 18-day-old SD rat fetuses.

7. 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 reagents.

8. The method according to claim 1, characterized in that In step 5), primary neuronal cells are labeled with BO-SNAs probes based on a copper ion-catalyzed click chemistry reaction and a ring strain-driven alkyne-azide coupling reaction.

9. 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 min 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 h to label the BO-SNAs probe on the cell surface through the click reaction. The method for labeling primary neuronal cells with BO-SNAs probes based on ring strain-driven alkyne-azide coupling reaction includes: After removing the click reaction functional group modification reagent, BO-SNAs probes were directly added to the culture medium and incubated with the cells for 20 minutes to label the BO-SNAs probes on the cell surface. After removing the BO-SNAs probes, the cells were treated with 4% paraformaldehyde for 15 minutes to fix the cells.

10. The method according to claim 1, characterized in that In step 6), the imaging method for primary neuronal cells includes: confocal imaging or STXM imaging.

Images