A composite nanomaterial of graphdiyne oxide-coated gold nanospheres, its preparation method and application

Abstract

The present invention discloses a composite nanomaterial of gold nanospheres wrapped with graphdiyne oxide and its preparation method and application. The composite nanomaterial of gold nanospheres wrapped with graphdiyne oxide has a core-shell structure, wherein the core is a gold nanosphere with a particle size of 90-110 nm, and the shell is a thin layer of graphdiyne oxide sheet with a thickness of 1.90-1.95 nm and a particle size of 250-300 nm. The presence of gold nanoparticles in the composite nanomaterial of gold nanospheres wrapped with graphdiyne oxide obtained by the present invention significantly enhances the surface-enhanced Raman signal of the outer layer of graphdiyne oxide of the material, and the presence of the graphdiyne oxide shell layer can also effectively isolate the adsorption of substances in the external environment on the gold core. More importantly, since the Raman scattering peak of the diyne bond carried by graphdiyne oxide itself is located in the Raman silent region of cells. Therefore, the composite nanomaterial is expected to be used as a SERS probe to achieve ultra-bright, fast and accurate cell Raman imaging.

Description

Technical Field

[0001] The present invention belongs to the field of material chemistry, and particularly relates to a composite nanomaterial of graphdiyne oxide-coated gold nanospheres, a preparation method thereof, and an application thereof. Background Art

[0002] Surface-enhanced Raman scattering (SERS) is a powerful spectroscopic technique that can provide non-destructive and ultrasensitive characterization at the single-molecule level, comparable to single-molecule fluorescence spectroscopy. At the same time, due to its good sensitivity, high spectral resolution for multiplex detection, excellent resistance to photobleaching and self-filtering, and non-invasive sampling, etc., it has received extensive attention in the field of bioimaging. However, the direct detection of SERS often requires the signal of Raman beacon molecules for judgment. Common Raman beacon molecules include rhodamine 6G molecules, carbon nanomaterials, etc.

[0003] Among them, graphene is a natural thin two-dimensional carbon nanomaterial. Due to its excellent physical and chemical properties and its strong Raman scattering signal, it has attracted extensive attention from researchers. However, when using graphene for cell detection, the Raman signal peaks of graphene overlap with those of cell components, making it difficult to distinguish. Therefore, we considered whether graphdiyne could be used as a Raman beacon molecule.

[0004] Graphdiyne is a new type of carbon allotrope. Because it is very similar to graphene in structure and is a two-dimensional planar material. However, the Raman signal of graphdiyne itself is very weak, and it is difficult to obtain a relatively obvious Raman signal peak of the alkyne peak of graphdiyne in biological Raman detection. Summary of the Invention

[0005] Object of the Invention: Aiming at the problems existing in the prior art, the present invention provides a composite nanomaterial of graphdiyne oxide-coated gold nanospheres. In this nanomaterial, graphdiyne is coated on the surface of gold nanoparticles. Graphdiyne itself has diyne bonds, so that when using graphdiyne for cell Raman detection, there is no need to add additional beacon molecules. At the same time, the electromagnetic field enhancement of gold can effectively improve the Raman signal of graphdiyne, effectively solving the problem that obvious characteristic peaks cannot be obtained for graphdiyne in biological Raman detection.

[0006] The present invention also provides a preparation method and an application of the composite nanomaterial of graphdiyne oxide-coated gold nanospheres.

[0007] Technical Solution: To achieve the above object, the composite nanomaterial of graphdiyne oxide-coated gold nanospheres described in the present invention has a core-shell structure. Its core is a gold nanosphere with a particle size of 90 - 110 nm, and its shell is a thin layer of graphdiyne oxide sheet with a thickness of 1.90 - 1.95 nm and a particle size of 250 - 300 nm.

[0008] The present invention uses thin-layer graphdiyne sheets for Raman imaging of cells. Considering the problem of the material entering the cells, the size of the gold core cannot be too large or too small. If it is too small, the electromagnetic field enhancement effect is weak; if it is too large, it cannot enter the cells. Therefore, considering comprehensively, a gold core size of about 90-110 nm of gold nanospheres is more appropriate. And the thickness of graphdiyne is that of a single-layer carbon material. The main consideration is the size of the thin-layer graphdiyne sheets. If the graphdiyne sheets are too large, the gold core and graphdiyne are in a non-wrapped state, but the gold spheres are loaded on the graphdiyne; if the graphdiyne sheets are too small, they cannot be completely wrapped. Therefore, according to the size of the gold core, the thin-layer graphdiyne sheets have a thickness of 1.90-1.95 nm and a particle size of 250-300 nm.

[0009] Preferably, the graphdiyne oxide-coated gold nanosphere composite nanomaterial is a core-shell structure, the core of which is a gold nanosphere with a particle size of about 100 nm, and the shell of which is a thin graphdiyne oxide sheet with a thickness of about 1.92 nm. The graphdiyne oxide-coated gold nanosphere composite nanomaterial of the present invention includes the following steps:

[0010] (1) Preparation of thin-layer graphdiyne oxide sheets: Take GDY powder, concentrated HNO3, and concentrated H2SO4 and place them in an ice-water bath. Add KMnO4 powder under stirring. Heat the obtained mixture in an oil bath to obtain a suspension. After cooling to room temperature, adjust the pH and then centrifuge to obtain a powder. Wash and disperse it to obtain a thin-layer graphdiyne oxide sheet solution;

[0011] (2) Preparation of graphdiyne oxide-coated gold nanosphere composite nanomaterials: Take the gold nanosphere solution and the thin-layer graphdiyne oxide sheet solution and mix them. After stirring, centrifuge to take the precipitate and finally redisperse it to obtain the graphdiyne oxide-coated gold nanosphere composite nanomaterials.

[0012] Among them, in step (1), take 10-11 mg of GDY powder, 1-2 mL of concentrated HNO3, and 3-6 mL of concentrated H2SO4 and place them in a flask under an ice-water bath. Add 10-11 mg of KMnO4 powder under vigorous stirring. Heat the obtained mixture in an oil bath at 75-85 °C for 24-30 hours to obtain a brown suspension. After cooling to room temperature, neutralize it with NaOH to pH 8.0-8.5 and then centrifuge to obtain a black powder. Wash it with deionized water and disperse it in 9-10 mL of water to obtain a brown thin-layer graphdiyne oxide sheet solution.

[0013] Among them, the particle size of the thin-layer graphdiyne oxide sheets prepared in step (1) is 250-300 nm.

[0014] Among them, in step (2), 1-2 mL of gold nanosphere solution and 6-12 mL of thin-layer graphdiyne sheet solution are mixed. After stirring for 1-2 h, centrifugation is carried out, and finally it is redispersed in 9-10 mL of water to obtain the graphdiyne-coated gold nanosphere composite nanomaterial.

[0015] Among them, the preparation of gold nanospheres in step (2) includes: (a) Preparation of gold seeds: Aqueous chloroauric acid solution is added to CTAC solution, and then freshly prepared sodium borohydride solution is added under vigorous stirring to obtain gold seeds; (b) Preparation of gold spheres: Take gold seed solution and ascorbic acid solution and add them to CTAC solution, then add chloroauric acid solution under vigorous stirring, and let it stand to obtain gold spheres; (c) Seed growth: Take gold sphere solution and ascorbic acid solution and add them to CTAC solution, then add chloroauric acid solution under vigorous stirring, and let it stand to obtain irregularly shaped gold nanoparticles; (d) Oxidative etching: Under rapid stirring, sodium hypochlorite solution is added to the solution of the irregularly shaped gold nanoparticles, and then chloroauric acid solution is added during stirring, and let it stand to obtain smooth gold nanospheres. In the existing preparation of gold nanospheres, in order to obtain smooth gold spheres, there is no oxidation step, and tools such as syringe pumps are used to control the amount and speed of adding aqueous chloroauric acid solution, which is troublesome and time-consuming; the present invention does not require the use of a syringe pump and directly uses sodium hypochlorite solution for oxidation, with simple steps and time saving.

[0016] Among them, the preparation of the graphdiyne-coated gold nanosphere composite nanomaterial in step (2): Take 1-2 mL of gold nanosphere solution with a particle size of 90-100 nm and a dispersion concentration of 450-550 μg / mL; and 6-12 mL of graphdiyne fragment solution with a particle size of 250-300 nm and a dispersion concentration of 90-100 μg / mL, mix them, stir for 1-2 h, and after centrifugation, redisperse in 9-10 mL of water to obtain the graphdiyne-coated gold nanosphere composite nanomaterial.

[0017] Preferably, in step (2), the preparation of the graphdiyne-coated gold nanosphere composite nanomaterial: Take 1-2 mL of gold nanosphere solution with a particle size of 100 nm and a dispersion concentration of 500 μg / mL; and 6-12 mL of graphdiyne fragment solution with a particle size of 250-300 nm and a dispersion concentration of 100 μg / mL, mix them, stir for 1-2 h, and after centrifugation, redisperse in 9-10 mL of water to obtain the graphdiyne-coated gold nanosphere composite nanomaterial.

[0018] Application of the graphdiyne-coated gold nanosphere composite nanomaterial described in the present invention in cellular Raman imaging.

[0019] Among them, the application process is as follows: Centrifuge and disperse the composite nanomaterial of graphitic carbon nitride wrapped with gold nanospheres in cell culture medium. After incubating well-grown cells with the above nanomaterial, digest them, and then transfer the cells to a cell culture dish containing a silicon wafer so that the cells can grow on the silicon wafer. After the cells grow on the silicon wafer, suck out the culture medium in the culture dish, add a fixing solution to fix the cell morphology, and then wash the silicon wafer and dry it naturally, and then the Raman imaging detection can be carried out.

[0020] Generally, the size and shape of gold nanoparticles will affect the strength of SERS signals. Considering that the spherical shape is the most stable and easiest to be coated, gold nanospheres are selected for preparation. Currently, the size of most of the used gold nanoparticles is 20-30 nm. In this invention, gold nanospheres of about 100 nm are used, and it is found that their SERS enhancement effect is the best. Therefore, gold nanospheres of about 100 nm in size will be prepared. However, when the gold nanospheres grow to more than 100 nm, it is always difficult to avoid sharp corners or edges. Therefore, the method of seed-mediated growth combined with sodium hypochlorite oxidation etching is used to obtain gold spheres with a smooth surface.

[0021] Preferably, the preparation of the gold nanospheres of the present invention includes the following steps:

[0022] Preparation of gold seeds: Add 40-60 μL of 0.05 mol / L chloroauric acid aqueous solution to 4-6 mL of 0.1 mol / L CTAC solution, and then add 100-300 μL of freshly prepared 0.02 mol / L sodium borohydride solution under vigorous stirring to obtain gold seeds;

[0023] Preparation of 10 nm gold spheres: Take 800-1000 μL of gold seed solution and 30-50 μL of 0.1 mol / L ascorbic acid solution and add them to 9-10 mL of 0.025 mol / L CTAC solution, and then add 40-60 μL of 0.05 mol / L chloroauric acid solution under vigorous stirring, and let it stand for 30-60 min to obtain 10 nm gold spheres;

[0024] Seed growth: Take 2-3 μL of 10 nm gold sphere solution and 30-50 μL of 0.1 mol / L ascorbic acid solution and add them to 9-10 mL of 0.025 mol / L CTAC solution, and then add 40-60 μL of 0.05 mol / L chloroauric acid solution under vigorous stirring, and let it stand for 1-2 h to obtain irregularly shaped gold nanoparticles;

[0025] Oxidative etching: Under rapid stirring, 9 - 10 μL of sodium hypochlorite solution (containing 1 - 1.5 wt% available chlorine) was added to 9 - 10 ml of the above - mentioned solution; after 5 - 6 min, 6 - 7 μL of 0.05 mol / L chloroauric acid solution was added during stirring. After standing for 2 - 4 h, smooth gold nanospheres with a particle size of about 100 nm were obtained. (Hereinafter referred to as "Au NSs")

[0026] In order for the graphitic carbon nitride nanosheets to successfully coat the gold nanoparticles, the relative size ratio of the diameter of the gold nanoparticles to the lateral size of the graphitic carbon nitride nanosheets should neither be too small nor too large. Therefore, according to the size of the currently synthesized gold nanospheres, it can be estimated that the size of the graphitic carbon nitride nanosheets should be about 250 - 300 nm. However, the size of the prepared graphitic carbon nitride ranges from several hundred nanometers to several micrometers. Therefore, the GDY nanosheets were fragmented by an oxidation method.

[0027] Preferably, the preparation of the thin - layer graphitic carbon nitride nanosheets comprises the following steps:

[0028] (1) Fragmentation of graphitic carbon nitride (hereinafter referred to as "GDY") - Preparation of graphitic carbon nitride oxide:

[0029] Take 10 - 11 mg of GDY powder, 1 - 2 mL of concentrated HNO3, and 3 - 6 mL of concentrated H2SO4 and place them in a flask in an ice - water bath. Add 10 - 11 mg of KMnO4 powder under vigorous stirring. Heat the resulting mixture in an oil bath at 80 °C for 24 - 30 hours to obtain a brown suspension. After cooling to room temperature, neutralize it with NaOH to pH 8.0 - 8.5, then centrifuge at 10000 rpm for 10 min to obtain a black powder. After washing with deionized water, disperse it in 9 - 10 mL of water to obtain a brown graphitic carbon nitride oxide solution (hereinafter referred to as "GDYO").

[0030] Preferably, the preparation of the graphitic carbon nitride oxide - coated gold nanosphere composite nanomaterial comprises the following steps:

[0031] Take 1 - 2 mL of the prepared gold nanospheres and mix them with 6 - 12 mL of the prepared graphitic carbon nitride oxide solution. After stirring for 1 - 2 h, centrifuge (8000 rpm for 10 min) twice, and finally redisperse it in 9 - 10 mL of water to obtain the graphitic carbon nitride oxide - coated gold nanosphere composite nanomaterial (hereinafter referred to as "Au - GDYO").

[0032] Preferably, the present method also provides the application of the above Au-GDYO in cell Raman imaging. It includes the following steps: centrifugally disperse the synthesized Au-GDYO in the cell culture medium DMEM; after incubating well-grown cells with the above-treated nanomaterials at 37 °C and 5% CO2 for 24 hours, digest the cells with trypsin. Subsequently, transfer the cells to a cell culture dish with a silicon wafer so that the cells can grow on the silicon wafer. After the cells grow on the silicon wafer, suck out the culture medium in the dish, add an appropriate amount of Karnovsky fixative (pH 7.3) to fix the cell morphology. Subsequently, wash the silicon wafer 2 times with phosphate-buffered saline (PBS) solution and then dry it naturally, and then the Raman imaging detection can be carried out.

[0033] It can be known from the experiment that all Raman signals are distributed in the whole cytoplasm, and no signal appears in the nucleus, indicating that Au-GDYO is distributed in the cytoplasm. Using the core-shell structure to strongly and rapidly enhance the Raman signal is a key advantage of Au-GDYO as a Raman probe for cell imaging.

[0034] The present invention first prepares gold nanospheres with a particle size of about 100 nm and thin-layer graphdiyne sheets with a particle size of about 250 - 300 nm respectively; mix the two in a suitable volume ratio under stirring at room temperature to obtain the graphdiyne-coated gold nanosphere composite nanomaterial. The experimental results show that the obtained graphdiyne-coated gold nanosphere composite nanomaterial has a unique core-shell structure, its core is a gold nanosphere with a particle size of about 100 nm, and its shell is a graphdiyne thin sheet with a thickness of about 1.92 nm. The presence of gold nanoparticles significantly enhances the surface-enhanced Raman signal of the outer graphdiyne of the material; the presence of the graphdiyne shell layer can also effectively isolate the adsorption of substances in the external environment on the gold core. More importantly, since the Raman scattering peak of the diacetylene bond carried by graphdiyne itself is located in the Raman silent region of cells (1800 - 2800 cm -1 ). Therefore, this composite nanomaterial is expected to be used as a SERS probe to achieve ultra-bright, fast and accurate cell Raman imaging. The preparation method of the whole graphdiyne-coated gold nanosphere composite nanomaterial is convenient to operate and has simple steps.

[0035] The present invention designs a novel shell-core structured graphdiyne-wrapped gold nanocomposite for Raman imaging detection of cells. Considering that graphene itself does not contain alkynyl bonds, when using graphene for cell Raman detection, it is necessary to additionally connect a beacon molecule containing alkynyl bonds or other signal peaks that can be clearly distinguished from cell components, making the experiment time-consuming and complex. Graphdiyne, as an allotrope of graphene, is very similar to graphene in structure. The difference is that graphdiyne itself has diacetylene bonds, so that when using graphdiyne for cell Raman detection, there is no need to additionally add a beacon molecule, and the experimental steps are simpler.

[0036] The present invention prepares a novel carbon nanomaterial - graphdiyne-wrapped gold nanoparticles. Graphdiyne itself has diacetylene bonds, and the Raman peak of the acetylene peak is located in the Raman silent region of cells. Thus, when performing Raman detection, the signal of the acetylene peak will not overlap with the Raman peaks of intracellular components and is difficult to distinguish. In the currently published relevant literature, only graphene-wrapped gold nanoparticles are used for cell imaging applications. However, since graphene does not contain alkynyl bonds, a signal molecule containing alkynyl bonds will be additionally connected to the surface of graphene to effectively simplify such experimental steps.

[0037] The present invention innovatively uses the diacetylene bonds inherent in graphdiyne for the first time. The Raman peak of the acetylene peak is located in the Raman silent region of cells. Thus, when performing Raman detection, the signal of the acetylene peak will not overlap with the Raman peaks of intracellular components and is difficult to distinguish. However, further research finds that its characteristic peaks are not obvious. The present invention further utilizes gold nanoparticles of a specific size, and the electromagnetic field enhancement of gold can effectively improve the Raman signal of graphdiyne, effectively solving the problem that graphdiyne cannot obtain obvious characteristic peaks in biological Raman detection. In addition, compared with additionally connecting a substance containing alkynyl bonds to graphene, the present invention only directly uses graphdiyne, which is more convenient in experimental steps; at the same time, when an alkynyl bond is externally connected to graphene, this alkynyl bond connection may not be stable and will fall off during the reaction process, and is not as stable as the alkynyl bond inherent in graphdiyne.

[0038] Beneficial effects: Compared with the prior art, the present invention has the following advantages:

[0039] The present invention for the first time uses a new carbon nanomaterial, graphdiyne, to prepare a gold nanocomposite wrapped with oxidized graphdiyne, which effectively overcomes the problem that when graphene is used for cell Raman detection, an additional beacon molecule containing an alkyne bond or other signal peaks that can be clearly distinguished from cell components must be connected, making the experiment time-consuming and complex. Moreover, its Raman signal is stronger than that of pure graphdiyne. At the same time, the presence of the oxidized graphdiyne shell can effectively isolate the adsorption of substances in the external environment on the gold core, effectively overcoming the problem that the direct adsorption of gold nanoparticles on the surface of the graphdiyne thin layer may still cause the gold core to be affected by the external environment, and it can be effectively applied to Raman imaging detection. Since graphdiyne itself has a diacetylene bond, there is no need to additionally connect a beacon molecule containing an alkyne bond on the surface of the outer shell material. Therefore, the preparation method is simple and time-saving. Description of the Drawings

[0040] Figure 1 is the transmission electron microscope image of the Au NSs prepared in Example 1;

[0041] Figure 2 is the transmission electron microscope image of the GDY prepared in Example 1;

[0042] Figure 3 is the transmission electron microscope image of the GDYO prepared in Example 1;

[0043] Figure 4 is the transmission electron microscope image of the Au-GDYO prepared in Example 1; where a) is the high-resolution transmission electron microscope image of the particles, and b) is the ultra-high-resolution transmission electron microscope image of the particles;

[0044] Figure 5 is the ultraviolet-visible absorption spectrum (a) and Zeta potential map (b) of the Au-GDYO prepared in Example 1;

[0045] Figure 6 is the cytotoxicity detection map of the Au-GDYO prepared in Example 1;

[0046] Figure 7 is the Raman spectrum of the Au-GDYO prepared in Example 1;

[0047] Figure 8 is the Raman imaging map of Au-GDYO in MCF-7 cells in Example 2. Detailed Embodiments

[0048] According to the following embodiments, the present invention can be better understood. However, those skilled in the art can easily understand that the content described in the embodiments is only used to illustrate the present invention and should not and will not limit the present invention detailed in the claims.

[0049] For the materials, reagents, etc. used in the following examples, unless otherwise specified, they can all be obtained commercially. For the experimental methods without specific conditions noted in the examples, they are generally carried out under conventional conditions or according to the conditions recommended by the manufacturer.

[0050] Example 1

[0051] Preparation of Au - GDYO

[0052] 1. Preparation of 100 nm gold nanospheres

[0053] a) Preparation of gold seeds: Add 50 μL of 0.05 mol / L chloroauric acid aqueous solution to 5 mL of 0.1 mol / L cetyltrimethylammonium chloride (CTAC) solution, and then add the newly prepared 200 μL of 0.02 mol / L sodium borohydride solution to the above solution under vigorous stirring. After 2 h, dilute the mixed solution 10 - fold with 0.1 mol / L CTAC solution to obtain solution A;

[0054] b) Preparation of 10 nm gold spheres: Take 900 μL of solution A and 40 μL of 0.1 mol / L ascorbic acid solution and add them to 10 mL of 0.025 mol / L CTAC solution, and then inject 50 μL of 0.05 mol / L chloroauric acid solution into it under vigorous stirring. Then keep the mixed solution undisturbed at room temperature for 30 min to obtain solution B;

[0055] c) Seed growth: Take 2.5 μL of solution B and 40 μL of 0.1 mol / L ascorbic acid solution and add them to 10 mL of 0.025 mol / L CTAC solution. Then inject 50 μL of 0.05 mol / L chloroauric acid solution into it under vigorous stirring. Then let the mixed solution stand at room temperature for 1 h to obtain solution C;

[0056] d) Oxidative etching: Under rapid stirring, add 10 μl of 10% sodium hypochlorite solution by mass to 10 mL of solution C; after five minutes, add 6.25 μL of 0.05 mol / L chloroauric acid solution during stirring. Let the mixed solution stand at room temperature until the oxidation is completed (the color of the solution will change during the oxidation process: the solution color will change from blue - purple to pink - gold slowly, and when there is no other obvious color change, the oxidation is completed). After the oxidation is completed, centrifuge and wash the above - mentioned mixed solution twice with deionized water (8000 rpm), and finally disperse all the obtained solid precipitates in 10 mL of water to obtain 100 nm gold nanospheres. As Figure 1 shown, the surface of the gold nanospheres is smooth and their average particle size is 100 nm.

[0057] 2. Preparation of graphdiyne (GDY)

[0058] 50 mg of hexakis(trimethylsilylethynyl)benzene (HEB-TMS) and 15 mL of tetrahydrofuran were successively added to a round-bottom flask and dissolved. The flask was placed in an ice-water bath at 0 °C and stirred for 1 h under Ar gas protection. While maintaining the temperature at 0 °C and under Ar gas protection, 1 mL of tetrabutylammonium fluoride (TBAF, concentration 1 mol / L in THF) was added, and the reaction was stirred for 15 min. At this time, the mixture should be light pink or purple depending on the purity of the reactants. Ethyl acetate was added to the flask to dilute the solution to 30 mL. The diluted mixture was added to a separatory funnel and washed with 30 mL of saturated brine and separated. The upper organic phase was taken. The washing with saturated brine was repeated 3 times, and then 20 mg of anhydrous magnesium sulfate was added to remove the excess water and filtered. The filtered mixture was transferred to a rotary evaporator and rotary evaporated at 40 °C to remove the solvent to obtain yellow solid hexaethynylbenzene (HEB). The whole process needs to be carried out under light protection.

[0059] The pre-prepared copper foil with dimensions of 2 cm × 2 cm × 10, which had been pickled in 1 mol / L hydrochloric acid solution for 24 h, was ultrasonically cleaned in water, ethanol, and acetone for 15 min respectively. Subsequently, the copper foil, 100 mL of acetone, 5 mL of pyridine, and 1 mL of tetramethylethylenediamine were added to a round-bottom flask wrapped with tin foil and mixed and stirred under Ar gas protection and heated to 50 °C and maintained for 1 h.

[0060] All the prepared HEB was dissolved in 50 mL of acetone and transferred to a constant-pressure separatory funnel. The solution in the separatory funnel was slowly added to the flask prepared in the previous step within 4 h. After the addition, stirring was maintained and the reaction was carried out at 50 °C for 36 h to obtain a black mixture. After the reaction was completed, the black mixture was rotary evaporated at 40 °C to remove the excess acetone, and then centrifuged at a speed of 10000 rpm for 12 min, and the black precipitate was taken. The black solid was successively centrifugally washed with acetone, N,N-dimethylformamide (DMF), ethanol, and water at least twice to wash away the excess organic matter. Subsequently, the washed black solid was added to a round-bottom flask and added to 1 mol / L hydrochloric acid solution, heated and pickled under reflux at 90 °C for 12 h and cooled to room temperature. The pickled product was centrifugally washed with secondary distilled water until the pH was 7. The washed solid was dried in a vacuum drying oven at 70 °C for 7 h to obtain graphdiyne powder. As Figure 2 shown, the prepared GDY is a large single-layer structure.

[0061] 3. Preparation of graphdiyne oxide (GDYO)

[0062] Take the 10 mg of the prepared GDY powder, 1 mL of 65% concentrated HNO3, and 3 mL of 98% concentrated H2SO4 and place them in a flask under an ice-water bath. Add 10 mg of KMnO4 powder while stirring vigorously. Heat the resulting mixture in an oil bath at 80 °C for 24 hours to obtain a brown suspension. After cooling to room temperature, neutralize it with NaOH to pH 8.0, then centrifuge at 10000 rpm for 10 min to obtain a black powder. Wash it with deionized water and disperse it in 10 mL of water to obtain a brown GDYO solution with a concentration of 100 μg / mL. As Figure 3 shown, the average particle size of the prepared GDYO is about 250 nm - 300 nm.

[0063] 4. Preparation of Au-GDYO

[0064] Take 1 mL of the prepared 500 μg / mL gold nanoparticles and 6 mL of the prepared 100 μg / mL GDYO solution. Stir at room temperature for 1 h, then centrifuge (8000 rpm for 10 min) twice. Finally, take the precipitate and redisperse it in 10 mL of water to prepare a 400 μg / mL Au-GDYO solution for subsequent experiments.

[0065] Figure 4 The morphology and composition of Au-GDYO were characterized by transmission electron microscopy (TEM) and high-resolution transmission electron microscopy (HR-TEM), clearly showing that the Au core was wrapped by oxidized graphdiyne flakes. Among them, the average diameter of Au-GDYO is 100 nm; the thickness of the oxidized graphdiyne shell layer is about 1.92 nm.

[0066] Figure 5 The Au NSs solution, GDYO solution, and Au-GDYO were characterized by ultraviolet-visible absorption spectroscopy and Zeta potentiometer, respectively. Figure 5 a is the ultraviolet-visible absorption spectra of the three. The figure clearly shows that Au-GDYO simultaneously has the characteristic peak of gold nanoparticles at 540 nm and the characteristic peak of oxidized graphdiyne at 220 nm; Figure 5 b is the Zeta potential diagram. It can be seen from the figure that the prepared gold spheres carry a positive potential of 14 mV, oxidized graphdiyne carries a negative potential of 22 mV, and Au-GDYO carries a negative potential of 14 mV. The above data further illustrate the successful synthesis of Au-GDYO.

[0067] Figure 6The cytotoxicity of Au-GDYO was studied by the MTT method. MCF-7 human breast cancer cells in the logarithmic phase were collected, the cell suspension concentration was adjusted, and 100 μL of the cell suspension was added to each well of a 96-well plate. The plate was seeded so that the density of the cells to be tested was approximately 5000 / well; incubated at 5% CO2 and 37 °C until the cells adhered to the wall. Subsequently, 10 μL of Au-GDYO solutions at concentrations of 12.5, 25, 50, 100, 150, and 200 μg / mL were added to each well (3 replicates were set for each concentration gradient); then incubated at 5% CO2 and 37 °C for 24 hours, 10 μL of MTT solution was added to each well, gently mixed, and the culture was terminated after continuing to culture for 4 hours. The culture medium in the wells was carefully aspirated; 100 μL of dimethyl sulfoxide solution was added to each well, and the plate was placed on a shaker and shaken at low speed for 10 minutes to fully dissolve the crystals; finally, the absorbance values of each well were measured at 570 nm using an enzyme-linked immunosorbent assay detector. After organizing and comparing the absorbance value data of each group, we found that Au-GDYO has good biocompatibility. The inhibitory effect of Au-GDYO on the proliferation of MCF-7 human breast cancer cells can be ignored, and subsequent Raman imaging experiments can be carried out.

[0068] Figure 7 These are the Raman spectra of GDYO and Au-GDYO. 0.8 mL of the prepared 100 μg / mL GDYO solution and 0.2 mL of the 400 μg / mL Au-GDYO solution were respectively dropped onto a clean silicon wafer of 5 mm × 5 mm. After drying, Raman detection was performed using a confocal Raman spectrometer (LabRam HR Evolution). The wavelength of the Raman spectrometer was selected as 633 nm, and the laser power on the sample plane was 10%. As can be seen from the figure, GDYO has three significant Raman signal peaks, which are the graphite carbon (G) peak at around 1580 cm -1 -1, the disordered (D) peak at around 1370 cm -1 -1, and the diyne peak at around 2150 cm -1 -1. The Raman signal of Au-GDYO has been greatly enhanced compared to the Raman signal of pure GDYO, indicating that Au-GDYO is a good Raman label for cell imaging and an excellent SERS substrate for bioimaging and biosensing. Since the bands where the D peak and G peak are located overlap with the Raman peaks of intracellular components, while the diyne peak is located in the Raman silent region of cells, it can be used as a characteristic peak for cell and tissue imaging.

[0069] Example 2

[0070] Application of Au-GDYO in Raman imaging of cells

[0071] The Au-GDYO dispersion prepared in Example 1 was centrifuged (8000 rpm for 10 min), and the precipitate was finally dispersed in 5 mL of cell culture medium DMEM such that the concentration of Au-GDYO in DMEM was 400 μg / mL.

[0072] When the cells grew to 50% of the cell flask, the cells were washed with PBS to remove dead cells or cells that did not adhere firmly. Then, 1 mL of the Au-GDYO solution in DMEM and 6 mL of the DMEM solution were added to the cell flask and incubated at 37 °C and 5% CO2 for 24 hours until the cells grew to 90% of the cell flask. The cells were then washed twice with PBS solution to remove the nanomaterials that did not enter the cells and dead cells. Then, the cells were digested with trypsin containing EDTA, the trypsin was removed, and 2 mL of DMEM cell culture medium was added to pipette the cells to disperse them into single cells. Subsequently, 0.5 mL of the above cell suspension was taken into a clean cell culture dish, and a sterilized silicon wafer was placed in the culture dish in advance so that the cells could grow on the silicon wafer. Then, 5 mL of the DMEM solution was added to the cell culture dish, and the culture dish was incubated at 37 °C and 5% CO2 for 12 h.

[0073] After the cells adhered and grew completely, the culture medium in the culture dish was aspirated, and an appropriate amount of Karnovsky fixative (pH 7.3) was added until the fixative completely covered the cells to well fix the cell morphology. The silicon wafer with the fixed cells was washed twice with PBS solution and then air-dried, and then it could be taken for Raman imaging detection.

[0074] A confocal Raman spectrometer (LabRam HR Evolution) was used to perform Raman imaging detection on the cells. The wavelength of the Raman spectrometer was selected as 633 nm, and the laser power was 10% on the sample plane. As Figure 8 is the Raman imaging diagram of the diacetylene peak of MCF-7 breast cancer cells. It can be seen from the figure that all Raman signals are distributed in the whole cytoplasm, and no signals appear in the nucleus and outside the cell, indicating that Au-GDYO is distributed in the cytoplasm.

[0075] In summary, the strong and rapid enhancement of Raman signals by Au-GDYO using the core-shell structure is a key advantage as a Raman probe for cell imaging.

Claims

1. A preparation method of a composite nanomaterial of graphitic carbon nitride-wrapped gold nanospheres, characterized in that, It includes the following steps: (1) Preparation of thin-layer graphdiyne oxide sheets: Take graphdiyne GDY powder, concentrated HNO3, and concentrated H2SO4 and place them in an ice-water bath. Add KMnO4 powder while stirring. Heat the resulting mixture in an oil bath to obtain a suspension. After cooling to room temperature, adjust the pH and then centrifuge to obtain a powder. Wash and disperse it to obtain a thin-layer graphdiyne oxide sheet solution; (2) Preparation of graphdiyne oxide-coated gold nanosphere composite nanomaterials: Take a gold nanosphere solution and a thin-layer graphdiyne oxide sheet solution, mix them, stir, centrifuge to collect the precipitate, and finally redisperse it to obtain graphdiyne oxide-coated gold nanosphere composite nanomaterials; In step (1), take 10 - 11 mg of GDY powder, 1 - 2 mL of concentrated HNO3, and 3 - 6 mL of concentrated H2SO4 and place them in a flask in an ice-water bath. Add 10 - 11 mg of KMnO4 powder while stirring. Heat the resulting mixture in an oil bath at 75 - 85 °C for 24 - 30 hours to obtain a brown suspension. After cooling to room temperature, neutralize it with NaOH to a pH of 8.0 - 8.5, then centrifuge to obtain a black powder. Wash it with deionized water and disperse it in 9 - 10 mL of water to obtain a brown thin-layer graphdiyne oxide sheet solution; In step (2), take 1 - 2 mL of a gold nanosphere solution and 6 - 12 mL of a thin-layer graphdiyne oxide sheet solution, mix them, stir for 1 - 2 h, centrifuge, and finally redisperse it in 9 - 10 mL of water to obtain graphdiyne oxide-coated gold nanosphere composite nanomaterials.

2. The preparation method according to claim 1, wherein The particle size of the thin-layer graphdiyne oxide sheets prepared in step (1) is 250 - 300 nm.

3. The preparation method according to claim 1, characterized in that, Preparation of graphdiyne oxide-coated gold nanosphere composite nanomaterials in step (2): Take 1 - 2 mL of a gold nanosphere solution with a particle size of 90 - 110 nm and a dispersion concentration of 450 - 550 μg / mL; and 6 - 12 mL of a graphdiyne oxide fragment solution with a particle size of 250 - 300 nm and a dispersion concentration of 90 - 100 μg / mL, mix them, stir for 1 - 2 h, centrifuge, and then redisperse it in 9 - 10 mL of water to obtain graphdiyne oxide-coated gold nanosphere composite nanomaterials.

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