Preparation method of fluorescent nano material Si-CDs (at) SiO2 (at) Protein A and method for specifically marking cells by using fluorescent nano material Si-CDs (at) SiO2 (at) Protein A
By preparing Si-CDs@SiO2@Protein A, the problems of carbon dots' optical properties being easily affected by the environment and having poor modifiability were solved, and stable cell fluorescence labeling and imaging effects were achieved.
Patent Information
- Application Number
- CN202510826331.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-06-19
- Publication Date
- 2025-10-10
AI Technical Summary
The optical and physicochemical properties of carbon dots (CDs) are easily affected by the external environment and have poor modifiability, which limits their application in the biomedical field.
Fluorescent nanomaterial Si-CDs@SiO2@Protein A was prepared. Si-CDs were synthesized by a one-pot hydrothermal method, coated with SiO2 by a modified Stöber method, and the surface was carboxylated and coupled with Protein A to form a stable fluorescent nanomaterial.
The optical and physicochemical properties of Si-CDs@SiO2@Protein A are stable, and it can specifically label cells and perform fluorescence imaging, with good resistance to photofading and easy modification.
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Abstract
Description
Technical Field
[0001] The present invention relates to the field of nanomaterials, and in particular to a fluorescent nanomaterial of silica-coated carbon dots coupled with Protein A protein, a preparation method of the fluorescent nanomaterial and an application thereof in cell-specific fluorescent labeling. Background Art
[0002] Carbon dots (CDs) are fluorescent carbon nanomaterials with sizes less than 10 nm. Their preparation methods are diverse and simple, the raw materials required are widely available, and they exhibit stable optical properties and strong resistance to photobleaching. Compared to semiconductor quantum dots, CDs do not contain heavy metals, resulting in lower biotoxicity. Their good biocompatibility has led to their widespread biomedical applications. CDs contain a rich surface area of functional groups that determine their physical, chemical, and fluorescent properties, including quantum yield and emission wavelength, aggregation-induced emission / quenching, fluorescence lifetime, and specific material responses.
[0003] CDs have good water solubility, and interactions with small molecules such as metal ions and drugs can quench or enhance their fluorescence. The mechanism by which small molecules quench CD fluorescence is currently unknown. Surface states, quantum effects, and the carbon core are the primary factors influencing the optical properties of CDs, with the main quenching mechanisms including aggregation quenching, internal filtering effects, and photoinduced electron transfer. Furthermore, some CDs can be used to monitor environmental and intracellular pH. It is currently believed that the fluorescence response of CDs to pH changes may be related to the protonation or deprotonation of their surface groups. These characteristics make CDs inherently useful as fluorescence sensors for detecting trace amounts of small molecules and pH changes. However, this also means that the optical properties of some CDs are susceptible to interference from external factors, making direct surface modification of CDs difficult. With the increasing breadth and depth of research and application of CDs, researchers are placing increasing demands on the stability and modifiability of CDs in their applications. This has led to the continued development of CD-based composite materials to address inherent limitations of CDs, such as fluorescence quenching due to environmental influences and poor modifiability. We have developed a method for preparing a fluorescent nanomaterial of silica-coated carbon dots coupled with Protein A protein and using it to specifically label cells. This fluorescent nanomaterial is easy to prepare and has good resistance to photofading. Summary of the Invention
[0004] To address the concerns that the optical and physicochemical properties of carbon dots (CDs) are susceptible to environmental interference and have poor modifiability, the present invention provides a method for preparing and applying a fluorescent nanomaterial, Si-CDs@SiO2@Protein A. This fluorescent nanomaterial, Si-CDs@SiO2@Protein A, exhibits stable optical and physicochemical properties. The surface-coupled protein, Protein A, synergizes with antibodies to enable specific fluorescent labeling of cells. The preparation method established in this invention yields spherical Si-CDs@SiO2@Protein A with a particle size of 10-50 nm, suitable for cell labeling.
[0005] In order to achieve the above object, the present invention is implemented through the following technical solutions: An object of the present invention is to provide a fluorescent nanomaterial Si-CDs@SiO2@Protein A for specific labeling and imaging of cells for non-therapeutic purposes.
[0006] Another object of the present invention is to provide a method for preparing the fluorescent nanomaterial Si-CDs@SiO2@Protein A for specific cell labeling and imaging, the specific steps being as follows: (1) Preparation of silicon-doped carbon dots (Si-CDs) with good fluorescence performance; (2) coating the Si-CDs obtained in step (1) with SiO2 to prepare fluorescent nanomaterial Si-CDs@SiO2; (3) Carboxylating the surface of Si-CDs@SiO2 obtained in step (2) to prepare Si-CDs@SiO2-COOH; (4) Activating the carboxyl group of Si-CDs@SiO2-COOH obtained in step (3) and coupling it with protein A to obtain fluorescent nanomaterial Si-CDs@SiO2@Protein A that can bind to antibodies; Furthermore, the precursor for preparing Si-CDs in step (1) is a silane coupling agent. N-β -(Aminoethyl)- gamma -Aminopropylmethyldimethoxysilane (KH602) and m-phenylenediamine (MPD).
[0007] Furthermore, the preparation method of Si-CDs in step (1) is a one-pot hydrothermal method.
[0008] The method for preparing Si-CDs in step (1) is a one-pot hydrothermal method: the precursors KH602 and MPD are mixed with pure water and placed in a high-pressure reactor. After the hydrothermal reaction, the supernatant of the reaction solution is collected by centrifugation and dialyzed in pure water to obtain a Si-CDs aqueous solution.
[0009] The mass ratio of KH602 to MPD is 20-40.
[0010] The hydrothermal reaction temperature is 150-180 °C, and the hydrothermal reaction time is 5-12 h.
[0011] Furthermore, the method of coating Si-CDs with SiO2 in step (2) is a modified Stöber method.
[0012] In step (2), SiO2 coating of Si-CDs is a modified Stöber method: Si-CDs are mixed with tetraethyl orthosilicate (TEOS) and cetyltrimethylammonium bromide (CTAB) and stirred at 70-80 °C for 1 h under the action of NaOH. TEOS is hydrolyzed to form a SiO2 matrix and the Si-CDs are coated to obtain spherical fluorescent nanomaterials Si-CDs@SiO2 with a particle size of about 10-50 nm.
[0013] In the present invention, cetyltrimethylammonium bromide (CTAB) is a surfactant, which is a reagent added to make the product particles uniform in size and does not participate in the main reaction.
[0014] The method for carboxylating Si-CDs@SiO2 in step (3) is as follows: weigh the surface modifier 3-aminopropyltriethoxysilane (APTES), then weigh the same amount of succinic anhydride, and evenly disperse the two in N,N -Dimethylformamide (DMF) solvent, add Si-CDs@SiO2 and stir for 3-4 h to complete the carboxyl modification of the Si-CDs@SiO2 surface to obtain Si-CDs@SiO2-COOH.
[0015] APTES and succinic anhydride are used in equimolar amounts. The mass ratio of APTES to Si-CDs@SiO2 is 1-2:1.
[0016] The method for coupling Protein A to Si-CDs@SiO2 in step (4) is as follows: Si-CDs@SiO2-COOH (1-3 mg / mL) is mixed with 1-2% 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and 1-2% N-The carboxyl groups were activated by reacting with hydroxysuccinimide (NHS) for 30 min. The carboxyl-activated Si-CDs@SiO2-COOH was obtained by centrifugation, washed with pure water, and resuspended in 0.5 mL of HEPES buffer. 0.5-1.0 mL of Protein A (50-150 μg / mL) was added and mixed, and the mixture was gently shaken at room temperature for 3-4 h. The Si-CDs@SiO2@Protein A was collected by centrifugation, washed with PBS buffer (0.01% NaN3), and stored until use.
[0017] Furthermore, the protein Protein A in step (4) is a protein present in Staphylococcus aureus ( Staphylococcus aureus ) cell wall protein with a molecular weight of 42 kDa. It specifically binds to mammalian immunoglobulins (Ig), typically at the Fc region of the immunoglobulin. Protein A was conjugated to carboxyl-activated Si-CDs@SiO2-COOH to create Si-CDs@SiO2@Protein A, which can bind to the IgG antibody used in routine biomedical experiments. The fluorescent nanomaterial Si-CDs@SiO2@Protein A prepared by the method described in the present invention has an excitation wavelength of 355-385 nm and an emission wavelength of 450-470 nm.
[0018] The fluorescent nanomaterial Si-CDs@SiO2@Protein A is used in the preparation of a reagent for cell-specific fluorescent imaging for non-therapeutic purposes.
[0019] The Si-CDs@SiO2@Protein A was synergistically used with keratin 8 antibody to specifically fluorescently label immortalized mouse thymic epithelial cells (iTEC) for non-therapeutic purposes, and then fluorescence imaging was performed using a laser confocal microscope.
[0020] Furthermore, the method for specific labeling and fluorescence imaging of iTEC cells is as follows: the cells are fixed with 4% paraformaldehyde (w / v) for 15 min, and then 0.2% Triton X-100 (v / v) is added to permeabilize the cells for 10 min; the cells are blocked in PBS containing 5% BSA (w / v) for 30 min and then incubated with keratin 8 antibody (0.1 ~ 10 μg / mL) at 4°C overnight to ensure that the antibody is fully bound to keratin 8 in the iTEC cells; after washing away the antibody that is not bound to the cells, the Si-CDs@SiO2@Protein A in step (4) is added and incubated with the cells at room temperature for 2 h to bind to the antibody located in the cells; after washing away the Si-CDs@SiO2@Protein A that is not bound to the antibody, the cells are fluorescently imaged under a laser confocal microscope.
[0021] Compared with the prior art, the present invention has the following beneficial effects: (1) The Si-CDs in the present invention have good compatibility with the silicon matrix and do not undergo obvious fluorescence quenching after being coated with SiO2.
[0022] (2) The present invention uses as little SiO2 precursor TEOS as possible to coat Si-CDs, and finally obtains spherical Si-CDs@SiO2 with a particle size of about 10-50 nm. This small-sized nanomaterial is suitable for labeling cells.
[0023] (3) Si-CDs@SiO2 has stable physical and chemical properties and good resistance to photobleaching.
[0024] (4) The surface of Si-CDs@SiO2 is inert SiO2, so it is easy to modify its surface and couple it with other molecules to achieve functionalization. Therefore, Si-CDs@SiO2 can be used as a universal fluorescent nanomaterial and can be modified and coupled according to different needs.
[0025] (5) After carboxylation and coupling with protein A, Si-CDs@SiO2 retains high-intensity fluorescence and can bind to the Fc segment of IgG antibody through Protein A. Therefore, the fluorescent nanomaterial Si-CDs@SiO2@Protein A can cooperate with antibodies to specifically label cells. BRIEF DESCRIPTION OF THE DRAWINGS
[0026] Figure 1 The morphology and particle size of Si-CDs prepared in Example 1 under high-resolution transmission electron microscopy (HRTEM) (the inset shows the lattice structure of Si-CDs).
[0027] Figure 22 is the fluorescence emission spectrum of Si-CDs prepared in Example 1 (the inset shows that Si-CDs emit blue fluorescence under 365 nm ultraviolet excitation).
[0028] Figure 3 is the UV-visible absorption spectrum of Si-CDs prepared in Example 1.
[0029] Figure 4 is the Fourier transform infrared (FTIR) spectrum of the Si-CDs prepared in Example 1. Figure 5 The morphology and particle size of Si-CDs@SiO2 prepared in Example 2 under transmission electron microscopy (TEM).
[0030] Figure 6 2 is the fluorescence emission spectrum of Si-CDs@SiO2 prepared in Example 2 (the inset shows that Si-CDs@SiO2 emits blue fluorescence under 365 nm ultraviolet excitation).
[0031] Figure 7 This is the Fourier transform infrared (FTIR) spectrum of Si-CDs@SiO2 prepared in Example 2.
[0032] Figure 8 This is the Fourier transform infrared spectrum (FTIR) of Si-CDs@SiO2-COOH prepared in Example 3.
[0033] Figure 9 This is the fluorescence emission spectrum of Si-CDs@SiO2@Protein A prepared in Example 4 (the inset shows that Si-CDs@SiO2@Protein A emits blue fluorescence under 356 nm ultraviolet excitation).
[0034] Figure 10 is the protein concentration coupled to Si-CDs@SiO2@Protein A prepared in Example 4 (Bradford method).
[0035] Figure 11 This is the fluorescence imaging of iTEC cells specifically labeled by Si-CDs@SiO2@Protein A under a laser confocal microscope in Example 5.
[0036] Figure 12 The relative fluorescence intensity of Si-CDs@SiO2 prepared in Example 2 is compared with that of the commercial small molecule fluorescent dye Alexa fluor 594 when stored under non-strict light protection conditions.
[0037] Figure 13 This is the process of preparing Si-CDs@SiO2@Protein A of the present invention. DETAILED DESCRIPTION
[0038] The following examples illustrate certain embodiments of the present invention and should not be construed as limiting the scope of the present invention. The present disclosure may be modified in terms of materials, methods, and reaction conditions, all of which are intended to fall within the spirit and scope of the present invention. Unless otherwise specified, the reagents used in the examples of the present invention are all commercially available. Experimental methods in the following examples, where specific conditions are not specified, generally follow conventional conditions or the conditions recommended by the manufacturer.
[0039] Because the optical and physicochemical properties of carbon dots (CDs) are easily interfered with by external environmental factors, their application in many fields is limited. Therefore, the present invention provides a fluorescent nanomaterial Si-CDs@SiO2@ProteinA, which has a core composed of silicon-doped carbon dots (Si-CDs) and a shell composed of silicon dioxide (SiO2), and is coupled to the surface of protein A through carboxylation. Compared with the original Si-CDs, the optical and physicochemical properties of Si-CDs are protected by the inert SiO2 shell and become easily modifiable. Further carboxylation of the SiO2 shell surface and coupling with protein A yields the functionalized fluorescent nanomaterial Si-CDs@SiO2@ProteinA, which enables specific fluorescent labeling and imaging of iTEC cells.
[0040] The precursor for preparing Si-CDs in the present invention is a silane coupling agent N-β -(Aminoethyl)- gamma Silicon-doped carbon dots (Si-CDs) were synthesized using a simple one-pot hydrothermal method using aminopropylmethyldimethoxysilane (KH602) and m-phenylenediamine (MPD), with KH602 acting as a silicon dopant and MPD acting as a carbon source. The Si-CDs were then coated with SiO2 by hydrolyzing tetraethyl orthosilicate (TOES) under alkaline conditions using a modified Stöber method.
[0041] Si-CDs have good compatibility with silicon substrates. In our preliminary experiments, CDs prepared using silicon-free reagents as precursors exhibited significant fluorescence quenching after SiO2 coating, suggesting that silicon-free CDs may have poor silicon substrate compatibility, resulting in fluorescence quenching by SiO2.
[0042] Furthermore, the preparation of Si-CDs@SiO2@Protein A in the present invention requires drastic changes in ion concentration and pH value, but the resulting Si-CDs@SiO2@Protein A still produces stable and high-intensity fluorescence. This is due to the protection of the physical, chemical, and optical properties of Si-CDs by the inert shell SiO2, so environmental changes do not cause significant negative interference with its fluorescence properties.
[0043] Given the feasibility of SiO2-coated Si-CDs, the particle size and morphology of Si-CDs@SiO2 are key parameters. Regular morphology and sufficiently small particle size favor Si-CDs@SiO2 applications in biomedical applications, such as fluorescent cell labeling. The SiO2-coated Si-CDs method of this invention yields spherical Si-CDs@SiO2 with a particle size of approximately 10-50 nm, suitable for cell labeling, with reasonable yields.
[0044] Since the shell of Si-CDs@SiO2 is inert SiO2, the surface of Si-CDs@SiO2 is easily modified with other groups to functionalize it. In the present invention, the surface of Si-CDs@SiO2 is carboxylated and coupled with protein A to specifically label cells in conjunction with antibodies, thereby realizing fluorescence imaging of iTEC cells under a microscope.
[0045] Example 1: Synthesis of Si-CDs The present invention utilizes a one-pot hydrothermal method to synthesize Si-CDs. A silane coupling agent is added to the precursor to dope the CDs with silicon, thereby obtaining Si-CDs with good silicon matrix compatibility. In this example, the Si-CDs were synthesized using the following steps: 10.9 mL silane coupling agent N-β -(Aminoethyl)- gamma -Aminopropylmethyldimethoxysilane (KH602) and 0.5 g m-phenylenediamine (MPD) were used as precursors and stirred evenly in 19.1 mL of pure water. The mixture was then transferred to an autoclave and heated at 160 °C for 10 h. After cooling naturally to room temperature, the product was centrifuged at 10,000 RPM for 30 min to collect the supernatant. The supernatant was filtered through a 0.22 μm filter membrane and dialyzed in a dialysis bag (1000 Da) for 48 h to obtain an aqueous solution of Si-CDs. Characterization of Si-CDs: The morphology and size of Si-CDs were characterized by transmission electron microscopy (TEM). Figure 1 The results showed that Si-CDs had good uniformity and dispersion, with an average particle size of 2 ± 0.5 nm. Figure 1 The inset shows the high-resolution transmission electron microscopy (HRTEM) image of Si-CDs, which shows that the lattice spacing of Si-CDs is 0.2 nm, which is consistent with the sp2The (100) plane spacing of carbon is consistent. The fluorescence spectrum (FL) of aqueous Si-CDs is as follows Figure 2 As shown, the excitation and emission wavelengths at the highest fluorescence intensity are 419 nm and 459 nm, respectively. Figure 2 The inset shows that Si-CDs emit bright blue fluorescence under 365 nm UV light excitation. Figure 3 Ultraviolet-visible (UV-Vis) spectroscopy showed that Si-CDs had two characteristic peaks at 288 nm and 355 nm in the ultraviolet region, corresponding to the n-π* transitions of unsaturated groups C=N / C=O and / or CN on the surface of Si-CDs. Figure 4 Fourier transform infrared spectroscopy (FT-IR) showed that the −1 The broad absorption at 2,983 cm is derived from the stretching vibration of OH / NH. −1 The absorption at 1634 cm comes from the stretching vibration of CH, which is due to the preservation of the hydrogen-rich atomic structure outside the benzene ring. −1 The absorption at 1043 cm comes from the C=C stretching vibration of polycyclic aromatic hydrocarbons, while the absorption at 1043 cm −1 The absorption at is attributed to the stretching vibration of CO (alcohol or phenol). The above results indicate that Si-CDs with lattice structure were successfully prepared, and their surfaces have a variety of chemical groups including hydroxyl, carboxyl and amine groups.
[0046] Example 2: Synthesis of Si-CDs@SiO2 The present invention utilizes a modified Stöber method to coat Si-CDs with SiO2. Under alkaline conditions, tetraethyl orthosilicate (TEOS) hydrolyzes, forming SiO2 nanospheres while simultaneously encapsulating the Si-CDs within them. This creates an inert SiO2 protective layer on the Si-CD surface, preserving the optical and physicochemical properties of the Si-CDs while also achieving surface modification. The specific method for preparing Si-CDs@SiO2 in this example is as follows: To 50 mL of pure water, 9.0 mL of the Si-CDs solution prepared in Example 1 was added, followed by 0.2 g of CTAB and 0.15 mL of 10 M NaOH. The mixture was stirred at 80 °C for 1 h. 300 μL of TEOS was slowly added dropwise, and the reaction was stirred at 80 °C for 3 h. An equal volume of 60 mL of anhydrous ethanol was added and the reaction solution was extracted. The mixture was centrifuged at 10,000 RPM for 10 min, the lower layer was removed and dispersed in 60 mL of anhydrous ethanol, and then 15 mL of glacial acetic acid was added for neutralization. The organic residue was extracted by stirring at 60 °C for 6 h. The lower layer was removed by centrifugation at 10,000 RPM for 10 min, and the mixture was washed three times with anhydrous ethanol to obtain the final product, Si-CDs@SiO2, dispersed in ethanol. The solid Si-CDs@SiO2 was then dried under vacuum at 80 °C. Characterization of Si-CDs@SiO2: Figure 5 a shows that Si-CDs@SiO2 are regular spherical under TEM, with an average particle size of 10-50 nm (scale bar = 50 nm). Figure 5 b shows that the surface of Si-CDs@SiO2 is rough under TEM, but it is impossible to directly observe that Si-CDs are embedded in the SiO2 matrix (scale = 20 nm). Without changing other reaction parameters, the amount of TEOS in the reaction system obviously affects the size of Si-CDs@SiO2. In previous experiments, when the amount of TEOS added was reduced to 200 μL, centrifugal collection was difficult due to the low yield of Si-CDs@SiO2. When the amount of TEOS added was increased to 300 μL, regular spherical small-sized Si-CDs@SiO2 with a reasonable yield could be prepared. Si-CDs@SiO2 with an average particle size of 10-50 nm prepared with 300 μL TEOS is the smallest particle that can be prepared by the present invention. Obviously, this small-sized material is more suitable for biomedical research. The optimal fluorescence emission spectrum of aqueous Si-CDs@SiO2 is shown in Figure 2. Figure 6 As shown, the excitation and emission wavelengths at their highest fluorescence intensity are 365 nm and 463 nm, respectively. The inset shows that under 365 nm UV excitation, Si-CDs@SiO2 emit distinct blue fluorescence compared to pure SiO2 spheres, which emit almost no fluorescence. Compared to the Si-CDs in Example 1, Si-CDs@SiO2 also produce high-intensity fluorescence, indicating that SiO2 coating does not significantly reduce the fluorescence of Si-CDs.
[0047] Example 3: Synthesis of Si-CDs@SiO2-COOH The principle of surface modification of Si-CDs@SiO2 with carboxyl groups is to introduce carboxyl groups onto the Si-CDs@SiO2 surface through the reaction of a carboxylating agent with SiO2. Carboxylating agents are typically compounds containing active carboxyl groups, such as carboxylic acids and acyl chlorides. Chemical bonds are formed between the carboxylating agent and SiO2, firmly fixing the carboxyl groups to the SiO2 surface. In this example, the specific method for surface carboxylation of Si-CDs@SiO2 to obtain Si-CDs@SiO2-COOH is as follows: Succinic anhydride (400 mg) and equimolar 3-aminopropyltriethoxysilane (APTES) were uniformly dispersed in N, N -dimethylformamide (DMF) (50 mL), magnetically stirred at 37 ° C for 3 h, and then about 10 mL of DMF suspension containing Si-CDs@SiO2 (160 mg) dispersed by ultrasonic was added to the system, and then 2 mL of deionized water was added for catalysis. After continuing magnetic stirring at 60 ° C for 5 h, Si-CDs@SiO2 was separated by centrifugation at 10,000 RPM. After washing with anhydrous ethanol three times, carboxyl-modified Si-CDs@SiO2-COOH was obtained, and finally vacuum dried at 60 ° C to obtain powder. By Figure 7 ) and Si-CDs@SiO2-COOH( Figure 8 ) FTIR spectrum comparison, at 1652 cm -1 The characteristic absorption peak of carboxyl group appeared at , which proved that the carboxylation of Si-CDs@SiO2 surface was completed and Si-CDs@SiO2-COOH was obtained.
[0048] Example 4: Synthesis of Si-CDs@SiO2@Protein A Using EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide) and NHS ( N -hydroxysuccinimide) activates the carboxyl group, allowing it to form a covalent amide bond with the amino group of other molecules. Therefore, after EDC and NHS activate the carboxyl group on the surface of Si-CDs@SiO2-COOH, it can be coupled with the amino groups of proteins, antibodies, nucleic acids and other small molecules to obtain functionalized Si-CDs@SiO2. Protein A is a protein found in Staphylococcus aureus ( Staphylococcus aureus) cell wall protein with a molecular weight of 42 kDa. Protein A specifically binds to the Fc region of mammalian immunoglobulins (Ig). Therefore, coupling carboxyl-activated Si-CDs@SiO2-COOH to Protein A allows for specific labeling of biological targets using antibodies. The specific method for coupling Si-CDs@SiO2-COOH to Protein A in this example is as follows: 0.4 mL of Si-CDs@SiO2-COOH (1 mg / mL, MES buffer: 0.01 M, pH = 6.0) was added to 9.6 mL of pure water and magnetically stirred at room temperature for 30 min. 4 mg of EDC and 6 mg of NHS were added to react at room temperature for 15 min to activate the carboxyl groups on the surface of Si-CDs@SiO2-COOH. The product was washed three times with pure water and dispersed in 0.5 mL of HEPES buffer (0.01 M, pH = 7.4). 0.5 mL of Protein A (150 μg / mL) was added and incubated with the activated Si-CDs@SiO2-COOH for 3 h at room temperature (gently shaken on a shaker). Si-CDs@SiO2@Protein A was obtained by centrifugation, washed three times with HEPES buffer, and dispersed in 1 mL of HEPES buffer (0.01 M, pH = 7.4) and stored at 4°C. The optimal fluorescence emission spectrum of A is Figure 9 As shown, the excitation and emission wavelengths when the fluorescence intensity is the highest are 375 nm and 460 nm, respectively. Figure 9 The illustration shows that under 365 nm ultraviolet excitation, Si-CDs@SiO2@Protein A emits bright blue fluorescence, while pure SiO2 spheres emit almost no light. The Bradford protein concentration assay kit (Biyuntian) was used to analyze the concentration of protein A coupled to Si-CDs@SiO2@Protein A. Figure 10 The standard curve showed that the concentration of coupled Protein A was 0.7183 mg / mL, proving that the synthesis of Si-CDs@SiO2@Protein A was completed. Example 5: Comparison of the fluorescent labeling effects of Si-CDs@SiO2@Protein A and commercial small molecule fluorescent secondary antibodies on cells Since Si-CDs@SiO2@Protein A is a fluorescent nanomaterial, and the coupled Protein A can bind to the Fc segment of the antibody, it can be used to specifically label cells in cooperation with the antibody and to perform fluorescent imaging under a laser confocal microscope. The specific method for using Si-CDs@SiO2@Protein A for cell-specific fluorescent imaging in this embodiment is as follows: After the iTEC cells were fixed with 4% paraformaldehyde (w / v) for 15 min, 0.2% Triton X-100 (v / v) was added to permeate the cells for 10 min; after gentle washing with PBS, the cells were blocked in PBS containing 5% BSA (w / v) for 30 min, and then incubated with keratin 8 antibody (0.1 ~ 10 μg / mL) at 4 ℃ overnight to ensure that the antibody was fully combined with the keratin 8 in the iTEC cells; after removing the unbound antibody, the cells were gently washed with PBS; Si-CDs@SiO2@Protein A (50 ~ 100 μg / mL) was added to incubate with the iTEC cells at room temperature for 2 h to bind with the keratin 8 antibody in the cells; after removing the unbound Si-CDs@SiO2@Protein A, the labeled iTEC cells were imaged under a laser confocal microscope to evaluate the effect of specific fluorescent labeling of the iTEC cells by Si-CDs@SiO2@Protein A. The experimental results are shown in Figure 11 As shown in FIG. 6, no photoluminescence phenomenon occurred in the blank control group, the iTEC cells labeled with Alexa fluor 594 fluorescent secondary antibody and DAPI nuclear staining became clear and visible, and the cell nucleus presented blue under an excitation wavelength of 405 nm, and the cytoplasm presented green and red under excitation wavelengths of 488 and 543 nm, respectively. Figure 11The iTEC cells labeled with Si-CDs@SiO2@Protein A (without nuclear staining) showed multi-color luminescence properties. Under excitation at 405, 488 and 543 nm, the cytoplasm showed blue, green and red light, respectively, while the nucleus did not emit light. This shows that Si-CDs@SiO2@Protein A is similar to the Alexa fluor 594 fluorescent secondary antibody. Both can specifically label keratin 8 in the iTEC cytoplasm by binding to the primary antibody (keratin 8 antibody), and thus localize in the cytoplasm and emit specific fluorescence. These results show that Si-CDs@SiO2@Protein A can pass through the cell membrane as easily and quickly as commercial fluorescent secondary antibodies and localize in the cell by binding to the primary antibody. It is worth noting that Si-CDs@SiO2 exhibits strong photostability, no blinking phenomenon is observed, and photobleaching is also rare. Compared with the commercial small molecule fluorescent dye Alexafluor 594, it has excellent optical stability and can be stored at room temperature without deliberately avoiding light. Figure 12 It shows that Si-CDs@SiO2 still maintains a very high fluorescence intensity after 6 months, while the fluorescence of Alexa fluor 594 is basically completely quenched after 2-3 months, which indicates that the optical stability of Si-CDs@SiO2 is significantly stronger than that of commercial small molecule fluorescent dyes.
[0049] The above description is only a preferred specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any technician familiar with the technical field, within the technical scope disclosed by the present invention, who makes equivalent replacements or changes based on the technical solution and inventive concept of the present invention, should be covered by the scope of protection of the present invention.
Claims
1. A method for preparing fluorescent nanomaterial Si-CDs@SiO2@Protein A, characterized in that: The method comprises the following preparation steps: (1) Preparation of silicon-doped carbon dots (Si-CDs); (2) coating the Si-CDs obtained in step (1) with SiO2 to prepare fluorescent nanomaterial Si-CDs@SiO2; (3) Carboxylating the surface of Si-CDs@SiO2 obtained in step (2) to prepare Si-CDs@SiO2-COOH; (4) The carboxyl group of Si-CDs@SiO2-COOH obtained in step (3) is activated and coupled with protein A to obtain fluorescent nanomaterial Si-CDs@SiO2@Protein A that can bind to antibodies.
2. The method for preparing the fluorescent nanomaterial Si-CDs@SiO2@Protein A according to claim 1, characterized in that: In step (1), the silane coupling agent N-β -(Aminoethyl)- γ -Aminopropylmethyldimethoxysilane KH602 and m-phenylenediamine (MPD) were mixed and subjected to hydrothermal reaction to obtain silicon-doped carbon dots Si-CDs.
3. The method for preparing the fluorescent nanomaterial Si-CDs@SiO2@Protein A according to claim 2, characterized in that: The mass ratio of KH602 and MPD is 20-40; The hydrothermal reaction temperature is 150-180 °C, and the hydrothermal reaction time is 5-12 h.
4. The method for preparing the fluorescent nanomaterial Si-CDs@SiO2@Protein A according to claim 3, characterized in that: Preparation method of fluorescent nanomaterial Si-CDs@SiO2: After mixing the obtained silicon-doped carbon dots Si-CDs with tetraethyl orthosilicate TEOS, add alkali hydrolysis to make TEOS form a SiO2 matrix and coat the Si-CDs to obtain spherical fluorescent nanomaterial Si-CDs@SiO2 with a particle size of 10-50 nm.
5. The method for preparing the fluorescent nanomaterial Si-CDs@SiO2@Protein A according to claim 4, characterized in that: Preparation method of fluorescent nanomaterial Si-CDs@SiO2-COOH: 3-aminopropyltriethoxysilane APTES and succinic anhydride are dispersed in a solvent, spherical fluorescent nanomaterial Si-CDs@SiO2 is added, and stirred to make the surface of Si-CDs@SiO2 carboxylated to obtain Si-CDs@SiO2-COOH.
6. The method for preparing the fluorescent nanomaterial Si-CDs@SiO2@Protein A according to claim 5, characterized in that: Preparation method of fluorescent nanomaterial Si-CDs@SiO2@Protein A: Si-CDs@SiO2-COOH was mixed with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide EDC and N- The carboxyl group was activated by the hydroxysuccinimide NHS reaction to obtain carboxyl-activated Si-CDs@SiO2-COOH.
7. The method for preparing the fluorescent nanomaterial Si-CDs@SiO2@Protein A according to claim 6, characterized in that: The carboxyl-activated Si-CDs@SiO2-COOH was suspended in a buffer solution, and Protein A was added. After mixing, the mixture was shaken at room temperature for coupling reaction, and Si-CDs@SiO2@Protein A was obtained by centrifugation.
8. The fluorescent nanomaterial Si-CDs@SiO2@ProteinA prepared according to the method according to any one of claims 1 to 7, characterized in that: The prepared fluorescent nanomaterial Si-CDs@SiO2@Protein A has an excitation wavelength of 355-385 nm and an emission wavelength of 450-470 nm.
9. Use of the fluorescent nanomaterial Si-CDs@SiO2@Protein A according to claim 8 in the preparation of a reagent for cell-specific fluorescence imaging for non-therapeutic purposes.
10. The use according to claim 9, characterized in that The Si-CDs@SiO2@Protein A is synergistically used with keratin 8 antibodies to specifically fluorescently label immortalized mouse thymic epithelial cells iTEC for non-therapeutic purposes, and then fluorescence imaging is performed using a laser confocal microscope.