Silk fibroin fluorescent microsphere and preparation method thereof

Abstract

The invention relates to the technical field of biological materials, in particular to a silk fibroin fluorescent microsphere and a preparation method thereof.The preparation method comprises the following steps that a silk fibroin water solution and an ethanol solution serve as a water phase 1 and a water phase 2 respectively; an oil phase is cut into water-in-oil micro-droplets containing silk fibroin and ethanol through a microfluidic device containing a double-tube injection capillary glass tube, and the silk fibroin fluorescent microspheres are obtained through co-induction molding of temperature and ethanol. According to the invention, the problems that the regulation and control capability of the microsphere structure morphology is limited and a solid is easily formed to block a microchannel due to rapid reaction between a reagent and silk fibroin when the silk fibroin microsphere is prepared by adopting a liquid drop microfluidic technology in the prior art are solved, and the regulation and control on the forming time, the particle size, the morphology and the fluorescence characteristic of the silk fibroin fluorescent microsphere can be realized.

Description

Technical Field

[0001] The present invention relates to the technical field of biomaterials, in particular to a silk fibroin fluorescent microsphere and a preparation method thereof. Background Art

[0002] In recent years, with the rapid development of biomedicine and materials science, the demand for high-performance microsphere materials has been increasing. Microspheres, with their unique physical and chemical properties, have shown great application potential in various fields such as cell culture, drug delivery, bioimaging, immunomedicine, and high-throughput drug screening.

[0003] Silk fibroin is the main protein in silk. It has the advantages of low toxicity, biocompatibility, good processability, unique and regular chemical structure, etc. It can be hydrolyzed by enzymes in the body into harmless small molecules, which are easily absorbed or metabolized by the body. It has good biocompatibility, biodegradability and excellent mechanical properties. It is widely used in tissue engineering, drug delivery, biosensors, fine chemicals and bionic materials.

[0004] However, traditional methods for preparing silk fibroin microspheres (such as spray drying, solvent evaporation, emulsification precipitation, and salting-out methods) have problems such as uneven particle size and limited ability to control the structure and morphology of the microspheres. Droplet microfluidics, as an emerging technology for studying droplet generation, manipulation, and application at the microscale, can generate monodisperse, size-controllable droplets, providing a new approach for the precise preparation of microspheres. However, when preparing silk fibroin microspheres on existing microfluidic chips, the aqueous phase often contains various additives and surfactants, and is limited by the high viscosity of the silk fibroin solution and the complex hydrogel formation mechanism, resulting in insufficient control over the size, structure, and morphology of the prepared microspheres. Although there are reagents that can induce conformational changes in silk fibroin molecules to cause gelation, these reagents react quickly with silk fibroin to form solid silk fibroin, which easily clogs the microchannels, greatly limiting the application of droplet microfluidics in silk fibroin microspheres. Summary of the Invention

[0005] In order to solve the defects in the prior art, the present invention provides a silk fibroin fluorescent microsphere and a preparation method thereof, so as to solve the problems existing in the prior art of using droplet microfluidics technology to prepare silk fibroin microspheres, such as limited ability to control the microsphere structure and morphology, and rapid reaction between reagents and silk fibroin that easily forms solids to block the microchannels. In addition, the preparation method of the present invention can regulate the molding time, particle size, morphology and fluorescence characteristics of the silk fibroin fluorescent microspheres by regulating the ethanol concentration and temperature.

[0006] To achieve the above object, the present invention adopts the following technical solutions:

[0007] In a first aspect, the present invention provides a method for preparing silk fibroin fluorescent microspheres, comprising the following steps:

[0008] Silk fibroin aqueous solution and ethanol solution were used as aqueous phase 1 and aqueous phase 2, respectively. The oil phase was sheared into W / O (water-in-oil) microdroplets containing silk fibroin and ethanol through a microfluidic device containing a double-tube injection capillary glass tube. Silk fibroin fluorescent microspheres were obtained by induced molding through temperature and ethanol.

[0009] Preferably, the concentration of the silk fibroin solution is 2 wt %.

[0010] Preferably, the volume concentration of the ethanol solution is 10% to 60%. Exemplarily, the volume concentration of the ethanol solution is any one of 10%, 25%, 40%, 50%, 60%, or a value in between. By adjusting the concentration of ethanol, the microdroplet size, microsphere formation time, particle size, morphology, and fluorescence intensity can be controlled.

[0011] Preferably, the volume concentration of ethanol in the W / O (water-in-oil) microdroplets containing silk fibroin and ethanol is 5-30%. Exemplarily, the volume concentration of ethanol in the collected liquid of the microdroplets is any one of 5%, 12.5%, 20%, 25%, 30%, or a value in between.

[0012] Preferably, the volume ratio of the aqueous phase 1 to the aqueous phase 2 is 1:1.

[0013] Preferably, the oil phase is selected from one or more of soybean oil, mineral oil, corn oil, dimethyl silicone oil, and fluorinated liquid.

[0014] Preferably, the oil phase contains a suitable surfactant. In some embodiments of the present invention, the surfactant is PGPR. Uniform preparation of W / O microdroplets can be achieved during the preparation process without adding other surfactants to the aqueous phase.

[0015] Preferably, during the shearing process, the flow rate ratio of aqueous phase 1 to aqueous phase 2 is 1:1.

[0016] Preferably, during the shearing process, the flow rate ratio of the aqueous phase 1, the aqueous phase 2 and the oil phase is 1:1:(13-40).

[0017] Preferably, the temperature of the induction molding process is 20-60°C. Exemplarily, the temperature of the induction molding process is any one of 20°C, 30°C, 40°C, 50°C, and 60°C, or a value in between. By adjusting the induction molding temperature, the microsphere molding time, particle size, and photoluminescence phenomenon can be controlled.

[0018] In a second aspect, the present invention provides a silk fibroin fluorescent microsphere prepared by the above preparation method.

[0019] Preferably, the silk fibroin fluorescent microspheres have enzymatic degradation properties.

[0020] Preferably, the silk fibroin fluorescent microspheres are biocompatible.

[0021] The beneficial effects of the present invention are:

[0022] The present invention provides a method for preparing silk fibroin fluorescent microspheres. The method employs a method in which an aqueous phase 1 and an aqueous phase 2 are separately introduced into a capillary glass microfluidic device, thereby simultaneously shearing the silk fibroin and ethanol, forming droplets, and mixing them. This prevents the problem of excessive cross-linking of the silk fibroin and ethanol, which may clog the microchannel. The present invention utilizes droplet microfluidics combined with ethanol-temperature induced molding technology. The microfluidic technology can uniformly form microdroplets to form uniform microspheres. The temperature and ethanol can induce the secondary structure of the silk fibroin to transform into a β-pleated structure, thereby promoting the formation of microspheres from the silk fibroin droplets. Microsphere molding can be achieved without the addition of other substances to the aqueous phase or freeze-drying. Furthermore, the particle size, morphology, molding time, and fluorescence properties of the silk fibroin microspheres can be controlled by regulating the ethanol concentration and temperature.

[0023] The silk fibroin microspheres prepared by this invention have a β-pleated structure, uniform particle size, and excellent biocompatibility. Their fluorescence intensity decreases with protease degradation, making them suitable as probes for protease activity detection. They exhibit a unique photoluminescence phenomenon, achieving autofluorescence emission without the need for exogenous labeling substances, and possess excellent photostability and resistance to photobleaching. This innovative fluorescent material overcomes the problems of traditional fluorescent microspheres, such as fluorescence leakage caused by chemical coupling, limited biocompatibility, and photosensitivity degradation, demonstrating significant advantages in fields such as biological detection and dynamic tracing.

[0024] The silk microspheres developed in this invention can be expanded to other fields such as drug delivery systems, organoid construction, dynamic tracing of biomarkers, and development of molecular imaging probes due to their excellent biocompatibility and controllable functional properties, showing the potential for interdisciplinary applications in cutting-edge directions such as intelligent responsive medical materials, implantable diagnostic and treatment devices, and time-space controlled release systems. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 Schematic diagram of preparing silk fibroin microdroplets for glass capillary microfluidic device.

[0026] Figure 2The preparation and characterization diagram of the silk fibroin microspheres in Example 1 (a. Schematic diagram of the preparation of silk fibroin microspheres using a microfluidic device and high-resolution real-time images of the generated microdroplets; b. Formation mechanism of the silk fibroin β-pleated structure; c. Microscopic image of silk fibroin microdroplets; d. Microscopic image (in oil) and electron microscope image of microspheres; e. Particle size distribution of silk fibroin microdroplets and microspheres).

[0027] Figure 3 These are optical microscope images of the silk fibroin microdroplets in Example 2 during the incubation and molding process at different temperatures.

[0028] Figure 4 Figure 2 is an investigation of the effect of temperature on silk fibroin microspheres in Example 2 (a. Flow chart of the preparation of silk fibroin microspheres; b. Microscopic images of microspheres prepared at different temperatures; c. Images of particle size changes of droplet shrinkage during microsphere curing at different temperatures; d. Droplet shrinkage before and after silk fibroin curing at different temperatures).

[0029] Figure 5 These are characterization diagrams of the photoluminescence performance of silk fibroin microspheres at different temperatures in Example 2 (a. laser confocal microscopic images of silk fibroin microspheres at different excitation wavelengths; b. fluorescence intensity of silk fibroin microspheres at different excitation wavelengths).

[0030] Figure 6 Characterization diagrams of silk fibroin droplets and microspheres prepared at different ethanol concentrations in Example 3 (a. Schematic diagram of the preparation of silk fibroin microparticles with low and high ethanol concentrations; b. Microscopic images of droplets and microspheres prepared at different ethanol concentrations; c. Graph of particle size change of droplets shrinking during microsphere curing at different ethanol concentrations; d. Graph of particle size of microspheres before and after curing at different ethanol concentrations; e. Graph of droplet shrinkage before and after curing at different ethanol concentrations; f. SEM images of microspheres at different ethanol concentrations, f1: 5%, f2: 12.5%, f3: 20%, f4: 30%; g is a partially enlarged image of Figure f).

[0031] Figure 7 These are optical microscope images of the coagulation process of silk fibroin microdroplets in Example 3 at different ethanol concentrations.

[0032] Figure 8 Characterization diagram of the photoluminescence performance of silk fibroin microspheres with different ethanol concentrations in Example 3 (a. laser confocal microscopic images of silk fibroin microspheres at different excitation wavelengths; b. fluorescence intensity of silk fibroin microspheres at different excitation wavelengths).

[0033] Figure 9 Characterization diagram of the enzymatic degradation performance of silk fibroin microspheres (a. Laser confocal microscopy image of the degradation process of silk fibroin microspheres; b. Changes in fluorescence intensity during the degradation process of silk fibroin microspheres).

[0034] Figure 10 The biocompatibility characterization diagram of silk fibroin microspheres (a. CCK-8 assay to quantify the effect of silk fibroin microspheres on cell survival and proliferation; b. Live / dead cell staining to evaluate the biocompatibility of microspheres, the ethanol concentration is the concentration in the microdroplet collection fluid). DETAILED DESCRIPTION

[0035] In order to enable those skilled in the art to better understand the technical solution of the invention, the present invention is further described in detail below in conjunction with specific implementation methods.

[0036] In the present invention, the silk fibroin is a natural high-molecular-weight fiber protein extracted from silk. The preparation method of the silk fibroin used in the following examples is as follows: the cocoons are cleaned, cut into small pieces, added to a 0.5wt% Na2CO3 solution, heated for 0.5h, then washed with deionized water to remove the sericin on the surface of the silk, repeatedly degummed, and dried in an oven. 1g of degummed silk is dissolved in 5ml of a ternary solvent system (CaCl2 / C2H5OH / H2O) solution, stirred at 72°C for 2h to fully dissolve the silk to obtain a crude silk fibroin solution. After the crude silk fibroin solution is cooled, it is transferred to a dialysis bag (MWCO 10kDa) and dialyzed with deionized water for three days. After the dialysis is completed, the silk fibroin solution is repeatedly centrifuged (7000rpm, 10min) to remove the solid matter in the solution, and the supernatant is freeze-dried to obtain silk fibroin, which is then stored in a refrigerator at 4°C.

[0037] The process of preparing silk fibroin microdroplets using a glass capillary microfluidic device in the present invention is shown in Figure 1 : Water phase 1 is injected from inlet 1 of the device, water phase 2 is injected from inlet 2 of the device, and oil phase is injected from inlet 3 of the device as shear phase. At the shear port, the oil phase shears water phase 1 and water phase 2 at the same time to form microdroplets of silk fibroin mixed with ethanol.

[0038] The materials and equipment used in the following examples are all commercially available products in the art.

[0039] Example 1 Preparation of silk fibroin microspheres

[0040] The steps for preparing the silk fibroin microspheres in this embodiment are as follows:

[0041] (1) Preparation of solution: Silk fibroin was added to deionized water and stirred at room temperature (stirring speed 800 rpm, stirring time 10 min) to dissolve to obtain a silk fibroin solution (aqueous phase 1), wherein the silk fibroin concentration was 2 wt%; deionized water was mixed with anhydrous ethanol and diluted to obtain an ethanol solution (aqueous phase 2), wherein the volume concentration of ethanol was 50%.

[0042] (2) Preparation of silk fibroin microspheres: Soybean oil containing 10 wt% PGPR was used as the oil phase, and the oil phase was used to shear the aqueous phase 1 and aqueous phase 2 at the same time (the volume ratio of aqueous phase 1 to aqueous phase 2 was 1:1). The speeds of aqueous phase 1 and aqueous phase 2 were controlled to be 50 μL / h and 50 μL / h, respectively, and the oil phase speed was 2000 μL / h. After obtaining a collection liquid containing microdroplets mixed with silk fibroin and ethanol (the volume concentration of ethanol was 25%), the collection liquid was placed in an incubator at 50°C to promote the formation of microdroplets into microspheres. After replacing the oil phase solution around the microspheres with n-hexane, the microspheres were washed with deionized water, and the microspheres were collected and freeze-dried to obtain silk fibroin microspheres.

[0043] The process of silk fibroin microspheres generation is shown in Figure 2 a. By introducing silk fibroin and ethanol separately, they are sheared simultaneously and mixed after forming droplets. This avoids the silk fibroin and ethanol mixing to form solids before shearing to form droplets. Real-time images of the preparation process show that there is no channel blockage. Figure 2 b It can be seen that the present invention can induce the secondary structure of silk fibroin to transform into a β-pleated structure, thereby promoting the formation of microspheres from silk fibroin microdroplets, and the microsphere formation can be achieved without adding other substances or freeze-drying. Figure 2 It can be seen that the particle size of the microdroplets prepared in this example is 102.58±1.13μm, and the monodispersity coefficient (CV value) is 1.1%. The particle size of the silk fibroin microspheres obtained by induced molding is 32.86±0.95μm, and the CV value is 2.9%. The morphology of the prepared silk fibroin microspheres was observed using an optical microscope and a scanning electron microscope. The results are shown in FIG. Figure 2 c and Figure 2 As shown in Figure d, the prepared microdroplets and microspheres have uniform particle size and morphology.

[0044] Example 2 Effect of Temperature on Droplets and Microspheres

[0045] The preparation method of silk fibroin microspheres is the same as that of Example 1, except that the concentration of the silk fibroin solution is fixed at 2wt%, the volume concentration of ethanol is fixed at 50%, and the temperature is adjusted (adjustment range is 20-60°C). Soybean oil containing 10wt% PGPR is used as the oil phase, and the oil phase is used to shear the aqueous phase 1 and aqueous phase 2 at the same time. The speeds of aqueous phase 1 and aqueous phase 2 are controlled to be 50μL / h and 50μL / h, respectively, and the oil phase speed is 1000μL / h. After obtaining a collection liquid containing microdroplets mixed with silk fibroin and ethanol, the collection liquid is placed in an incubator at 20°C, 30°C, 40°C, 50°C, and 60°C, respectively, and the microdroplets are formed into silk fibroin microspheres. The oil phase solution around the microspheres can be replaced with n-hexane and then washed with deionized water to obtain silk fibroin microspheres dispersed in water for easy subsequent processing and application.

[0046] The morphology of the prepared silk fibroin microspheres was observed using an optical microscope. Figure 3 and Figure 4 As shown in the figure, the silk fibroin droplets shrink as the solvent gradually diffuses and evaporates under static conditions, and the β-pleated structure induced by ethanol and temperature gradually aggregates to form microspheres. As the temperature increases from 20°C to 60°C, the time it takes for the droplets to form microspheres gradually decreases from 180 minutes to 20 minutes, and the particle size of the prepared microspheres also gradually decreases. The photoluminescence properties of the microspheres were observed using a laser confocal microscope, see Figure 5 , it can be found that the fluorescence intensity of the silk fibroin microspheres prepared in this example gradually increases with the increase of the induction molding temperature under 405nm laser excitation. These results show that adjusting the induction molding temperature can achieve the control of microsphere molding time, particle size and photoluminescence phenomenon.

[0047] Example 3 Effect of ethanol volume concentration on droplets and microspheres

[0048] The preparation method of silk fibroin microspheres is the same as that of Example 1, except that the concentration of the silk fibroin solution is fixed at 2 wt %, the incubator temperature is fixed at 40° C., the ethanol concentration is adjusted to 10%, 25%, 40%, and 60%, respectively. Soybean oil containing 10 wt % PGPR is used as the oil phase, and the oil phase is used to shear the aqueous phase 1 and the aqueous phase 2 at the same time. The speeds of the aqueous phase 1 and the aqueous phase 2 are controlled to be 150 μL / h and 150 μL / h, and the oil phase speed is 2000 μL / h. After obtaining a collection liquid containing microdroplets mixed with silk fibroin and ethanol (the concentrations of ethanol in the obtained microdroplet collection liquid are 5%, 12.5%, 20%, and 30%, respectively, and the ethanol concentrations involved in the following characterization are all the ethanol concentrations in the microdroplet collection liquid), the collection liquid is placed in an incubator at 40° C., and the microdroplets are formed into silk fibroin microspheres. The oil phase surrounding the microspheres can be replaced with n-hexane and then rinsed with deionized water to obtain water-dispersed silk fibroin microspheres, which are convenient for subsequent processing and application. The water-dispersed silk fibroin microspheres can be freeze-dried and then used for scanning electron microscopy.

[0049] Figure 6 a is a schematic diagram of the preparation of silk fibroin microparticles using low and high concentrations of ethanol. It can be seen that different ethanol concentrations have an effect on the morphology of the microspheres. Figure 6 b is the microscopic image of droplets and microspheres prepared with different ethanol concentrations (b1 is the droplet, the scale bar is 200 μm; b2 is the microsphere, the scale bar is 25 μm); Figure 6 c is the change of droplet size over time during the microsphere solidification process at different ethanol concentrations; Figure 6 d is the particle size diagram of droplets and microspheres before and after droplet solidification at different ethanol concentrations; Figure 6 e is the droplet shrinkage diagram before and after solidification of silk fibroin at different ethanol concentrations. It can be seen that with the increase of ethanol concentration, the particle size of the microdroplets obtained by shearing gradually decreases, and the particle size of the prepared microspheres also gradually decreases; Figure 6 f is the scanning electron microscope image of microspheres at different ethanol concentrations. Figure 6 g is Figure 6 From the local magnified surface image of f (f1 and g1: 5%, f2 and g2: 12.5%, f3 and g3: 20%, f4 and g4: 25%, f5 and g5: 30%), it can be seen that with the increase of ethanol concentration, the surface of the microspheres gradually becomes rough and the structure gradually becomes dense, which can realize the preparation of silk fibroin microcapsules to microspheres.

[0050] In addition, with the increase of ethanol concentration, the time for droplets to form microspheres gradually decreased from 90 min to 40 min, e.g. Figure 7 The photoluminescence phenomenon of the microspheres was observed by laser confocal microscopy. Figure 8 As shown in the figure, as the ethanol concentration increases, the fluorescence intensity of the microspheres under 405nm and 488nm laser excitation gradually increases. When the ethanol concentration is 20% (the concentration after mixing with the silk fibroin solution), the fluorescence intensity is the strongest. Further increasing the ethanol concentration, the fluorescence intensity decreases. These results show that adjusting the ethanol concentration can achieve the control of microdroplet size, microsphere formation time, particle size, morphology, and fluorescence intensity.

[0051] Example 4 Detection of enzymatic degradation performance of silk fibroin microspheres

[0052] The detection method is as follows:

[0053] (1) Preparation of Protease XIV Solution: Add Protease XIV powder to PBS solution and stir evenly to obtain 1.75 U / mL Protease XIV solution.

[0054] (2) 500 μg of the silk fibroin microspheres prepared in Example 1 were added to 200 μl of protease XIV solution, and the degradation process of the microspheres was observed using a laser confocal microscope at room temperature.

[0055] The degradation process was observed by laser confocal microscopy. Figure 9 It can be found that within 3 hours, as time increases, the silk microspheres gradually degrade and their fluorescence gradually decreases, showing protease-responsive fluorescence attenuation characteristics.

[0056] Example 5 Evaluation of cytotoxicity of silk fibroin microspheres

[0057] The biocompatibility of silk fibroin microspheres was evaluated by mouse fibroblast (NIH3T3 cell) proliferation assay. 4NIH 3T3 cell suspension was seeded into a 96-well plate and cultured at 37°C for 24 h to adhere to the wall. The silk fibroin microspheres prepared in Example 1 were then irradiated with UV light for 1 h and added to a DMEM culture medium (containing 10% fetal bovine serum (FBS), 1 U / mL penicillin, 1×10 -3 mg / mL streptomycin) to prepare a 0.5 mg / mL mixed solution. 100 μL of the mixed solution was then added to the wells containing cells as the experimental group, while the blank group was added to the wells without cells. The control group was added with DMEM medium (containing 10% fetal bovine serum (FBS), 1 U / mL penicillin, 1×10 - 3 Each group was incubated for 24 and 72 h, and the absorbance at 450 nm was measured using a microplate reader using a cell counting kit-8 (CCK8) to quantitatively determine cell activity.

[0058] In order to directly observe the effect of silk fibroin microspheres on cell viability, live / dead staining was used to assess the viability of cells after co-culture with silk fibroin microspheres. 4 NIH 3T3 cell suspension was seeded into 12-well plates and cultured at 37°C for 24 h to adhere to the wall. The silk fibroin microspheres were then irradiated with UV light for 1 h and added to DMEM medium (containing 10% fetal bovine serum (FBS), 1 U / mL penicillin, 1×10 -3 The researchers then added 100 μL of the mixed solution to the wells containing cells (the experimental group). The blank group consisted of cells without cells, while the control group consisted of cells containing DMEM medium without silk fibroin microspheres. Two replicates were performed for each group. After incubation for 72 hours, the stained cells were observed using a fluorescence microscope. Live cells appeared yellow-green (excited at 490 nm) and dead cells appeared red (excited at 545 nm).

[0059] from Figure 10 The test results show that after the silk fibroin microspheres were co-incubated with NIH 3T3 cells for 1 day and 3 days, the cell viability was higher than that of the control group, and the cell viability was significantly increased on the 3rd day (p<0.001). The live / dead staining results showed that the cell viability was comparable to that of the control group, with only a few cells showing red color, indicating that the silk fibroin microspheres provided by the present invention have good biocompatibility and have great application potential in many fields such as cell co-culture, drug delivery, tissue engineering, and regenerative medicine.

[0060] The above are merely preferred embodiments of the present invention. It should be noted that the above preferred embodiments should not be construed as limiting the present invention, and the scope of protection of the present invention should be determined by the scope defined in the claims. Persons skilled in the art will appreciate that improvements and modifications may be made without departing from the spirit and scope of the present invention, and such improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for preparing silk fibroin fluorescent microspheres, characterized in that: The following steps are involved: Silk fibroin aqueous solution and ethanol solution were used as aqueous phase 1 and aqueous phase 2, respectively. The oil phase was sheared into oil-in-water microdroplets containing silk fibroin and ethanol through a microfluidic device containing a double-tube injection capillary glass tube. Silk fibroin fluorescent microspheres were obtained by inducing molding through temperature and ethanol.

2. The method for preparing silk fibroin fluorescent microspheres according to claim 1, wherein: The concentration of the silk fibroin solution is 2 wt %.

3. The method for preparing silk fibroin fluorescent microspheres according to claim 1, wherein: The volume concentration of the ethanol solution is 10-60%.

4. The method for preparing silk fibroin fluorescent microspheres according to claim 1, wherein: The volume concentration of ethanol in the water-in-oil microdroplets containing silk fibroin and ethanol is 5-30%.

5. The method for preparing silk fibroin fluorescent microspheres according to claim 1, wherein: The volume ratio of the aqueous phase 1 to the aqueous phase 2 is 1:

1.

6. The method for preparing silk fibroin fluorescent microspheres according to claim 1, wherein: The oil phase is selected from one or more of soybean oil, mineral oil, corn oil, dimethyl silicone oil, and fluorinated liquid.

7. The method for preparing silk fibroin fluorescent microspheres according to claim 1 or 6, characterized in that: The oil phase contains a surfactant.

8. The method for preparing silk fibroin fluorescent microspheres according to claim 1, characterized in that: During the shearing process, the flow rate ratio of the water phase 1, the water phase 2 and the oil phase is 1:1:(13-40).

9. The method for preparing silk fibroin fluorescent microspheres according to claim 1, wherein: The temperature of the induction molding process is 20-60°C.

10. Silk fibroin fluorescent microspheres prepared by the preparation method according to any one of claims 1 to 9.

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