Large-scale controllable preparation method of super-bright fluorescent silicon microspheres based on fluorescent probe doping

Abstract

The invention discloses a large-scale controllable preparation method of super-bright fluorescent silicon microspheres based on fluorescent probe doping, and belongs to the technical field of nano material preparation. According to the method, firstly, a fluorescent probe is efficiently embedded into a silicon skeleton, so that a microsphere fluorescent signal is enhanced, a raindrop effect is avoided, and the problems of low brightness and non-uniform luminescence of a traditional fluorescent microsphere are solved; through multi-step refined modification, the interface compatibility of the fluorescent probe and a silica sol system is improved, and uniform doping and stable embedding are ensured; the fluorescent probe is doped into the silicon microspheres at high density, so that the unit signal intensity and the imaging consistency are improved, and the problem of outer surface modification is avoided. According to the preparation process, double injection pumps are adopted for synchronous feeding, a large-volume reaction system and constant-temperature stirring are adopted, 0.5-3 g of dry microspheres can be stably prepared in a single batch, and the preparation efficiency is improved. The prepared superbright fluorescent silicon microspheres have the advantages of high brightness, high stability, good dispersity, batch consistency and the like, and a feasible scheme is provided for batch production of fluorescent coding probes.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of nanomaterial preparation, and particularly relates to a large-scale controllable preparation method of super-bright fluorescent silicon microspheres based on fluorescent probe doping. BACKGROUND

[0002] Fluorescent probes, such as upconversion nanoparticles (UCNPs), colloidal gold nanoparticles (Au) and aggregation-induced emission dyes (AIE), are widely used in digital immunoassay, single molecule recognition imaging and high-throughput analysis platform, etc. These fluorescent probes, with their unique optical properties, provide the possibility for precise detection and analysis. However, in practical applications, the brightness of these fluorescent probes often cannot meet the needs of high sensitivity and high resolution detection, limiting their application effect and detection accuracy in complex detection scenarios. In order to improve the brightness and stability of the probes, preparing these fluorescent probes into microspheres becomes an effective solution. The microsphere structure can significantly improve the unit light intensity of the probes, enhance the signal-to-noise ratio, and thus improve the detection sensitivity and image resolution.

[0003] Among many microsphere materials, silicon microspheres become an ideal fluorescent probe carrier due to their excellent dispersibility, light transmittance, biocompatibility and easy surface functionalization. They not only can effectively encapsulate fluorescent probes and protect them from external environmental influences, but also can optimize the distribution and optical properties of the probes by precisely controlling the particle size and surface properties. In addition, the size controllability of silicon microspheres makes them have a wide range of applications in various biological detection platforms, especially in digital immunoassay, single molecule recognition imaging and high-throughput analysis, providing strong support for high sensitivity and high resolution detection.

[0004] However, the preparation of fluorescent microspheres with good dispersibility, precise particle size control and strong fluorescence uniformity still faces many technical challenges. Traditional microsphere preparation methods (such as sol-gel reaction), although widely used in laboratories, have obvious limitations. The particle size distribution of the microspheres prepared by these methods is uneven, resulting in large performance differences at different detection locations; the fluorescence intensity is inconsistent, making the detection signal unstable, which directly affects the performance and application effect of the microspheres. In addition, many existing methods can only be specifically designed for a single probe, lack of universality, and are difficult to migrate to the preparation of other probe types, which greatly limits the diversified development of high-performance fluorescent microspheres.

[0005] With the increasing demand of detection technology for the performance of fluorescent microspheres, especially the high brightness, precise particle size control and large-scale production, the existing preparation technology has been difficult to meet. Therefore, a new construction strategy is urgently needed, which can realize the efficient and uniform doping of fluorescent probes in silicon microspheres, ensure the consistency of fluorescence intensity and particle size, and have good process scalability to adapt to the needs of batch production, so as to promote the application and development of fluorescent microspheres in a wider field. SUMMARY

[0006] In view of the problems of uneven distribution of fluorescent probes in silicon microspheres, inaccurate particle size control and poor stability of synthesis process in the prior art, the present application aims to provide a large-scale controllable preparation method of super-bright fluorescent silicon microspheres based on fluorescent probe doping, which innovatively introduces the interface assembly and multi-step temperature control coating strategy, successfully solves the problems of insufficient brightness, uneven particle size and poor process scalability, and provides a reliable solution for the large-scale application of fluorescent microspheres.

[0007] In order to achieve the above purposes, the present application adopts the following technical solutions: The present application provides a large-scale controllable preparation method of super-bright fluorescent silicon microspheres based on fluorescent probe doping, comprising: S1, the acid pretreated fluorescent probe is sequentially subjected to amination and silanization surface modification to obtain a surface modified fluorescent probe; S2, a reaction system is constructed, which contains potassium chloride, anhydrous ethanol, water, ammonia water and tetraethyl orthosilicate; S3, under the protection of nitrogen, the surface modified fluorescent probe solution and tetraethyl orthosilicate are synchronously injected into the reaction system, and stirred to react, then 3-aminopropyl triethoxysilane solution is injected to continue the reaction, followed by centrifugation, washing and drying to obtain super-bright fluorescent silicon microspheres doped with up-conversion nanoparticles.

[0008] The fluorescent probe is any one of up-conversion nanoparticles (UCNPs), colloidal gold nanoparticles (Au), aggregation-induced emission dye (AIE), quantum dots, fluorescent dye and fluorescent protein.

[0009] In S1, the amination specifically includes surface acidification pretreatment of the fluorescent probe, reaction of the acid pretreated fluorescent probe with an amination reagent, centrifugation after reaction, washing and drying to obtain amination modified fluorescent probe; the amination reagent is any one of phosphoethanolamine, amino modified polyethylene glycol and amino silane reagent.

[0010] Preferably, the amination reagent is phosphoethanolamine.

[0011] Preferably, the molar ratio of the fluorescent probe to the amination reagent is 1:5-15, the reaction temperature is 280-320℃, and the reaction time is 1-5 h.

[0012] Preferably, the acidification pretreatment is to mix, shake and react the fluorescent probe solution, acid and ethanol, and then centrifuge to obtain the pretreated fluorescent probe.

[0013] Further preferably, the volume ratio of the fluorescent probe solution, acid and ethanol is 1:1-2:1-2, the shaking reaction temperature is 20-30℃, the rotation speed is 100-150 rpm, and the time is 2-3 h.

[0014] In S1, the silanization specifically comprises the following steps: under nitrogen protection, mixing the amination-modified fluorescent probe with 3-aminopropyl triethoxysilane to react in the dark, centrifuging, washing, and resuspending to obtain the silanized fluorescent probe.

[0015] Preferably, the molar ratio of the amination-modified fluorescent probe to 3-aminopropyl triethoxysilane is 1:1-2, the reaction temperature is 10-20℃, and the reaction time is 15-25 h.

[0016] In S2, the reaction system is constructed by sequentially stirring and uniformly mixing KCl, anhydrous ethanol, water, ammonia and TEOS; and the mass ratio of KCl, anhydrous ethanol, water, ammonia and tetraethyl orthosilicate is 1:35-40:10-20:7-8:8-9.

[0017] In S3, the volume ratio of the surface-modified fluorescent probe solution to tetraethyl orthosilicate is 1:2-3, the injection speed is 0.5-2 mL / h, the stirring speed is 200-300 rpm, and the reaction time is 10-14 h.

[0018] Preferably, the solvent used for the surface-modified UCNPs solution is anhydrous ethanol.

[0019] In S3, the volume ratio of the 3-aminopropyl triethoxysilane solution to tetraethyl orthosilicate is 1:3-5, the injection speed is 0.5-2 mL / h, and the injection time is 3-4 h before the reaction ends.

[0020] Preferably, the solvent of the 3-aminopropyl triethoxysilane solution is n-butanol.

[0021] The fluorescent probe doped super-bright fluorescent silica microspheres obtained by the large-scale controllable preparation method based on the fluorescent probe doped super-bright fluorescent silica microspheres.

[0022] Compared with the prior art, the present application has the following technical effects: This invention provides a large-scale, controllable preparation method for ultrabright fluorescent silicon microspheres based on fluorescent probe doping. Through amination-silanization dual modification, the fluorescent probe forms covalent bonds with the silicon matrix, avoiding leakage and quenching caused by traditional physical adsorption, significantly improving fluorescence intensity and stability. Simultaneous injection of the surface-modified fluorescent probe solution and TEOS into the reaction system ensures that the fluorescent probe is encapsulated in the silicon microspheres from the early stages of growth, achieving uniform doping and preventing fluorescent probe aggregation on the surface of the silicon microspheres, thus improving the uniformity and stability of the fluorescence signal. By controlling the reaction system, the morphology of the microspheres can be precisely controlled, and secondary injection... The addition of 3-aminopropyltriethoxysilane further crosslinks the silane layer, sealing surface defects in the microspheres, reducing non-radiative transitions, and improving optical signal acquisition efficiency. Precise particle size control is achieved by adjusting the injection rate of the surface-modified fluorescent probe solution and tetraethyl orthosilicate, meeting the needs of different application scenarios. This method, through surface modification, reaction system regulation, and simultaneous injection technology, achieves synergistic optimization of fluorescence performance, microsphere morphology, and production efficiency, while also meeting the requirements of industrial production. The solution reaction system is simple to operate and easy to scale up, providing a feasible solution for the large-scale preparation of ultra-bright fluorescent silicon microspheres.

[0023] Furthermore, the fluorescent probe after acid pretreatment is modified with amination reagents such as ethanolamine phosphate to introduce amino groups (-NH2). The amino groups can undergo condensation reactions with the siloxy groups in the subsequent silanizing reagents (such as 3-aminopropyltriethoxysilane, APTES) to form stable chemical bonds, thereby firmly fixing the fluorescent probe inside the silicon microspheres.

[0024] Furthermore, the fluorescent probe is modified with a silanizing agent to introduce siloxy groups. These siloxy groups can undergo a condensation reaction with silicic acid (Si(OH)4) generated by the hydrolysis of tetraethyl orthosilicate (TEOS) to form a silicon dioxide (SiO2) network structure, which encapsulates the fluorescent probe. This chemical bonding method can effectively prevent leakage of the fluorescent probe during the preparation process and improve the stability of the silicon microspheres.

[0025] Furthermore, potassium chloride, as an electrolyte, can adjust the ionic strength of the reaction system, affecting the polymerization rate of silicic acid (Si(OH)4) and the particle size distribution of silica microspheres. Ammonia, as a catalyst, can promote the hydrolysis and condensation reaction of TEOS, accelerate the formation of silica microspheres, and also adjust the pH value of the reaction system. By controlling the concentration and injection rate of TEOS, the growth rate and final particle size of silica microspheres can be adjusted.

[0026] Further, the surface modified fluorescent probe solution and TEOS are synchronously added by using double injection pumps, a large volume reaction system (60-100 mL) is matched, and constant temperature stirring conditions are provided, so that 0.5-3 g of dry microspheres can be stably prepared in a single batch while the particle size and fluorescence uniformity are maintained, the preparation efficiency and application expansion ability are significantly improved compared with the traditional microsphere preparation limited to microgram / milligram level, the method has good scale-up adaptability, the reaction conditions are mild, and the operation steps are standardized, so that continuous and stable synthesis of more than gram level can be realized, and a reliable preparation basis is provided for the large-scale application of fluorescent microspheres in on-site detection, biological imaging and multiple diagnosis.

[0027] The super-bright fluorescent silica microspheres based on the doping of fluorescent probes provided by the application have the comprehensive advantages of high brightness, high stability, good dispersibility and batch consistency. By efficiently embedding the fluorescent probes into the silica framework, the fluorescence signal intensity of the unit particle of the microspheres is significantly enhanced, the "raindrop effect" is effectively avoided, and the recognition quality of the fluorescence image and the reliability of the digital probe are greatly improved, thereby breaking through the problems of low brightness and uneven light emission of traditional fluorescent microspheres. By doping the fluorescent probes at a high density in the silica microspheres, the super-bright fluorescent microspheres with high brightness, low background and high light stability are constructed, compared with the traditional probes in which fluorescent dyes or quantum dots are modified on the outer surface of the microspheres, the unit signal intensity and imaging consistency are greatly improved, and the problems of fluorescence quenching, agglomeration and batch fluctuation commonly occurring in the outer surface modification are avoided; the microspheres can simultaneously carry multiple fluorescence signals, thereby improving the multiplicity and flexibility of detection, meeting the demand for multiple marker detection in complex biological detection, and serving as an ideal marker or carrier in flow cytometry, microfluidic chip and other technologies, thereby providing strong support for the development and application of these technologies and further expanding the application range. BRIEF DESCRIPTION OF DRAWINGS

[0028] Figure 1 The super-bright fluorescent silica microspheres doped with UCNPs are characterized, wherein (a) is a transmission electron microscope image of the silica microspheres with particle sizes of 0.2, 0.5 and 1 µm, and the scale is 2 µm; (b) is a transmission electron microscope image of the silica microspheres with particle sizes of 2 and 5 µm, and the scale is 5 µm, (c) is a distribution mapping diagram of Si, O, Na, Y, F and other elements in the silica microspheres, and (d) is a comparison diagram of the emission spectra of UCNPs and the doped fluorescent silica microspheres, wherein the green color represents the emission spectrum of the silica microspheres, and the red color represents the emission spectrum of UCNPs; Figure 2Characterization of Au-doped ultrabright fluorescent silicon microspheres: (a) is a transmission electron microscope image of Au, with a scale bar of 200 nm; (b) is a transmission electron microscope image of Au-doped silicon microspheres, with a scale bar of 5 µm; (c) is the absorption curve of Au and Au-doped ultrabright fluorescent silicon microspheres; orange represents the absorption curve of Au, and blue represents the absorption curve of Au-doped ultrabright fluorescent silicon microspheres. Figure 3 Characterization of AIE dye-doped ultrabright fluorescent silicon microspheres: (a) is a transmission electron microscope image of AIE dye-doped silicon microspheres with a scale bar of 5 µm; (b) is the fluorescence spectrum of AIE dye and AIE dye-doped silicon microspheres, with blue representing the fluorescence spectrum of AIE dye and red representing the fluorescence spectrum of AIE dye-doped silicon microspheres. Detailed Implementation

[0029] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0030] Unless otherwise specified, all test materials and reagents used in the following examples are commercially available. Where specific techniques or conditions are not specified in the examples, they can be performed according to the techniques or conditions described in the literature or the product instructions.

[0031] Example 1 This embodiment provides a large-scale controllable preparation method for ultrabright fluorescent silicon microspheres based on upconversion nanoparticle doping, specifically including the following steps: 1. Preparation of green fluorescent upconversion nanoparticles (UCNPs) UCNPs were synthesized using a thermal coprecipitation method to prepare β-NaYF4:Yb20%,Er2%. Acetate was dissolved at 160 °C under nitrogen protection, followed by the addition of sodium hydroxide and ammonium fluoride, and then the temperature was raised to 300 °C for reaction. The nanoparticles were then precipitated and collected. To improve fluorescence intensity, a core-shell structure method was used. β-NaYF4:Yb20%,Er2% was first used as a seed to coat the outer shell of NaYF4:Nd20%, and further processed through a high-temperature reaction to finally obtain core-shell structured nanoparticles with high fluorescence intensity.

[0032] 2. Modification and alteration of UCNPs (1) Preprocessing of UCNPs To 10 mL of the cyclohexane solution containing UCNPs, 15 mL of hydrochloric acid and 15 mL of absolute ethanol were added to obtain a mixed solution, and the mixed solution was placed in a shaker (set at 25 °C and 120 rpm) and shaken for two hours. After shaking, the solution was centrifuged at 10 500 rpm for 30 min, and the supernatant was discarded, and the precipitated UCNPs were retained. The precipitated UCNPs were added to 10 mL of deionized water and mixed for use.

[0033] (2) Amination of UCNPs In a round flask containing a magnet, 10 mL of a phosphoethanolamine (PEA) solution (concentration of 8 mg / mL) was added, and the above-mentioned UCNPs aqueous solution was slowly added to the PEA solution using a syringe pump (syringe speed of 20 mL / h). After the injection was completed, nitrogen was introduced into the system to build an inert system and was sealed. The reaction was carried out at 20 °C for 60 min to allow the UCNPs to fully react with the PEA to form an aminated structure on the surface. After the reaction was completed, the product was collected by centrifugation at 10 500 rpm for 30 min and was placed in a 4 °C refrigerator for use.

[0034] (3) Silanization The aminated UCNPs were allowed to stand at room temperature to restore the temperature to room temperature, and then 10 mL of absolute dimethylformamide (DMF) was added to the precipitate, and ultrasonic assistance was used to completely dissolve the UCNPs to obtain a DMF solution of the UCNPs. 500 μL of 3-aminopropyltriethoxysilane (APTES) was dissolved in 0.5 mL of absolute DMF to obtain a DMF solution of APTES. The DMF solution of APTES was slowly added to the DMF solution of UCNPs, and ultrasonic assistance was continuously used to completely mix the two. After the mixing was completed, nitrogen was injected into the reactor and the bottle body was wrapped with tin paper to create a light-proof environment. The silanization reaction was carried out on a stirrer at room temperature overnight. After the silanization reaction was completed, the reaction product was transferred to a centrifuge tube and centrifuged at 10 500 rpm for 30 min. The supernatant was discarded, and the precipitate was resuspended with 10 mL of absolute ethanol.

[0035] 3. Preparation of fluorescent silica microspheres (1) Preparation of super-bright fluorescent silica microspheres Silanized UCNPs pretreatment: Silanized UCNPs were taken, diluted with an equal volume of absolute ethanol, and then 1.25 mL of ammonia water was added. The mixed solution was placed on a magnetic stirrer and stirred at 240 rpm for use. Reaction system construction: in a 100 mL beaker, 0.0048 g of potassium chloride (KCl), 13.1 mL of anhydrous ethanol, 1.88 mL of water, 1.25 mL of ammonia water, and 0.35 mL of tetraethyl orthosilicate (TEOS) were sequentially added, covered with plastic wrap, and placed on a stirrer to mix evenly; The reaction system was continuously stirred, and the solution state was observed. When it was observed that the solution in the beaker completely turned milky white, 4 mL of TEOS and 10 mL of an ethanol solution of UCNPs were injected into the beaker at a speed of 1 mL / h through a double-channel injection pump. After the injection was completed, the plastic tube was promptly extracted, nitrogen was passed through the bottle opening for 30 s for deoxidation, the plastic wrap was sealed, the beaker was wrapped with tin paper, and the beaker was continuously placed on a magnetic stirrer for overnight stirring at 240 rpm to allow the reaction to proceed fully. Four hours before the end of the reaction, 1 mL of a n-butanol solution of APTES with a concentration of 0.2 mg / mL was injected into the reaction system at a speed of 1 mL / h. After the reaction was completed, the reaction solution was transferred to a centrifuge tube, centrifuged at 10 500 rpm for 30 min to collect the precipitate, and the washed product was placed in a vacuum drying oven for vacuum drying, thereby obtaining 2 g of UCNPs-doped super-bright fluorescent silica microspheres.

[0036] Example 2 On the basis of Example 1, the preparation of small-particle-size UCNPs-doped super-bright fluorescent silica microspheres (0.8-1.2 μm) was provided, and the specific steps were as follows: according to the method for preparing the reaction system in the preparation of the super-bright fluorescent silica microspheres described above, 0.0048 g of potassium chloride, 13.1 mL of anhydrous ethanol, 1.88 mL of water, and 1.25 mL of ammonia water were sequentially added to a 100 mL beaker, but the total amount of TEOS was reduced to 2 mL. Similarly, after the solution turned milky white, 4 mL of TEOS and 10 mL of an ethanol solution of UCNPs were injected into the beaker at a speed of 2 mL / h through a double-channel injection pump, and the operations after the injection were the same as those in the preparation of the super-bright fluorescent silica microspheres, i.e., the plastic tube was extracted, deoxidation was performed by passing nitrogen through the bottle opening for 30 s, the plastic wrap was sealed, the beaker was wrapped with tin paper, and overnight stirring was performed. A n-butanol solution of APTES was injected four hours before the end of the reaction, the reaction was centrifuged, washed, and dried after the reaction, thereby obtaining UCNPs-doped super-bright fluorescent silica microspheres with a particle size in the range of 0.8-1.2 μm.

[0037] Example 3 On the basis of embodiment 1, large particle size UCNPs doped super-bright fluorescent silica microspheres (up to 5 μm) are prepared, and the specific steps are as follows: 0.0048 g of potassium chloride, 13.1 mL of anhydrous ethanol, 1.88 mL of water, and 1.25 mL of ammonia water are sequentially added into a 100 mL beaker, TEOS is raised to 6 mL, and ammonia water is moderately reduced to 0.8 mL; after the solution becomes milky white, 4 mL of TEOS and 10 mL of UCNPs ethanol solution are injected into the beaker at a speed of 0.5 mL / h through a double-channel injection pump; the subsequent extraction plastic tube, nitrogen for 30 s, sealing of preservative film, wrapping with tin paper, overnight stirring, injection of APTES solution, centrifugation, washing, and drying are consistent with the above method, and finally UCNPs doped super-bright fluorescent silica microspheres with a particle size of up to 5 μm are obtained.

[0038] After the obtained silica microsphere precipitate is washed with anhydrous ethanol for three times, dry fluorescent silica microsphere solid product with a single batch yield of up to 0.5-3 g can be obtained, which is much higher than the traditional trace level Stöber method preparation process, fully meeting the requirements of high batch consistency and material supply for downstream encoding probes, functional modification or chip loading. The system has good scale-up adaptability, mild reaction conditions, and standardized operation steps, which is convenient for realizing continuous and stable synthesis of more than 1 kg, and provides a reliable preparation basis for the large-scale application of fluorescent microspheres in on-site detection, biological imaging, and multiple diagnosis.

[0039] Referring to the attached Figure 1, a shows the UCNPs doped super-bright fluorescent silica microspheres with different particle sizes (0.2, 0.5 and 1 µm) prepared by the method of embodiment 2 of the present application, indicating that the method of the present application successfully realizes the regulation of the particle size of the silica microspheres; b shows the silica microspheres with particle sizes of 2 and 5 µm prepared by the method of embodiment 3 of the present application, further proving that the preparation method can prepare silica microspheres with a larger particle size range, and all the silica microspheres have a relatively regular spherical morphology and a smooth surface, which indicates that the silica microspheres prepared by the method provided in embodiments 2-3 of the present application can form a good spherical structure, and the silica microspheres are relatively uniformly dispersed without obvious agglomeration; the element distribution mapping can be used to preliminarily judge the doping of UCNPs in the silica microspheres, and each element has a certain distribution range in the silica microspheres, indicating that UCNPs are successfully doped into the silica microspheres and achieve dispersion; the emission spectra of UCNPs and the doped fluorescent silica microspheres are compared, and both curves show multiple emission peaks, indicating that both UCNPs and the doped fluorescent silica microspheres have upconversion fluorescence characteristics, and the emission spectrum intensity of the doped fluorescent silica microspheres is significantly higher than that of UCNPs, indicating that doping UCNPs into the silica microspheres can significantly enhance the fluorescence signal. The silica microspheres as carriers can effectively enhance the fluorescence performance of UCNPs, which has important significance for improving the sensitivity and accuracy of fluorescence detection, and makes the UCNPs doped fluorescent silica microspheres have a broader application prospect in the fields of biological imaging and optical sensing.

[0040] The silica microspheres with different particle sizes and good dispersity are observed by transmission electron microscopy; the element distribution mapping confirms that UCNPs are successfully and uniformly doped into the silica microspheres; the emission spectrum comparison shows that the fluorescence performance of the doped silica microspheres is significantly enhanced, which provides strong experimental support for the application of UCNPs doped super-bright fluorescent silica microspheres in the fields of biomedicine and optical detection, and indicates that the material has potential application value.

[0041] Embodiment 4 On the basis of embodiment 1, before the reaction starts, the silanized gold nanometer Au solution is diluted with an equal volume of anhydrous ethanol and stirred at 240 rpm for standby. In a 100 mL beaker, 0.0048 g of KCl, 13.1 mL of anhydrous ethanol, 1.88 mL of water, 1.25 mL of ammonia water, 0.35 mL of TEOS are sequentially added, covered with plastic wrap, and placed on a stirrer for stirring. When it is observed that the solution has completely turned into a milky white color, the raw material injection is prepared; 4 mL of TEOS and 10 mL of Au-containing ethanol solution are injected into the beaker through a double-channel injection pump at a speed of 1 mL / h; after the injection is completed, the plastic tube is promptly extracted, nitrogen gas is passed through the bottle opening for 30 seconds to remove oxygen in the system, and then the plastic wrap is covered again, wrapped with tin paper, and continued to be stirred on the magnetic stirrer at 240 rpm overnight.

[0042] 4 hours before the end of the reaction, 1 mL of APTES solution with a concentration of 0.2 mg / mL was injected into the reaction system at a speed of 1 mL / h; after the end of the reaction, the reaction product was transferred to a centrifuge tube, collected by centrifugation (10 500 rpm, 30 min), washed three times with anhydrous ethanol, and collected by drying in a vacuum drying box until the product was completely dried, to obtain the ultra-bright silicon microspheres doped with Au fluorescent probes.

[0043] Referring to the accompanying drawings Figure 2 The Au nanoparticles exhibit a dispersed state, and the particle size is relatively uniform; the Au-doped silicon microspheres exhibit a relatively regular spherical structure, and are relatively uniformly distributed, and the size of the silicon microspheres is in the micron level, indicating that the Au nanoparticles are successfully doped into the silicon microspheres or the surface; the absorption curve of the Au-doped ultra-bright fluorescent silicon microspheres changes in position and shape compared with the absorption curve of Au, due to the interaction between the Au nanoparticles and the silicon microspheres, resulting in a change in optical properties.

[0044] Example 5 In this example, based on Example 1, before the reaction, the silanized AIE solution was diluted with an equal volume of anhydrous ethanol and stirred at 240 rpm for standby. In a 100 mL beaker, 0.0048 g of KCl, 13.1 mL of anhydrous ethanol, 1.88 mL of water, 1.25 mL of ammonia water, and 0.35 mL of TEOS were sequentially added, covered with plastic wrap, and placed on a stirrer for stirring. When the solution was observed to be completely milky white, the raw material injection was prepared; 4 mL of TEOS and 10 mL of AIE-containing ethanol solution were injected into the beaker at a speed of 1 mL / h by a double-channel injection pump; after the injection was completed, the plastic tube was promptly removed, nitrogen was passed through the bottle opening for 30 seconds to remove oxygen in the system, and then the plastic wrap was covered again, wrapped in tin paper, and continued to be stirred on the magnetic stirrer at 240 rpm overnight.

[0045] 4 hours before the end of the reaction, 1 mL of APTES solution with a concentration of 0.2 mg / mL was injected into the reaction system at a speed of 1 mL / h; after the end of the reaction, the reaction product was transferred to a centrifuge tube, collected by centrifugation (10 500 rpm, 30 min), washed three times with anhydrous ethanol, and collected by drying in a vacuum drying box until the product was completely dried, to obtain the ultra-bright silicon microspheres doped with Au fluorescent probes.

[0046] Referring to the accompanying drawings Figure 3The AIE dye-doped silicon microspheres present a relatively regular spherical shape, and are relatively uniformly distributed in the field of view. The microspheres are uniform in size and smooth in surface, without obvious agglomeration or abnormal structure. Observation of the surface morphology of the microspheres can preliminarily determine that the AIE dye is uniformly doped inside the silicon microspheres, and the surface is smooth without obvious dye aggregation, indicating that the doping is successful, and the dye is uniformly distributed in the microspheres. Compared with the fluorescence spectrum of the AIE dye, the emission peak position of the AIE dye-doped silicon microspheres is shifted, and the fluorescence intensity is significantly enhanced. The shift of the emission peak position means that there is an interaction between the AIE dye and the silicon microspheres, which changes the electronic structure of the dye molecules, thereby affecting the fluorescence emission wavelength. Since the silicon microspheres provide a limited microenvironment for the AIE dye, the motion of the dye molecules is limited, the probability of non-radiative transition is reduced, and the fluorescence quantum yield is improved. This fluorescence enhancement effect is of great significance for improving the sensitivity and accuracy of fluorescence detection, and makes the AIE dye-doped silicon microspheres have a broader application prospect in the fields of biological sensing and optical imaging.

[0047] The above characterization results not only verify that the AIE dye is successfully doped into the silicon microspheres, but also show the significant advantages of the doped silicon microspheres in fluorescence performance, providing important experimental basis for improving the fluorescence performance of silicon microspheres and expanding their applications in various fields.

[0048] In summary, the application discloses a large-scale controllable preparation technology method of super-bright silicon microspheres doped with fluorescent probes. By constructing an integrated fluorescent probe surface modification system of oleic acid removal-ammoniation-silane coating, high compatibility and high uniform doping efficiency of the system with silica sol system are realized, and the problems of traditional doping process such as fluorescence probe aggregation, uneven distribution and serious "raindrop effect" are solved. For the first time, in the sol-gel system, a synchronous dynamic dropping strategy of TEOS and fluorescent probe dispersion liquid is realized by using a double injection pump, which significantly improves the uniformity of microsphere nucleation-embedding and provides a basic guarantee for high-brightness probe construction. By systemically regulating the TEOS dosage, ammonia concentration, ethanol dilution ratio and stirring rate, the particle size can be accurately controlled in the range of 0.8-5 μm, which meets the differentiated needs of different application scenarios for the size of fluorescent microspheres. The high-yield preparation of 0.5-3 g of dried microspheres in a single batch breaks through the bottleneck of low yield and difficult scale-up of traditional microsphere synthesis methods, and has good process amplification and industrialization potential. Compared with the traditional method of modifying fluorescent dyes or quantum dots on the outer surface of microspheres, the fluorescent microspheres prepared by the method realize the complete embedding of the fluorescence source through internal doping, which not only effectively avoids the problems of dye falling off, surface quenching and background interference, but also exhibits higher brightness consistency and clear spot outline in imaging, greatly improving the image recognition friendliness and signal repeatability, and is especially suitable for high-precision digital counting, automatic image processing and stable detection requirements in complex biological systems.

[0049] The above merely illustrates the technical idea of the present application, and cannot limit the protection scope of the present application. Any modification made according to the technical idea of the present application, on the basis of the technical scheme, falls within the protection scope of the present application.

Claims

1. A method for large-scale controllable preparation of ultrabright fluorescent silicon microspheres based on fluorescent probe doping, characterized in that, include: S1, the acid-pretreated fluorescent probe is sequentially subjected to amination and silanization surface modification to obtain a surface-modified fluorescent probe; S2, construct a reaction system containing potassium chloride, anhydrous ethanol, water, ammonia, and tetraethyl orthosilicate; S3, under nitrogen protection, the surface-modified fluorescent probe solution and tetraethyl orthosilicate were simultaneously injected into the reaction system, stirred and reacted, and then 3-aminopropyltriethoxysilane solution was injected to continue the reaction. After centrifugation, washing and drying, fluorescent probe-doped ultrabright fluorescent silicon microspheres were obtained.

2. The method for large-scale controllable preparation of ultrabright fluorescent silicon microspheres based on fluorescent probe doping according to claim 1, characterized in that, The fluorescent probe is any one of upconversion nanoparticles, colloidal gold nanoparticles, aggregation-induced emission dyes, quantum dots, fluorescent dyes, and fluorescent proteins.

3. The method for large-scale controllable preparation of ultrabright fluorescent silicon microspheres based on fluorescent probe doping according to claim 1, characterized in that, In S1, the amination specifically includes: pretreating the fluorescent probe with surface acidification, reacting the pretreated fluorescent probe with an amination reagent, centrifuging, washing, and drying after the reaction to obtain the amination-modified fluorescent probe; the amination reagent is any one of ethanolamine phosphate, amino-modified polyethylene glycol, and aminosilane reagents.

4. The method for large-scale controllable preparation of ultrabright fluorescent silicon microspheres based on fluorescent probe doping according to claim 3, characterized in that, The molar ratio of the fluorescent probe to the amination reagent is 1:5~15, the reaction temperature is 280~320 ℃, and the reaction time is 1~5 h.

5. The method for large-scale controllable preparation of ultrabright fluorescent silicon microspheres based on fluorescent probe doping according to claim 1, characterized in that, In S1, the silanization specifically includes mixing the aminated fluorescent probe with 3-aminopropyltriethoxysilane under nitrogen protection and reacting in the dark, followed by centrifugation, washing, and resuspending to obtain the silanized fluorescent probe.

6. The method for large-scale controllable preparation of ultrabright fluorescent silicon microspheres based on fluorescent probe doping according to claim 5, characterized in that, The molar ratio of the aminated upconversion nanoparticles to 3-aminopropyltriethoxysilane is 1:1~2, the reaction temperature is 10~20 ℃, and the reaction time is 15~25 h.

7. The method for large-scale controllable preparation of ultrabright fluorescent silicon microspheres based on fluorescent probe doping according to claim 1, characterized in that, In S2, the reaction system is obtained by sequentially mixing potassium chloride, anhydrous ethanol, water, ammonia, and TEOS; the mass ratio of potassium chloride, anhydrous ethanol, water, ammonia, and tetraethyl orthosilicate is 1: 35~40: 10~20: 7~8: 8~9.

8. The method for large-scale controllable preparation of ultrabright fluorescent silicon microspheres based on fluorescent probe doping according to claim 1, characterized in that, In S3, the volume ratio of the surface-modified fluorescent probe solution to tetraethyl orthosilicate is 1:2~3, the injection rate is 0.5~2 mL / h, the stirring speed is 200~300 rpm, and the reaction time is 10~14 h.

9. The method for large-scale controllable preparation of ultrabright fluorescent silicon microspheres based on fluorescent probe doping according to claim 1, characterized in that, In S3, the volume ratio of the 3-aminopropyltriethoxysilane solution to tetraethyl orthosilicate is 1:3~5, the injection rate is 0.5~2 mL / h, and the injection time is 3~4 hours before the end of the reaction.

10. Ultrabright fluorescent silicon microspheres doped with fluorescent probes obtained by a large-scale controllable preparation method according to any one of claims 1 to 9.

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