Preparation method of high monodisperse micron-sized silica microspheres, microspheres and chip
By adding stabilizers and inorganic salts in a specific order and adding silicon source monomers in two stages at a constant flow rate, highly monodisperse micron-sized silica microspheres were prepared. This method solves the problems of uneven microsphere particle size and difficulty in mass production in existing technologies, and achieves the preparation of highly monodisperse and low-cost microspheres. When applied to solid-phase chips, it exhibits excellent customized site conversion rate and low cost.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- SUZHOU LASSO BIOCHIP TECH CO LTD
- Filing Date
- 2026-05-07
- Publication Date
- 2026-06-05
AI Technical Summary
Existing technologies struggle to prepare micron-sized silica microspheres with high monodispersity, low cost, and ease of mass production. Existing methods suffer from limitations in particle size range, poor monodispersity, or difficulty in mass production.
By adding stabilizers and inorganic salts in a specific order, adding silicon source monomers in two batches at a constant flow rate, controlling reaction conditions, using mechanical stirring to form a laminar flow field, and precisely controlling the feeding time and speed, highly monodisperse micron-sized silica microspheres can be prepared.
Micron-sized silica microspheres with uniform particle size and excellent monodispersity were successfully prepared. When applied to solid-phase chips, they exhibited higher conversion rates for customized sites and lower costs. Moreover, the preparation process was mild and reproducible.
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Figure CN122144747A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of chip fabrication, and more particularly to a method for preparing highly monodisperse micron-sized silica microspheres, the microspheres, their application in solid-state chip fabrication, and chips fabricated using the microspheres. Background Technology
[0002] Silica microspheres, as an important inorganic material, are widely used in biomedicine, fillers, coatings, cosmetics, electronic packaging, optics and biochips due to their unique physicochemical properties, such as low dielectric constant, low density, easy chemical modification, high specific surface area, excellent thermal stability and strong chemical stability.
[0003] Common methods for preparing silica microspheres include sol-gel method, microemulsion method, precipitation method, and seed growth method. Stöber et al. (Stöber W, Fink A, Ernst Bohn. Controlled growth of monodisperse silica spheres in the micron size range [J]. Journal of Colloid & Interface Science, 1968, 26, 1, 62-69) first disclosed a method for preparing silica microspheres via a sol-gel process. This method uses tetraethyl orthosilicate as the silicon source and ammonia as the catalyst, directly hydrolyzing and condensing the silicon source to synthesize silica microspheres in a one-step reaction. However, the microspheres prepared by this method have a small particle size, making it difficult to reach the micron scale, and the product is prone to agglomeration.
[0004] The invention patent with publication number CN118908232A uses diethanolamine to prepare an alkaline solution and adjusts the solution environment to catalyze the hydrolysis reaction of tetraethyl orthosilicate. It also controls the particle size of silica microspheres by changing the amount of surfactant (CTAB) in the reaction system. However, as can be seen from its examples, even slight changes in the surfactant content can lead to problems such as microsphere agglomeration and irregular surface morphology. In addition, if the CTAB template is not completely removed by calcination, carbon residue will result, causing the microspheres to turn yellow / black. Furthermore, the method has a long settling time and low production efficiency.
[0005] Fujiwara et al. (Fujiwara M, Shiokawa K, Tanaka Y, et al. Preparation and formation mechanism of silica microcapsules (hollow sphere) by water / oil / water in terfacial reaction [J]. Chemistry of Materials, 2004, 16, 25, 5420-5426) disclosed a method for preparing silica microspheres via a microemulsion method. This method uses sodium silicate as the silica source and forms a W / O / W emulsion system with a surfactant. By changing the silica source concentration and stirring speed, silica microspheres were successfully prepared. However, the microspheres prepared by this method have a wide particle size distribution and must undergo strict screening before use.
[0006] Zheng Jing et al. (Zheng Jing, Lin Yinqin. Synthesis of ultrafine silica by chemical precipitation method [J]. Bulletin of the Chinese Ceramic Society, 2016, 35(09), 2941-2945) disclosed a method for preparing silica microspheres by precipitation. The method uses sodium silicate as the silicon source and ethyl acetate as the acidifying agent. High-purity silica microspheres were successfully prepared by calcination at 600℃ for 4 hours. However, the microspheres prepared by this method are mostly irregular in morphology, have poor uniformity, poor repeatability and low yield.
[0007] Invention patents with publication numbers CN104724714A and CN115477307A disclose methods for preparing silica microspheres by seed growth. These methods first use the Stöber method to prepare nanoscale silica microspheres as seeds, and then continue to add silicon sources to eventually grow the nanoscale silica microspheres to the micrometer scale, thereby making the microsphere particle size controllable. However, this method is prone to secondary nucleation and aggregation of microspheres, requiring purification before further application, thus limiting its mass production.
[0008] In summary, although current technologies have made some progress in the preparation of monodisperse micron-sized silica, existing methods have significant limitations: the synthesized silica microspheres either have limited particle size ranges, poor monodispersity, or are difficult to mass-produce, making it difficult to meet the demands of solid-state chips and other applications for high monodispersity and low cost. Therefore, there is an urgent need to develop a method for preparing silica microspheres that offers high monodispersity, low cost, and ease of mass production. Summary of the Invention
[0009] Therefore, to solve the above problems, the present invention provides a method for preparing highly monodisperse micron-sized silica microspheres, as well as the microspheres and chips.
[0010] This invention is achieved through the following technical solution: A method for preparing highly monodisperse micron-sized silica microspheres includes the following steps: S1: An alkaline hydrolysate is obtained by mixing organic solvent, alkaline catalyst, ultrapure water and stabilizer evenly. S2: A certain mass of silicon source monomer is dispersed in an organic solvent to form a monomer solvent; S3: Under stirring conditions, the monomer solvent is added to the alkaline hydrolysate in two portions at a preset constant flow rate. The first addition of the monomer solvent takes 10 to 25 minutes. During the interval between the two additions of the monomer solvent, a certain mass of inorganic salt is added and stirred evenly. After reaction and aging, a silica dispersion is obtained. S4: The silica dispersion was centrifuged, washed, and freeze-dried to obtain micron-sized silica microspheres.
[0011] Preferably, in step S3, the monomer solvent is added to the alkaline hydrolysate at a constant flow rate of 80 mL / min to 120 mL / min using a microfluidic device.
[0012] Preferably, in step S3, the monomer solvent is added for the first time and then paused after 10 to 25 minutes; a certain mass of inorganic salt is added and stirred evenly, and then the monomer solvent is added a second time until all of it is added; then the reaction is continued to be stirred and aged to obtain a silica dispersion.
[0013] Preferably, in step S3, after the monomer solvent is completely added, the reaction continues for 3 to 5 hours at a stirring rate of 350 rpm to 750 rpm, and then aged for 5 to 7 hours to obtain a silica dispersion.
[0014] Preferably, in step S1, the organic solvent, alkaline catalyst, ultrapure water, and stabilizer are placed in a reaction vessel, the temperature of the reaction vessel is maintained at 25℃~50℃, and the mixture is mechanically stirred at a speed of 350rpm~750rpm to make it uniformly mixed into an alkaline hydrolysate.
[0015] Preferably, the organic solvent is one of methanol, ethanol, isopropanol, and n-propanol; the alkaline catalyst is one of sodium hydroxide, potassium hydroxide, and ammonium hydroxide; the stabilizer is one of polyethylene glycol, polyvinyl alcohol, and polyvinylpyrrolidone; and the silicon source monomer is one of methyl orthosilicate and tetraethyl orthosilicate.
[0016] Preferably, the inorganic salt is one of sodium chloride, ammonium chloride, potassium chloride, ammonium nitrate, sodium nitrate, and potassium nitrate.
[0017] Preferably, in the silica dispersion, the concentration of the organic solvent is 11 mol / L to 29 mol / L; the concentration of the alkaline catalyst is 0.8 mol / L to 3 mol / L; the concentration of the ultrapure water is 4 mol / L to 12 mol / L (excluding the amount of water in the ammonia solution); the concentration of the stabilizer is 0.02 mmol / L to 0.2 mmol / L; the concentration of the inorganic salt is 1.5 mmol / L to 5.6 mmol / L; and the concentration of the silicon source monomer is 0.3 mol / L to 0.7 mol / L.
[0018] Highly monodisperse micron-sized silica microspheres, used in solid-state chip fabrication, are prepared using the method described above.
[0019] The chip comprises highly monodisperse micron-sized silica microspheres as described above.
[0020] The beneficial effects of the technical solution of this invention are mainly reflected in: 1. This invention adds stabilizers and inorganic salts in a specific order, so that the inorganic salts and stabilizers work synergistically. The inorganic salts can reduce the electrostatic repulsion between particles, promote collision and growth, and effectively increase the particle size to the micron level. The stabilizers guide the uniform growth of particles, ensuring the sphericity and particle size uniformity of microspheres. This invention overcomes the defects of adding inorganic salts alone, which can easily lead to the aggregation of microspheres, or adding stabilizers alone, which can lead to the microspheres having a small particle size. It successfully prepares micron-sized silica microspheres with excellent monodispersity.
[0021] 2. This invention uses a preset speed to add the monomer solvent to the alkaline hydrolysate in two separate additions at a constant flow rate, achieving precise and stable feeding. This effectively avoids excessively high local concentrations of silicon source monomers, thereby significantly reducing heterogeneous nucleation phenomena and ensuring uniform and controllable particle growth. This is beneficial for obtaining silica microspheres with narrow particle size distribution. The laminar flow field formed by mechanical stirring provides gentle shear force, which promotes uniform dispersion and mass transfer of reactants in the system without damaging the spherical structure of the generated silica microspheres, thus ensuring the integrity and surface smoothness of the microspheres.
[0022] 3. This invention ensures that the reaction system achieves growth promotion at the appropriate stage by precisely controlling the addition time of inorganic salts, avoiding the formation of a large number of small-diameter microspheres or irregular aggregates in the later stage due to the addition of inorganic salts too early or too late, thus significantly improving the stability of the product. At the same time, the step-by-step feeding method and strict control of the feeding time and feeding speed achieve fine control of the nucleation and growth stages, making the entire preparation process mild, reproducible, and easy to mass-produce.
[0023] 4. The highly monodisperse micron-sized silica microspheres prepared by this invention exhibit excellent performance in solid-phase chip fabrication. When applied to solid-phase chip fabrication, their surface properties after probe fixation differ from existing products. They also exhibit lower adsorption during the quality inspection process, resulting in improved conversion rate of customized sites, as well as higher efficiency and lower cost. Attached Figure Description
[0024] Figure 1 This is a scanning electron microscope image of the highly monodisperse micron-sized silica microspheres prepared in Example 1; Figure 2 This is a scanning electron microscope image of the highly monodisperse micron-sized silica microspheres prepared in Example 2; Figure 3 This is a scanning electron microscope image of the highly monodisperse micron-sized silica microspheres prepared in Example 3; Figure 4 This is a scanning electron microscope image of the highly monodisperse micron-sized silica microspheres prepared in Example 4; Figure 5 This is a scanning electron microscope image of the highly monodisperse micron-sized silica microspheres prepared in Example 5; Figure 6 This is a scanning electron microscope image of the highly monodisperse micron-sized silica microspheres prepared in Example 6; Figure 7 This is a scanning electron microscope image of the silica microspheres prepared by Comparative Example 1; Figure 8 This is a scanning electron microscope image of the silica microspheres prepared by Comparative Example 2; Figure 9 This is a scanning electron microscope image of the silica microspheres prepared by Comparative Example 3; Figure 10 This is a scanning electron microscope image of the silica microspheres prepared by Comparative Example 4; Figure 11 This is a scanning electron microscope image of the silica microspheres prepared by Comparative Example 5; Figure 12 This is a scanning electron microscope image of the silica microspheres prepared by Comparative Example 6; Figure 13 This is a flowchart of a method for preparing highly monodisperse silica microspheres. Detailed Implementation
[0025] To make the objectives, advantages, and features of the present invention clearer and more detailed, the following non-limiting description of preferred embodiments will be illustrated and explained. The following embodiments are merely typical examples of applying the technical solutions of the present invention; any technical solutions formed by equivalent substitutions or equivalent transformations fall within the scope of protection claimed by the present invention.
[0026] I. Preparation of highly monodisperse micron-sized silica microspheres: Example 1 This embodiment provides a method for preparing highly monodisperse silica microspheres, including the following steps: S1: 10L of anhydrous methanol, 1.58L of ammonia (28% by mass), 2.2L of ultrapure water, and 6.5g of polyethylene glycol (molecular weight 4000) were added sequentially into a glass reactor with a liner. The reactor temperature was maintained at 40℃, and the mixture was mechanically stirred at 500rpm for 30min until homogeneous to obtain an alkaline hydrolysate. S2: Add 5L of anhydrous methanol and 1.5L of methyl orthosilicate to another container in sequence, and disperse evenly by ultrasonication to obtain the monomer solvent; S3: Under stirring conditions, the monomer solvent is added to the reactor at a constant flow rate of 90 mL / min using a microfluidic device. After 20 min, the feeding is stopped, 3.3 g of sodium chloride is added, and the mixture is stirred evenly. The monomer solvent is then added at a constant flow rate of 90 mL / min until all the solvent is added. After reacting for 3 h, the mixture is allowed to stand and age for 5 h to obtain a silica microsphere dispersion. S4: The silica microsphere dispersion is centrifuged, washed, and freeze-dried to obtain the following: Figure 1 The highly monodisperse silica microspheres shown have an average particle size of 1.46 μm and high particle size uniformity.
[0027] Example 2 This embodiment provides a method for preparing highly monodisperse silica microspheres, including the following steps: S1: 10L of anhydrous ethanol, 1.58L of ammonia (28% by mass), 2.2L of ultrapure water, and 6.5g of polyethylene glycol (molecular weight 4000) were added sequentially into a glass reactor with a liner. The reactor temperature was maintained at 40℃, and the mixture was mechanically stirred at 500rpm for 30min until homogeneous to obtain an alkaline hydrolysate. S2: Add 5L of anhydrous ethanol and 1.5L of methyl orthosilicate to another container in sequence, and disperse evenly by ultrasonication to obtain the monomer solvent; S3: Under stirring conditions, the monomer solvent is added to the reactor at a constant flow rate of 90 mL / min using a microfluidic device. After 20 min, the feeding is stopped, 3.3 g of sodium chloride is added, and the mixture is stirred evenly. The monomer solvent is then added at a constant flow rate of 90 mL / min until all the solvent is added. After reacting for 3 h, the mixture is allowed to stand and age for 5 h to obtain a silica microsphere dispersion. S4: The silica microsphere dispersion is centrifuged, washed, and freeze-dried to obtain the following: Figure 2The highly monodisperse silica microspheres shown have an average particle size of 1.64 μm and a high degree of uniformity in particle size.
[0028] Example 3 This embodiment provides a method for preparing highly monodisperse silica microspheres, including the following steps: S1: 10L of anhydrous methanol, 1.58L of ammonia (28% by mass), 2.2L of ultrapure water, and 6.5g of polyethylene glycol (molecular weight 4000) were added sequentially into a glass reactor with a liner. The reactor temperature was maintained at 40℃, and the mixture was mechanically stirred at 500rpm for 30min until homogeneous to obtain an alkaline hydrolysate. S2: Add 5L of anhydrous methanol and 1.5L of tetraethyl orthosilicate to another container in sequence, and disperse evenly by ultrasonication to obtain the monomer solvent; S3: Under stirring conditions, the monomer solvent is added to the reactor at a constant flow rate of 90 mL / min using a microfluidic device. After 20 min, the feeding is stopped, 3.3 g of sodium chloride is added, and the mixture is stirred evenly. The monomer solvent is then added at a constant flow rate of 90 mL / min until all the solvent is added. After reacting for 3 h, the mixture is allowed to stand and age for 5 h to obtain a silica microsphere dispersion. S4: The silica microsphere dispersion is centrifuged, washed, and freeze-dried to obtain the following: Figure 3 The highly monodisperse silica microspheres shown have an average particle size of 1.51 μm and a high degree of uniformity in particle size.
[0029] Example 4 This embodiment provides a method for preparing highly monodisperse silica microspheres, including the following steps: S1: 10L of anhydrous ethanol, 1.58L of ammonia (28% by mass), 2.2L of ultrapure water, and 6.5g of polyethylene glycol (molecular weight 4000) were added sequentially into a glass reactor with a liner. The reactor temperature was maintained at 40℃, and the mixture was mechanically stirred at 500rpm for 30min until homogeneous to obtain an alkaline hydrolysate. S2: Add 5L of anhydrous ethanol and 1.5L of tetraethyl orthosilicate to another container in sequence, and disperse evenly by ultrasonication to obtain the monomer solvent; S3: Under stirring conditions, the monomer solvent is added to the reactor at a constant flow rate of 90 mL / min using a microfluidic device. After 20 min, the feeding is stopped, 3.3 g of sodium chloride is added, and the mixture is stirred evenly. The monomer solvent is then added at a constant flow rate of 90 mL / min until all the solvent is added. After reacting for 3 h, the mixture is allowed to stand and age for 5 h to obtain a silica microsphere dispersion. S4: The silica microsphere dispersion is centrifuged, washed, and freeze-dried to obtain the following: Figure 4 The highly monodisperse silica microspheres shown have an average particle size of 1.26 μm and high uniformity.
[0030] Example 5 This embodiment provides a method for preparing highly monodisperse silica microspheres, including the following steps: S1: 10L of anhydrous ethanol, 1.58L of ammonia (28% by mass), 2.2L of ultrapure water, and 31.7g of polyvinyl alcohol (molecular weight 30000) were added sequentially into a glass reactor with a liner. The reactor temperature was maintained at 40℃, and the mixture was mechanically stirred at 500rpm for 30min until homogeneous to obtain an alkaline hydrolysate. S2: Add 5L of anhydrous ethanol and 1.5L of tetraethyl orthosilicate to another container in sequence, and disperse evenly by ultrasonication to obtain the monomer solvent; S3: Under stirring conditions, the monomer solvent is added to the reactor at a constant flow rate of 90 mL / min using a microfluidic device. After 20 min, the feeding is stopped, 3.3 g of sodium chloride is added, and the mixture is stirred evenly. The monomer solvent is then added at a constant flow rate of 90 mL / min until all the solvent is added. After reacting for 3 h, the mixture is allowed to stand and age for 5 h to obtain a silica microsphere dispersion. S4: The silica microsphere dispersion is centrifuged, washed, and freeze-dried to obtain the desired product. Figure 5 The highly monodisperse silica microspheres shown have an average particle size of 1.22 μm and a high degree of uniformity in particle size.
[0031] Example 6 This embodiment provides a method for preparing highly monodisperse silica microspheres, including the following steps: S1: 10L of anhydrous ethanol, 1.58L of ammonia (28% by mass), 2.2L of ultrapure water, and 42.2g of polyvinylpyrrolidone (molecular weight 40000) were added sequentially into a lined glass reactor. The reactor temperature was maintained at 40℃, and the mixture was mechanically stirred at 500rpm for 30min until homogeneous to obtain an alkaline hydrolysate. S2: Add 5L of anhydrous ethanol and 1.5L of tetraethyl orthosilicate to another container in sequence, and disperse evenly by ultrasonication to obtain the monomer solvent; S3: Under stirring conditions, the monomer solvent is added to the reactor at a constant flow rate of 90 mL / min using a microfluidic device. After 20 min, the feeding is stopped, 3.3 g of sodium chloride is added, and the mixture is stirred evenly. The monomer solvent is then added at a constant flow rate of 90 mL / min until all the solvent is added. After reacting for 3 h, the mixture is allowed to stand and age for 5 h to obtain a silica microsphere dispersion. S4: The silica microsphere dispersion is centrifuged, washed, and freeze-dried to obtain the following: Figure 6 The highly monodisperse silica microspheres shown have an average particle size of 1.14 μm and a high degree of uniformity in particle size.
[0032] Comparative Example 1 This comparative example provides a method for preparing silica microspheres, including the following steps: S1: 10L of anhydrous ethanol, 1.58L of ammonia (28% by mass) and 2.2L of ultrapure water are added sequentially to a glass reactor with a liner. The reactor temperature is maintained at 40℃ and the mixture is mechanically stirred at 500rpm for 30min until homogeneous to obtain an alkaline hydrolysate. S2: Add 5L of anhydrous ethanol and 1.5L of methyl orthosilicate to another container in sequence, and disperse evenly by ultrasonication to obtain the monomer solvent; S3: Under stirring conditions, the monomer solvent is added to the reactor at a constant flow rate of 90 mL / min using a microfluidic device. After 20 min, the feeding is stopped, 3.3 g of sodium chloride is added, and the mixture is stirred evenly. The monomer solvent is then added at a constant flow rate of 90 mL / min until all the solvent is added. After reacting for 3 h, the mixture is allowed to stand and age for 5 h to obtain a silica microsphere dispersion. S4: The silica microsphere dispersion is centrifuged, washed, and freeze-dried to obtain the following: Figure 7 The silica microspheres shown; Compared with the preparation method of Example 2, the difference of Comparative Example 1 is that no stabilizer was added. The silica microspheres prepared in Comparative Example 1 have an average particle size of 1.50 μm, but there are many irregular multinucleated aggregates.
[0033] Comparative Example 2 This comparative example provides a method for preparing silica microspheres, including the following steps: S1: 10L of anhydrous ethanol, 1.58L of ammonia (28% by mass) and 2.2L of ultrapure water are added sequentially to a glass reactor with a liner. The reactor temperature is maintained at 40℃ and the mixture is mechanically stirred at 500rpm for 30min until homogeneous to obtain an alkaline hydrolysate. S2: Add 5L of anhydrous ethanol and 1.5L of methyl orthosilicate to another container in sequence, and disperse evenly by ultrasonication to obtain the monomer solvent; S3: Under stirring conditions, the monomer solvent is added to the reactor at a constant flow rate of 90 mL / min using a microfluidic device until all solvent is added. After reacting for 3 hours, the mixture is allowed to stand and age for 5 hours to obtain a silica microsphere dispersion. S4: The silica microsphere dispersion is centrifuged, washed, and freeze-dried to obtain the following: Figure 8 The silica microspheres shown; Compared with the preparation method of Example 2, the difference of Comparative Example 2 is that no stabilizer and inorganic salt were added. Therefore, the average particle size of the silica microspheres prepared in Comparative Example 2 is 528.2 nm, which is relatively small, with uneven morphology, poor dispersibility, and a large number of irregular particles and multinucleated aggregates.
[0034] Comparative Example 3 This comparative example provides a method for preparing silica microspheres, including the following steps: S1: 10L of anhydrous ethanol, 1.58L of ammonia (28% by mass), 2.2L of ultrapure water, and 42.2g of polyvinylpyrrolidone (molecular weight 40000) were added sequentially into a lined glass reactor. The reactor temperature was maintained at 40℃, and the mixture was mechanically stirred at 500rpm for 30min until homogeneous to obtain an alkaline hydrolysate. S2: Add 5L of anhydrous ethanol and 1.5L of methyl orthosilicate to another container in sequence, and disperse evenly by ultrasonication to obtain the monomer solvent; S3: Under stirring conditions, the monomer solvent is added to the reaction vessel at a constant flow rate of 90 mL / min using a microfluidic device, along with 3.3 g of sodium chloride. The mixture is stirred until homogeneous, and the monomer solvent is added at a constant flow rate of 90 mL / min until all the solvent is added. After reacting for 3 hours, the mixture is allowed to stand and age for 5 hours to obtain a silica microsphere dispersion. S4: The silica microsphere dispersion is centrifuged, washed, and freeze-dried to obtain the following: Figure 9 The silica microspheres shown; Compared with the preparation method of Example 6, the difference of Comparative Example 3 is that the inorganic salt and monomer solvent are added to the alkaline aqueous solution at the same time. Therefore, the average particle size of the silica microspheres prepared in Comparative Example 3 is 1.35 μm, but a large number of small-sized microspheres and a large number of multinucleated aggregates are generated.
[0035] Comparative Example 4 This comparative example provides a method for preparing silica microspheres, including the following steps: S1: 10L of anhydrous ethanol, 1.58L of ammonia (28% by mass), 2.2L of ultrapure water, and 42.2g of polyvinylpyrrolidone (molecular weight 40000) were added sequentially into a lined glass reactor. The reactor temperature was maintained at 40℃, and the mixture was mechanically stirred at 500rpm for 30min until homogeneous to obtain an alkaline hydrolysate. S2: Add 5L of anhydrous ethanol and 1.5L of methyl orthosilicate to another container in sequence, and disperse evenly by ultrasonication to obtain the monomer solvent; S3: Under stirring conditions, the monomer solvent is added to the reactor at a constant flow rate of 90 mL / min using a microfluidic device. After 5 min, the feeding is stopped, 3.3 g of sodium chloride is added, and the mixture is stirred evenly. The monomer solvent is then added at a constant flow rate of 90 mL / min until all the solvent is added. After reacting for 3 h, the mixture is allowed to stand and age for 5 h to obtain a silica microsphere dispersion. S4: The silica microsphere dispersion is centrifuged, washed, and freeze-dried to obtain the following: Figure 10 The silica microspheres shown; Compared with the preparation method of Example 6, the difference in Comparative Example 4 is that the inorganic salt was added 5 minutes after the monomer solvent was added. At this time, the average particle size of the silica microspheres prepared in Comparative Example 4 was 1.29 μm, but a large number of small-diameter microspheres and a small amount of polynuclear aggregates were generated.
[0036] Comparative Example 5 This comparative example provides a method for preparing silica microspheres, including the following steps: S1: 10L of anhydrous ethanol, 1.58L of ammonia (28% by mass), 2.2L of ultrapure water, and 42.2g of polyvinylpyrrolidone (molecular weight 40000) were added sequentially into a lined glass reactor. The reactor temperature was maintained at 40℃, and the mixture was mechanically stirred at 500rpm for 30min until homogeneous to obtain an alkaline hydrolysate. S2: Add 5L of anhydrous ethanol and 1.5L of methyl orthosilicate to another container in sequence, and disperse evenly by ultrasonication to obtain the monomer solvent; S3: Under stirring conditions, the monomer solvent is added to the reactor at a constant flow rate of 90 mL / min using a microfluidic device. After 10 min, the feeding is stopped, 3.3 g of sodium chloride is added, and the mixture is stirred evenly. The monomer solvent is then added at a constant flow rate of 90 mL / min until all the solvent is added. After reacting for 3 h, the mixture is allowed to stand and age for 5 h to obtain a silica microsphere dispersion. S4: The silica microsphere dispersion is centrifuged, washed, and freeze-dried to obtain the following: Figure 11 The silica microspheres shown; Compared with the preparation method of Example 6, the difference in Comparative Example 5 is that the inorganic salt was added 10 min after the monomer solvent was added. At this time, the average particle size of the silica microspheres prepared in Comparative Example 5 was 1.15 μm, but a small number of small-sized microspheres were generated.
[0037] Comparative Example 6 This comparative example provides a method for preparing silica microspheres, including the following steps: S1: 10L of anhydrous ethanol, 1.58L of ammonia (28% by mass), 2.2L of ultrapure water, and 42.2g of polyvinylpyrrolidone (molecular weight 40000) were added sequentially into a lined glass reactor. The reactor temperature was maintained at 40℃, and the mixture was mechanically stirred at 500rpm for 30min until homogeneous to obtain an alkaline hydrolysate. S2: Add 5L of anhydrous ethanol and 1.5L of methyl orthosilicate to another container in sequence, and disperse evenly by ultrasonication to obtain the monomer solvent; S3: Under stirring conditions, the monomer solvent is added to the reactor at a constant flow rate of 90 mL / min using a microfluidic device. After 30 min, the feeding is stopped, 3.3 g of sodium chloride is added, and the mixture is stirred evenly. The monomer solvent is then added at a constant flow rate of 90 mL / min until all the solvent is added. After reacting for 3 h, the mixture is allowed to stand and age for 5 h to obtain a silica microsphere dispersion. S4: The silica microsphere dispersion is centrifuged, washed, and freeze-dried to obtain the following: Figure 12 The silica microspheres shown; Compared with the preparation method of Example 6, the difference in Comparative Example 6 is that the inorganic salt was added 30 min after the monomer solvent was added. At this time, the average particle size of the silica microspheres prepared in Comparative Example 6 was 1.0 μm, but a large number of small-sized microspheres and a lot of irregular agglomerates were generated.
[0038] In the above embodiments and comparative examples, the centrifugation, washing, and freeze-drying operations refer to: centrifuging the silica microspheres at 5000 rpm for 5 min and removing the supernatant; then adding deionized water and shaking for 5 min, centrifuging again to remove the supernatant, repeating this process twice, for a total of 3 times. At this time, the pH value of the supernatant is measured to be 6.8-7.2. Then, an appropriate amount of deionized water is added to the microspheres, shaken evenly, and stored at -20 degrees Celsius for 24 hours, and then placed in a freeze dryer for freeze-drying for 24 hours. In some other embodiments, other existing centrifugation, washing, and freeze-drying methods can also be used, which will not be described in detail here.
[0039] II. Application and Effect Comparison of Highly Monodisperse Micron-sized Silica Microspheres on Solid-Phase Chips: The highly monodisperse silica microspheres prepared according to the preparation methods of Examples 4, 5, and 6 above were used in the fabrication of solid-state chips, along with two commercially available silica microsphere products. The two commercially available silica microsphere products include Product 1 (purchased from Bangs Laboratories, Inc., brand name SS04000) and Product 2 (purchased from Suzhou Zhiyi Microsphere Technology Co., Ltd., brand name KBsphere®1Si).
[0040] In one embodiment, the highly monodisperse silica microspheres prepared in Examples 4, 5, and 6, as well as Products 1 and 2, were applied to solid-phase gene chip fabrication according to the following steps: Step A: Hydrophobic treatment of monocrystalline silicon substrate: First, a dense silicon nitride (SiN) layer with a thickness of about 1 μm is deposited on the monocrystalline silicon substrate.
[0041] Step B: Etching: After transferring the pattern from the photomask to the surface of the silicon nitride-coated monocrystalline silicon substrate using photoresist, plasma etching is used to transfer the pattern from the photoresist surface to the silicon nitride layer again, exposing the monocrystalline silicon substrate directly to the air. Excess photoresist on the silicon substrate surface is then removed using chemical methods. The etched silicon substrate is rinsed three times with copious amounts of deionized water, then cleaned three times with analytical grade ethanol, and finally air-dried with nitrogen (99.99% pure) for later use.
[0042] Step C: Loading silica microspheres: Inject the pretreated solid silica microspheres into analytical grade ultrapure water and prepare a monodisperse silica solution with a concentration of 0.1 mg / mL by ultrasonic dispersion (2 h, 80 W). Place the solution in a magnetically stirred container and maintain a certain flow rate of 2 rpm to ensure that the silica microspheres are uniformly distributed in the solution and do not sink to the bottom. Place the device on an air cushion for vibration isolation to prevent vibrations in the room from being transmitted to the device.
[0043] The single-crystal silicon plate was cut into the standard size of a glass slide (75mm*25mm*1.00mm), and the cut silicon plate was vertically inserted into a container containing a silica gel solution, so that the silicon plate was completely submerged in the silica gel solution. A large beaker was used to cover the device to prevent airflow and isolate dust. At the same time, the solution in the device was heated to 50°C and the liquid level was allowed to drop to a specified height H (the specified height H means that there are no etched holes on the chip surface below H). The silicon plate was then removed, rinsed with a large amount of deionized water, then rinsed with analytical grade ethanol, and dried with nitrogen. The above operation was repeated 3 times to obtain a solid-phase gene chip.
[0044] According to the above steps, chips 1, 2, and 3 were prepared using the highly monodisperse silica microspheres obtained in Examples 4, 5, and 6, respectively; and chips 4 and 5 were prepared using products 1 and 2. The conversion rates of customized sites of each chip were compared through chip quality inspection. Based on multiple comparisons, the conversion rates of customized sites of chips 1, 2, 3, 4, and 5 were 89%, 89%, 90%, 85%, and 87%, respectively. It can be seen that the highly monodisperse silica microspheres prepared by the present invention are superior to commercially available microspheres in terms of customized site conversion rate. This may be because the surface properties of the highly monodisperse silica microspheres prepared by the present invention are somewhat different from those of commercially available silica microspheres after the probe is fixed, resulting in lower adsorption during the quality inspection process.
[0045] In other embodiments, the highly monodisperse silica microspheres disclosed in this invention can also be used in the fabrication of solid-phase gene chips through other existing chip preparation methods, which will not be elaborated here.
[0046] This invention has many other embodiments, and all technical solutions formed by equivalent transformations or equivalent transformations fall within the protection scope of this invention.
Claims
1. A method for preparing highly monodisperse micron-sized silica microspheres, characterized in that: Includes the following steps: S1: An alkaline hydrolysate is obtained by mixing organic solvent, alkaline catalyst, ultrapure water and stabilizer evenly. S2: A certain mass of silicon source monomer is dispersed in an organic solvent to form a monomer solvent; S3: Under stirring conditions, the monomer solvent is added to the alkaline hydrolysate in two portions at a preset constant flow rate. The first addition of the monomer solvent takes 10 to 25 minutes. During the interval between the two additions of the monomer solvent, a certain mass of inorganic salt is added and stirred evenly. After reaction and aging, a silica dispersion is obtained. S4: The silica dispersion was centrifuged, washed, and freeze-dried to obtain micron-sized silica microspheres.
2. The method for preparing highly monodisperse micron-sized silica microspheres according to claim 1, characterized in that: In step S3, the monomer solvent is added to the alkaline hydrolysate at a constant flow rate of 80 mL / min to 120 mL / min.
3. The method for preparing highly monodisperse micron-sized silica microspheres according to claim 2, characterized in that: In step S3, the monomer solvent is added to the alkaline hydrolysate at a constant rate using a microfluidic device. The monomer solvent is added for the first time and then paused after 10 to 25 minutes. After adding a certain mass of inorganic salt and stirring evenly, the monomer solvent is added a second time until all of it is added. Then, the reaction is stirred and aged to obtain a silica dispersion.
4. The method for preparing highly monodisperse micron-sized silica microspheres according to claim 1, characterized in that: In step S3, after the monomer solvent is completely added, the reaction continues for 3 to 5 hours at a stirring rate of 350 rpm to 750 rpm, and then aged for 5 to 7 hours to obtain a silica dispersion.
5. The method for preparing highly monodisperse micron-sized silica microspheres according to claim 1, characterized in that: In step S1, organic solvent, alkaline catalyst, ultrapure water and stabilizer are placed in a reaction vessel. The temperature of the reaction vessel is maintained at 25℃~50℃, and the mixture is mechanically stirred at a speed of 350rpm~750rpm to make it uniformly mixed into an alkaline hydrolysate.
6. The method for preparing highly monodisperse micron-sized silica microspheres according to claim 1, characterized in that: The organic solvent is one of methanol, ethanol, isopropanol, and n-propanol; the alkaline catalyst is one of sodium hydroxide, potassium hydroxide, and ammonium hydroxide; the stabilizer is one of polyethylene glycol, polyvinyl alcohol, and polyvinylpyrrolidone; and the silicon source monomer is one of methyl orthosilicate and ethyl orthosilicate.
7. The method for preparing highly monodisperse micron-sized silica microspheres according to claim 1, characterized in that: The inorganic salt mentioned is one of sodium chloride, ammonium chloride, potassium chloride, ammonium nitrate, sodium nitrate, and potassium nitrate.
8. The method for preparing highly monodisperse micron-sized silica microspheres according to claim 1, characterized in that: In the silica dispersion, the concentration of the organic solvent is 11 mol / L to 29 mol / L; the concentration of the alkaline catalyst is 0.8 mol / L to 3 mol / L; the concentration of the ultrapure water is 4 mol / L to 12 mol / L; the concentration of the stabilizer is 0.02 mmol / L to 0.2 mmol / L; the concentration of the inorganic salt is 1.5 mmol / L to 5.6 mmol / L; and the concentration of the silicon source monomer is 0.3 mol / L to 0.7 mol / L.
9. Highly monodisperse micron-sized silica microspheres, used in solid-state chip fabrication, characterized by: The highly monodisperse micron-sized silica microspheres were prepared using the method described in any one of claims 1-8.
10. A chip, characterized in that: Including the highly monodisperse micron-sized silica microspheres as described in claim 9.