Preparation method and application of modified silica microspheres

Abstract

The application provides a preparation method and application of modified silica microspheres. First, amino PEG silane reagent is protected by reacting with fluorenylmethyloxycarbonyl chloride, then the fluorenylmethyloxycarbonyl-protected PEG silane reagent is mixed with PEG alkoxy silane reagent, the mixed reagent is used for modifying silica microspheres to obtain silica microspheres with surface amino modification, and finally, the fluorenylmethyloxycarbonyl protecting group is removed to obtain silica microspheres with exposed amino groups. Effect verification results show that the microspheres obtained by the application can improve the detection signal intensity and signal-to-noise ratio of the chip in the hybrid decoding process.

Description

Technical Field

[0001] This invention belongs to the field of chip fabrication technology, specifically relating to a method for preparing modified silica microspheres, chip microspheres prepared by the method, and chips containing the microspheres. Background Technology

[0002] In gene chips that use microbeads as detection units, each type of microbead is linked to an oligonucleotide chain. This oligonucleotide chain consists of a complex tag sequence and a probe sequence, with a unique correspondence between the complex tag sequence and the probe sequence. During chip fabrication, hundreds of thousands of microbeads are randomly scattered into the microwells of the chip substrate. At this stage, the information of each microbead in each well is unknown, i.e., which probe is linked to the microbead and which SNP site it is used to detect. Therefore, it cannot be used for detection experiments at this time.

[0003] The process of analyzing the information of microbeads in each well of the chip before detection experiments is called decoding. Sequences with different fluorescent modifications and complementary to the composite tag sequence are subjected to multiple rounds of hybridization, unwinding, and scanning with the chip. Each round of hybridization stains the microbeads a different color, and the fluorescence information obtained at each well in each round of hybridization scanning is recorded. Through data analysis, the probe sequence corresponding to each well can be obtained. The decoded chip is then ready for formal detection experiments.

[0004] The decoding and detection processes of the aforementioned chip both utilize the principle of hybridization. The quality of the hybridization process directly affects the decoding rate and detection accuracy. The main detection unit on the chip is the microbead, and both decoding and detection hybridization involve reactions with the microbead. Theoretically, when the microbead surface has better hydrophilicity and can prevent non-specific adsorption, the hybridization process is easier, the non-specificity of hybridization is lower, and the cleaning after hybridization is more thorough. Therefore, the surface properties of the microbead are crucial to the chip.

[0005] The silanol groups on the surface of silica microspheres have low reactivity. Therefore, when applying them to chromatographic packing materials, catalyst immobilization, drug controlled release, and biochips, it is necessary to modify the silica microspheres to increase their surface reactivity for further modification and application. Common modifying groups include amino, carboxyl, aldehyde, and epoxy groups, with amino modification being the most widely used method. Currently, the main modifying reagents used for amino-modified microspheres include 3-aminopropyltrimethoxysilane (APTMS), 3-aminopropyltriethoxysilane (APTES), 3-aminopropyl(diethoxy)methylsilane (APMDES), and 3-aminopropyldimethylethoxysilane (APDMES). We have found that existing amino-modified microspheres exhibit low brightness and signal-to-noise ratio during chip decoding, and the decoding rate of some microspheres is low. This may be due to the combined effects of low signal and non-specificity.

[0006] To address the aforementioned technical problems, numerous beneficial explorations have been conducted in existing technologies. For example, Zhihao Yang et al. (Langmuir, 1999, 15, 8405-8411) reported on the modification of glass surfaces with silanizing agents PEG-750 and PEG-5000. They found that PEG-750 exhibited a brush-like appearance on the glass surface, while PEG-5000 showed a mushroom-like appearance. Both silanizing agents effectively prevented protein adsorption on the glass surface. Wen-Yih Chen (Colloids and Surfaces B: Biointerfaces, 2022, 209, 112142) modified silicon nanowire field-effect transistors with silanizing agents Silane-PEG-NH2(1K) and Silane-PEG-OH(1K), then linked them with glutaraldehyde and immobilized DNA probes to detect miRNA-21. Experiments showed that when the ratio of Silane-PEG-NH2 to Silane-PEG-OH was 1:3, ultra-low concentrations of miRNA-21 could be detected. However, there are no existing reports on the use of the above-mentioned PEG silanizing agents for the modification of chip microspheres. Summary of the Invention

[0007] Based on the aforementioned research, this invention addresses the technical problem of low decoding rates caused by low signal strength and non-specificity of certain microspheres during decoding. Several aminoPEG silanizing reagents and mixed PEG silanizing reagents were selected as microsphere modification reagents. When these modified microspheres are applied in chip fabrication, they can improve the detection signal strength and signal-to-noise ratio.

[0008] The technical solution of this invention is as follows: An amino-PEG silanizing agent is first protected by reacting with fluorenemethyloxycarbonyl chloride. Then, the fluorenemethyloxycarbonyl-protected amino-PEG silanizing agent and a PEG silanizing agent are mixed to modify silica microspheres. Finally, the fluorenemethyloxycarbonyl protecting group is removed to obtain mixed-modified silica microspheres with exposed amino groups. Compared to microspheres modified with traditional silanizing agents or microspheres modified with simple amino-PEG silanizing agents, the mixed-modified microspheres in this invention show significantly improved signal intensity and signal-to-noise ratio, and the decoding analysis results are also significantly better than the former two.

[0009] The structural formula of the aminoPEG silanizing agent is shown below:

[0010]

[0011] The structural formula of the PEG silanizing agent is shown below:

[0012]

[0013] Wherein, R1 represents either methoxy or ethoxy; m and n represent the number of PEG monomers; and R2 represents either hydroxyl, carboxyl, or alkoxy groups.

[0014] The schematic diagram of the technical solution adopted in this invention is as follows:

[0015] (1) Amino protection of aminoPEG silanizing reagent

[0016]

[0017] (2) The microspheres were modified by mixing aminoPEG silanizing agents with different PEG silanizing agents.

[0018]

[0019] In Equations 1 to 5 above, R1 represents either methoxy or ethoxy; m and n represent the number of PEG monomers, where m may or may not be equal to n. The proportion of functional groups on the surface of the microspheres shown in the figure does not represent the actual proportion.

[0020] The specific steps are as follows:

[0021] 1. Synthesis of fluorene methoxycarbonyl-protected PEG silanizing reagent

[0022] Using dichloromethane as a solvent, the amino-PEG silanizing agent (Formula 1) and the sterically hindered amine N,N-diisopropylethylamine were added sequentially to a reaction flask in a molar ratio of 1:1.5–3. Under ice-water bath and nitrogen protection, a dichloromethane solution of fluorenemethyloxycarbonyl chloride was slowly added dropwise to the reaction system, wherein the molar ratio of the amino-PEG silanizing agent to fluorenemethyloxycarbonyl chloride was 1:1.5. The reaction was carried out for 30 minutes in an ice-water bath, followed by 2–5 hours at room temperature. After separation and purification, the fluorenemethyloxycarbonyl-protected PEG silanizing agent (Compound 2) was obtained.

[0023] Preferably, the aminoalkoxysilanizing agent is selected from any one of Silane-PEG-NH2(100), Silane-PEG-NH2(200), Silane-PEG-NH2(500), Silane-PEG-NH2(1000), Silane-PEG-NH2(5000), and Silane-PEG-NH2(8000), where the value in parentheses represents the number of PEG monomers.

[0024] 2. Hybrid modification of silica microspheres

[0025] Silica microspheres with a particle size of 0.5–5 μm were added to a mixed reaction solvent containing compounds of formula 2 and formulas 3, 4, or 5 in a molar ratio of 1–50:1. A catalyst was selectively used. The reaction was carried out at 25–100 °C for 2–15 hours. After centrifugation to remove the reaction solvent, the mixture was washed with ethanol and deionized water and dried to obtain mixed modified amino microspheres (compounds Bead-AmFmoc-PEG-OH, Bead-AmFmoc-PEG-COOH, Bead-AmFmoc-PEG-OMe).

[0026] The solvent is any one of toluene, xylene, ethanol, acetone, and ammonia; the catalyst is any one of triethylamine and N,N-diisopropylethylamine, or no catalyst is added; the total volume concentration of the compound of formula 2 and the compound of formula 3, 4, or 5 is less than 10%, and the solvent volume is 10-50 ml / g microspheres.

[0027] 3. Deprotection

[0028] Using a 20% piperidine DMF solution as a solvent, compounds Bead-AmFmoc-PEG-OH, Bead-AmFmoc-PEG-COOH, or Bead-AmFmoc-PEG-OMe were added to the reaction solvent and reacted at 20–40 °C for 20–60 minutes. After centrifugation to remove the solvent, the microspheres were washed with ethanol and dried to obtain deprotected amino-modified microspheres (compounds Bead-Am-PEG-OH, Bead-Am-PEG-COOH, and Bead-Am-PEG-OMe).

[0029] In a second aspect, the present invention provides chip microspheres prepared by the above-described method for preparing amino-modified silica microspheres.

[0030] In a third aspect, the present invention provides a chip containing the aforementioned microspheres.

[0031] In the specific embodiments section of this invention, the effects of microspheres Bead-Am-PEG-OH, Bead-Am-PEG-COOH, and Bead-Am-PEG-OMe were tested, as follows:

[0032] Three types of amino-based microspheres were coupled with oligonucleotide chains according to the method described in patent (CN112717842B). Each type of microsphere was coupled with 960 different oligonucleotide chains (960 beadtypes). These 960 microspheres with different oligonucleotide chains were mixed in a specific ratio and loaded into a chip. Hybridization and scanning were performed using a pre-designed fluorescent modification sequence complementary to a portion of the oligonucleotide complex tag. Simultaneously, microspheres modified with traditional silanizing reagents or microspheres modified with pure amino-PEG silanizing reagents were used as controls. Oligonucleotide chains were coupled with these microspheres using the same method, and they were loaded into the chip and subjected to hybridization and scanning in the same manner.

[0033] Comparative scanning reveals that the signal intensity and signal-to-noise ratio of microspheres modified with traditional silanizing reagents are low, with some dark or unclear points, which can easily lead to misjudgment during analysis. The brightness of microspheres modified with pure aminoPEG silanizing reagents is also low, but the number of unclear microspheres is greatly reduced. The signal intensity and signal-to-noise ratio of the hybrid modified microspheres of this invention are significantly improved, and the decoding analysis results are also significantly better than the former two.

[0034] The possible principle behind this invention's use of mixed silanizing agents to modify microspheres and improve signal intensity and signal-to-noise ratio is as follows:

[0035] 1. Using PEG as a linker can improve the hydrophilicity of the microsphere surface, and the hydrophilic surface helps to improve the efficiency of hybridization and related biological processes, thereby enhancing the signal. In addition, PEG itself can prevent the non-specific binding of fluorescent modified sequences or proteins to microspheres, and can also separate microspheres from biomolecules, reducing the interference of the surface on biological reactions, thereby increasing the signal intensity while reducing non-specificity.

[0036] 2. Modification with mixed silanizing reagents can reduce the complexity of the microsphere surface structure and decrease the non-specific binding of primers or proteins to the microspheres. In addition, it can reduce the probe density on the microsphere surface and avoid self-quenching caused by excessively high concentrations of fluorescent molecules during hybridization and detection.

[0037] Compared with the prior art, the technical effects of the present invention are as follows:

[0038] In terms of functionality, this invention uses both fluorene methoxycarbonyl-protected amino PEG silanizing reagent and PEG silanizing reagent as modifiers to modify microspheres, which significantly improves the signal intensity and signal-to-noise ratio of the microspheres, thereby improving the microsphere resolution rate during decoding and providing reliable raw materials for the subsequent preparation of biochips to improve detection accuracy.

[0039] In terms of preparation, the amino-PEG silanizing reagent and PEG silanizing reagent used in this invention can be obtained through purchase, and the amino protecting agent fluorene methoxycarbonyl chloride and the sterically hindered amine N,N-diisopropylethylamine are also readily available. The raw materials are easy to obtain, the reaction conditions are mild, and the process is easy to implement. Attached Figure Description

[0040] Figure 1 This is a hybridization scan image of 960beadtype prepared by coupling with amino-modified microspheres using the commonly used reagent 3-aminopropyltriethoxysilane (APTES) and loaded onto a chip.

[0041] Figure 2 This is a hybridization scan image of 960beadtype prepared by coupling microspheres modified with Silane-PEG-NH2 and loaded into a chip.

[0042] Figure 3 This is a hybridization scan image of 960beadtype prepared by coupling amino-modified microspheres according to the present invention and loaded into a chip. Detailed Implementation

[0043] The present invention will now be described in detail with reference to embodiments and accompanying drawings. However, the following embodiments should not be construed as limiting the scope of the present invention.

[0044] Example 1

[0045] Taking the modified microspheres Bead-Am-PEG-OH(1K) (m, n approximately 20) with the structure shown below as an example, the specific synthesis method is as follows:

[0046]

[0047] 1. Preparation of Silane-PEG-NHFmoc(1K)

[0048] 50 mL of dichloromethane, 10 g of aminopolyethylene glycol trimethoxysilane (Silane-PEG-NH2,1K), and 2.5 mL of hindered amine N,N-diisopropylethylamine were added sequentially to a 250 mL three-necked flask, and the mixture was cooled to 0 °C under nitrogen protection. 2.8 g of fluorenemethoxycarbonyl chloride was dissolved in 20 mL of dry dichloromethane solution and slowly added dropwise to the reaction system. After the addition was complete, the reaction was continued for 30 minutes in an ice-water bath, then the ice-water bath was removed, and the reaction was allowed to proceed at room temperature for 3 hours. After removing the solvent by rotary evaporation, the compound Silane-PEG-NHFmoc(1K) was purified by column chromatography using a mixture of dichloromethane and ethyl acetate in a volume ratio of 10:1 to 5:1 as the eluent, yielding the structure shown below.

[0049]

[0050] 2. Preparation of Bead-AmFmoc-PEG-OH(1K)

[0051] Add 20 mL of ethanol, 1 g of Silane-PEG-NHFmoc(1K), 2 g of Silane-PEG-OH(1K), and 1 g of silica microspheres with a particle size of 3 μm to a single-necked flask in sequence. React at 80 °C for 5 hours. After cooling to room temperature, centrifuge to remove the reaction solvent. Wash with ethanol and deionized water in sequence and dry to obtain Bead-AmFmoc-PEG-OH(1K) structure as shown below. It is generally stored in this state before further use.

[0052]

[0053] 3. Preparation of Bead-Am-PEG-OH(1K)

[0054] 8 mL of N,N-dimethylformamide, 2 mL of piperidine, and 1 g of Bead-AmFmoc-PEG-OH(1K) microspheres were added sequentially to a single-necked flask. The mixture was reacted at room temperature for 60 minutes. After centrifugation to remove the solvent, the mixture was washed with ethanol to obtain Bead-Am-PEG-OH(1K) surface-modified microspheres.

[0055] Example 2

[0056] Taking the amino-modified microspheres Bead-Am-PEG-COOH(5K) (m, n approximately 110) with the structure shown below as an example, the specific synthesis method is as follows:

[0057]

[0058]

[0059] 1. Preparation of Silane-PEG-NHFmoc(5K)

[0060] 50 mL of dichloromethane, 20 g of aminopolyethylene glycol triethoxysilane (Silane-PEG-NH2,5K), and 0.72 mL of hindered amine N,N-diisopropylethylamine were added sequentially to a 250 mL three-necked flask, and the mixture was cooled to 0 °C under nitrogen protection. 1.6 g of fluorenemethyloxycarbonyl chloride was dissolved in 20 mL of dry dichloromethane solution and slowly added dropwise to the reaction system. After the addition was complete, the reaction was continued for 30 minutes in an ice-water bath, then the ice-water bath was removed, and the reaction was allowed to proceed at room temperature for 5 hours. After removing the solvent by rotary evaporation, column chromatography was performed using a mixture of dichloromethane and ethyl acetate in a volume ratio of 5:1 to 1:1 as the eluent to obtain the structure of Silane-PEG-NHFmoc(5K), as shown below.

[0061]

[0062] 2. Preparation of Bead-AmFmoc-PEG-COOH(5K)

[0063] Add 20 mL of anhydrous ethanol, 1.5 g of Silane-PEG-NHFmoc(5K), 1.5 g of Silane-PEG-COOH(5K), 0.5 mL of N,N-diisopropylethylamine, and 1 g of silica microspheres with a particle size of 3 μm to a single-necked flask in sequence. React at room temperature for 2 hours, centrifuge to remove the reaction solvent, wash with ethanol and deionized water in sequence and dry to obtain the Bead-AmFmoc-PEG-COOH(5K) structure shown below. It is generally stored in this state before further use.

[0064]

[0065] 3. Preparation of Bead-Am-PEG-COOH(5K)

[0066] 8 mL of DMF, 2 mL of piperidine, and 1 g of Bead-AmFmoc-PEG-COOH(5K) microspheres were added sequentially to a single-necked flask. The mixture was reacted at room temperature for 40 minutes. After centrifugation to remove the solvent, the mixture was washed with ethanol to obtain Bead-Am-PEG-COOH(5K) surface-modified microspheres.

[0067] Example 3

[0068] Taking the surface-modified microspheres with the structure shown below as an example (m=6, n is approximately 20), the specific synthesis method is as follows:

[0069]

[0070] 1. Preparation of Silane-PEG-NHFmoc(1K)

[0071] 50 mL of dichloromethane, 10 g of aminopolyethylene glycol trimethoxysilane (Silane-PEG-NH2,1K), and 2.5 mL of hindered amine N,N-diisopropylethylamine were added sequentially to a 250 mL three-necked flask, and the mixture was cooled to 0 °C under nitrogen protection. 2.8 g of fluorenemethoxycarbonyl chloride was dissolved in 20 mL of dry dichloromethane solution and slowly added dropwise to the reaction system. After the addition was complete, the reaction was continued for 30 minutes in an ice-water bath, then the ice-water bath was removed, and the reaction was allowed to proceed at room temperature for 3 hours. After removing the solvent by rotary evaporation, the compound Silane-PEG-NHFmoc(1K) was purified by column chromatography using a mixture of dichloromethane and ethyl acetate in a volume ratio of 10:1 to 5:1 as the eluent, yielding the structure shown below.

[0072]

[0073]

[0074] 2. Preparation of Bead-AmFmoc-PEG-OMe(1K)

[0075] 20 mL of toluene, 1 g of Silane-PEG-NHFmoc(1K), 1.5 g of Silane-PEG-OMe(500), and 1 g of silica microspheres with a particle size of 3 μm were added sequentially to a single-necked flask. The mixture was reacted at 90 °C for 10 hours. After cooling to room temperature, the reaction solvent was removed by centrifugation. The mixture was then washed with ethanol and deionized water and dried to obtain the Bead-AmFmoc-PEG-OMe(1K) structure shown below. This structure is generally stored in this state before further use.

[0076]

[0077] 3. Preparation of Bead-Am-PEG-OMe(1K)

[0078] 8 mL of DMF, 2 mL of piperidine, and 1 g of Bead-AmFmoc-PEG-OMe(1K) microspheres were added sequentially to a single-necked flask. The mixture was reacted at room temperature for 30 minutes. After centrifugation to remove the solvent, the mixture was washed with ethanol to obtain Bead-Am-PEG-OMe(1K) surface-modified microspheres.

[0079] Comparative Example 1

[0080] Compared with Example 1, the specific synthesis method is as follows:

[0081] 1. Preparation of Bead-AmFmoc-PEG(1K)

[0082] 20 mL of ethanol, 1 g of Silane-PEG-NHFmoc(1K), and 1 g of silica microspheres with a particle size of 3 μm were added sequentially to a single-necked flask. The mixture was reacted at 80 °C for 5 hours. After cooling to room temperature, the reaction solvent was removed by centrifugation. The mixture was then washed with ethanol and deionized water and dried to obtain the Bead-AmFmoc-PEG(1K) structure shown below.

[0083]

[0084] 2. Preparation of Bead-Am-PEG(1K)

[0085] 8 mL of DMF, 2 mL of piperidine, and 1 g of Bead-AmFmoc-PEG(1K) microspheres were added sequentially to a single-necked flask. The mixture was reacted at room temperature for 30 minutes. After centrifugation to remove the solvent, the mixture was washed with ethanol to obtain the surface-modified Bead-Am-PEG(1K) microspheres shown below.

[0086]

[0087] Comparative Example 2

[0088] A conventional APTES-modified amino microspheres were prepared using the same method as in Comparative Example 1, and the surface-modified microspheres with the following structures were obtained:

[0089]

[0090] Effect comparison

[0091] The brightness and signal-to-noise ratio of conventional APTES-modified microspheres, single Silane-PEG-NH2-modified microspheres, and surface-modified microspheres synthesized by the method of this invention were compared. The specific method is as follows:

[0092] 10 grams each of the surface-modified microspheres from Comparative Example 1, Comparative Example 2, and Example 1 were activated. After activation, 960 10-milligram microspheres were separated, and each sample was coupled with an oligonucleotide chain to obtain 960 microspheres. 5 mg of the 960 microspheres were mixed and loaded into a chip. The chip was hybridized and scanned using the decoding hybridization solution of the 960 microspheres. The same hybridization solution and scanning parameters were used for all three types of microspheres.

[0093] Figure 1 The image shows a hybridization scan of 960beadtype prepared by coupling with amino-modified microspheres of 3-aminopropyltriethoxysilane (APTES) using the commonly used reagent in Comparative Example 2, after being loaded into a chip. Figure 2 The hybridization scan of 960beadtype prepared by coupling microspheres modified with Silane-PEG-NH2 in Comparative Example 1 and loaded into a chip is shown. Figure 3 The image shows a hybridization scan of 960beadtype prepared by coupling amino-modified microspheres in Example 1 of this invention and loaded onto a chip. The comparison shows that the signal intensity and signal-to-noise ratio of the modified microspheres in Comparative Example 2 are low, with some dark or unclear points, which can easily lead to misinterpretation during analysis. The brightness of the modified microspheres in Comparative Example 1 is also low, but the number of unclear microspheres is significantly reduced. The modified microspheres in Example 1 show significantly improved signal intensity and signal-to-noise ratio, and the decoding analysis results are significantly better than the former two.

[0094] The examples of the present invention have been specifically described above, but the present invention is not limited to the embodiments described. Those skilled in the art can make various equivalent modifications or substitutions without departing from the spirit of the present invention, and these equivalent modifications or substitutions are all included within the scope defined by the claims of this application.

Claims

1. A method for preparing modified silica microspheres, characterized in that, The amino-PEG silanizing agent is first protected by reacting a hindered amine with fluorenemethyloxycarbonyl chloride. Then, the fluorenemethyloxycarbonyl-protected amino-PEG silanizing agent and a PEG silanizing agent are mixed to modify silica microspheres. Finally, the fluorenemethyloxycarbonyl protecting group is removed to obtain mixed-modified silica microspheres with exposed amino groups. The structural formula of the aminoPEG silanizing agent is shown below: ， The structural formula of the PEG silanizing agent is shown below: ， R1 represents either methoxy or ethoxy; m and n represent the number of PEG monomers, ranging from 6 to 8000; and R2 represents either hydroxyl, carboxyl, or alkoxy groups.

2. The method for preparing modified silica microspheres according to claim 1, characterized in that: in, The aminoPEG silanizing agent is selected from Silane-PEG-NH2 with n of 20, 100, 200, 1000, 5000 or 8000. m is selected from 6 to 110.

3. The method for preparing modified silica microspheres according to claim 1, characterized in that, Includes the following steps: A. Fluorenemethyloxycarbonyl-protected amino PEG silanizing reagent Amino PEG silanizing reagent and sterically hindered amine were added sequentially to the reaction vessel at a molar ratio of 1:1.5~3. Under ice-water bath and nitrogen protection, fluorene methoxycarbonyl chloride solution was slowly added to the reaction system. After the reaction was carried out under ice-water bath, the reaction was carried out at room temperature. After separation and purification, fluorene methoxycarbonyl protected PEG silanizing reagent was obtained. B. Modification of silica microspheres Silica microspheres were added to a mixed solution of aminoPEG silanizing agent and PEG silanizing agent protected by fluorene methoxycarbonyl group, and a catalyst was selectively used. The reaction was carried out at 25-100℃ for 2-15 hours. After centrifugation to remove the reaction solvent, the mixture was washed with ethanol and deionized water and dried to obtain the mixed modified amino microspheres. C. Defluorination methoxycarbonyl protection The mixed modified amino microspheres obtained in step B were added to the deprotection reaction solvent and reacted at 20-40°C for 20-60 minutes. After centrifugation to remove the solvent, the microspheres were washed with ethanol and dried to obtain the deprotected amino modified microspheres.

4. The method for preparing modified silica microspheres according to claim 3, characterized in that: in, In step A, the solvents for the aminoPEG silanizing agent, the hindered amine, and the fluorene methoxycarbonyl chloride are all dry dichloromethane. The molar ratio of the aminoPEG silanizing agent and fluorenemethyloxycarbonyl chloride is 1:1.5, and the reaction time is 30 minutes in an ice-water bath followed by 2-5 hours at room temperature.

5. The method for preparing modified silica microspheres according to claim 3, characterized in that: in, In step A, the separation and purification of the reactants are as follows: after removing the solvent by rotary evaporation, column chromatography is performed using a mixture of dichloromethane and ethyl acetate in a volume ratio of 10:1 to 1:1 as the eluent; The sterically hindered amine is N,N-diisopropylethylamine.

6. The method for preparing modified silica microspheres according to claim 3, characterized in that: in, In step B, the molar ratio between the fluorene methoxycarbonyl-protected amino PEG silanizing agent and the PEG silanizing agent is 1~50:1; The reaction solvent is any one of toluene, xylene, ethanol, acetone, and ammonia. The catalyst is selected from any one of triethylamine and N,N-diisopropylethylamine, or no catalyst is added; The silica microspheres have a particle size of 0.5–5 μm, and the total volume concentration of the mixed solution of fluorene methoxycarbonyl-protected amino PEG silanizing agent and PEG silanizing agent is less than 10%, with a solvent volume of 10–50 mL / g microspheres. After the reaction was completed, the reaction solvent was removed by centrifugation, and the mixture was washed with ethanol and deionized water and dried to obtain mixed modified amino microspheres.

7. The method for preparing modified silica microspheres according to claim 3, characterized in that: in, In step C, the solvent for the deprotection reaction is a DMF solution containing 20% ​​piperidine.

8. A chip microsphere, characterized in that, The modified silica microspheres were prepared using the preparation method described in any one of claims 1 to 7.

9. A chip, characterized in that, Contains the chip microspheres as described in claim 8.

Images