Silicon hydroxyl magnetic bead and preparation method thereof

Abstract

According to the silicon hydroxyl magnetic bead and the preparation method thereof, an optimized solvothermal method and a reverse microemulsion method are combined, high-quality magnetic cores are prepared on a large scale through the optimized solvothermal method, an integrated process of high silicon hydroxyl coating is carried out through the improved reverse microemulsion method, and large-scale and stable preparation of the high-performance silicon hydroxyl magnetic bead is achieved. The prepared magnetic beads have the characteristics of strong magnetic responsiveness, uniform particle size, good dispersion stability, rich silicon hydroxyl and high activity, so that the efficiency and purity of nucleic acid extraction can be remarkably improved.

Description

Technical Field

[0001] This invention relates to a magnetic bead and its preparation method, specifically to a silicon hydroxyl magnetic bead and its preparation method, belonging to the field of functional nanomaterial preparation technology. Background Technology

[0002] Nucleic acid (DNA / RNA) extraction is a cornerstone technology in modern molecular biology, medical diagnostics, gene sequencing, forensic medicine, and other fields. High-efficiency, high-purity nucleic acids are crucial for the success of subsequent experiments (such as PCR and sequencing).

[0003] Magnetic separation technology based on silanol beads has become the mainstream method for nucleic acid extraction due to its advantages such as simple operation, speed, good automation compatibility, and avoidance of toxic organic solvents. Silanol beads can efficiently adsorb nucleic acids under specific salt concentrations and pH conditions.

[0004] However, the preparation of existing silanol magnetic beads faces the following technical bottlenecks: 1) Limitations on preparation scale: Existing methods for preparing silanol magnetic beads, such as the co-precipitation method and modified Stöber method, are often limited to small-batch or laboratory-scale production, making it difficult to meet the large-scale demand for magnetic beads in industrial or high-throughput applications. Furthermore, problems such as uneven particle size, incomplete coating, and decreased magnetic properties are prone to occur during scale-up.

[0005] 2) Insufficient coating uniformity and stability: The ability to achieve uniform and dense coating of the silicon shell on the magnetic core surface directly affects the dispersibility, specific surface area, silanol density, and adsorption / elution efficiency of nucleic acids for magnetic beads. Under large-scale conditions, existing preparation methods struggle to guarantee coating uniformity and the stability of silanol activity. Summary of the Invention

[0006] This invention proposes a silanol magnetic bead and its preparation method, aiming to overcome the above-mentioned shortcomings of the existing technology and achieve stable and high-quality preparation of large-scale high-performance silanol magnetic beads, which are particularly suitable for nucleic acid separation and purification in the biomedical field.

[0007] The technical solution of this invention is a method for preparing silanol-hydroxyl magnetic beads, combining an optimized solvothermal method and a reverse microemulsion method. The optimized solvothermal method is used to prepare high-quality magnetic cores in large quantities, while the improved reverse microemulsion method is used for a highly silanol-coated integrated process, achieving large-scale and stable preparation of high-performance silanol-hydroxyl magnetic beads. The prepared magnetic beads exhibit strong magnetic responsiveness, uniform particle size, good dispersion stability, abundant silanol groups, and high activity, thereby significantly improving the efficiency and purity of nucleic acid extraction.

[0008] Specifically, a method for preparing silanol magnetic beads includes the following steps: I. Preparation of bare ferrite magnets: 1) Weigh out 100g of PEG-2000 and 300g of anhydrous sodium acetate and mix them together; 2) Add the powder from step 1) to 1500ml of ethylene glycol and stir until completely dissolved to obtain solution A; 3) Weigh 150g of ferric chloride hexahydrate, add it to 1500ml of ethylene glycol, and stir until completely dissolved to obtain solution B; 4) Slowly pour solution B into solution A and stir continuously for 30 minutes to ensure thorough mixing; 5) The mixed solution is evenly transferred to several polytetrafluoroethylene-lined hydrothermal synthesis reactors; 6) Place the synthesis vessel in an oven and heat it for reaction; 7) After the reaction is complete, allow it to cool naturally to room temperature.

[0009] 8) Open the synthesis vessel, collect the black product, and wash it with anhydrous ethanol by centrifugation at least three times to remove residual reactants, solvents and byproducts; 9) The resulting product is bare ferrite magnetic particles, which can be used for later use or dried and stored.

[0010] II. Preparation of silanol magnetic beads.

[0011] Preferably, in step 1, 6), the reaction is carried out at 150°C for 8 hours. The resulting Fe3O4 exhibits good bare magnetic dispersion, high crystallinity, and strong magnetism.

[0012] Preferably, step two includes: 1) Weigh 20g of the bare ferrite magnetic particles prepared in step one; 2) Add 20g of bare magnet to 1000ml of cyclohexane; 3) Ultrasonic stirring and dispersion for 30 minutes to ensure that the bare magnets are uniformly dispersed in cyclohexane, forming suspension C; 4) Take another 1000ml of cyclohexane and add 12ml of emulsifier CO-520; 5) Sonicate for 30 minutes to completely dissolve and disperse CO-520 in cyclohexane, forming solution D; 6) Slowly pour solution D into suspension C; 7) Stir ultrasonically for 30 minutes to form a preliminary oil-in-magnetic-core emulsion system; 8) Add 200 ml of an aqueous solution containing 0.5% (w / v) sodium dodecylbenzenesulfonate to the above system; 9) Add 10 ml of 28% concentrated ammonia solution to the above system; 10) Ultrasonic stirring for 30 minutes to form a stable water core / oil reverse microemulsion system containing magnetic cores; 11) Take 40 ml of tetraethyl orthosilicate and dilute it with 160 ml of cyclohexane to obtain mixed solution E; 12) Slowly add mixed solution E dropwise to the reaction system of step 10) at a rate of 1 ml / min; 13) After the mixed solution E is completely added, continue to stir gently for 4 hours to form a uniform silanol shell on the surface of the magnetic core; 14) After the reaction is complete, transfer the product system out; 15) Cleaning: First, clean with anhydrous ethanol 3 times, then clean with purified water 3 times; 16) Finally, disperse the cleaned silanol magnetic beads in purified water to obtain the final product, and adjust the concentration as needed.

[0013] Advantages of this invention: 1) Significant mass production capability: It can directly achieve one-time mass synthesis (bare magnets and coated silanol magnetic beads) from raw materials (100g iron source), which can effectively solve the core problem of industrial scale-up and reduce the production cost per unit output; 2) Strong magnetism and rapid separation: The magnetic nuclei prepared by the solvothermal method have high crystallinity, high saturation magnetization, and rapid magnetic response; 3) High dispersibility and stability: PEG-2000 is used to regulate the dispersion of bare magnetic beads. The improved reverse microemulsion method uses a composite emulsifier combined with ammonia catalysis to achieve uniform and dense coating of the silicon shell, resulting in excellent suspension of the final magnetic beads (not easy to precipitate and agglomerate) and good colloidal stability in the aqueous phase. 4) High specific surface area and silanol activity: The uniform and dense silicon shell can provide a large specific surface area and abundant silanol (-Si-OH) sites, and the coating process is mild, which is conducive to maintaining the high reactivity of silanol. 5) High nucleic acid extraction efficiency and purity: The magnetic beads can be used to extract nucleic acids from different biological samples (blood, cells, tissues, etc.) with high extraction efficiency. At the same time, thanks to the excellent coating and washing steps, the extracted nucleic acid purity is higher, which meets the requirements of downstream high-end applications. 6) Stable and controllable process flow: The solvothermal conditions are mature and controllable. Key parameters such as the selection of emulsifiers and the feeding method (slow dripping and controlled stirring) in the reverse microemulsion method have been optimized to ensure the consistency of product performance between different batches. 7) Environmental friendliness: Compared with existing technologies that use strong acids, strong bases or other toxic reagents, the reagents used are relatively mild and can effectively recover some solvents (such as cyclohexane). Attached Figure Description

[0014] Figure 1 This is a TEM image of the bare ferrite magnet prepared in Example 1 of the present invention.

[0015] Figure 2This is a TEM image of the silanol magnetic beads prepared in Example 2 of this invention.

[0016] Figure 3 This is a particle size distribution diagram (DLS result) of the silanol magnetic beads prepared in Example 2 of the present invention. Detailed Implementation

[0017] The present invention will be further described in detail below with reference to embodiments and specific implementation methods.

[0018] Example 1: Preparation of large quantities of bare ferrite magnets (Fe3O4) Includes the following steps: 1) Weigh out 100g of PEG-2000 and 300g of anhydrous sodium acetate (NaAc) and mix them together; 2) Add the powder from step 1) to 1500ml of ethylene glycol (EG) and stir until completely dissolved to obtain solution A; 3) Weigh 150g of ferric chloride hexahydrate (FeCl3·6H2O), add it to 1500ml of ethylene glycol, and stir until completely dissolved to obtain solution B; 4) Slowly pour solution B into solution A and continuously stir magnetically (or mechanically) for 30 minutes to ensure thorough mixing; 5) Transfer the mixed solution evenly to several hydrothermal synthesis reactors with appropriate capacity and PTFE lining (ensure the filling degree is within the safe range). 6) Place the synthesis vessel in an oven and heat at 150°C for 8 hours; 7) After the reaction is complete, allow it to cool naturally to room temperature.

[0019] 8) Open the synthesis vessel, collect the black product, and wash it with anhydrous ethanol by centrifugation multiple times (at least three times) to remove residual reactants, solvents and byproducts; 9) The resulting product is high-quality Fe3O4 bare magnetic particles, which can be used for standby or dried and stored.

[0020] In the above steps, a solvothermal method was employed, using high-concentration raw materials (150g FeCl3·6H2O, 300g NaAc, 100g PEG-2000, and 3000ml EG system). By precisely controlling the reaction conditions (150℃, 8h), the obtained Fe3O4 bare magnets exhibited good dispersibility, high crystallinity, and strong magnetism. PEG-2000 served as a key dispersant and morphology modifier, enabling the large-scale preparation of magnetic cores. High-quality bare magnets in the hundreds of grams (depending on reactor capacity scaling) could be synthesized in a single step.

[0021] Example 2: Preparation of large quantities of silanol magnetic beads Includes the following steps: 1) Weigh 20g of the bare magnetic particles prepared in Example 1; 2) Add 20g of bare magnet to 1000ml of cyclohexane; 3) Ultrasonic stirring and dispersion for 30 minutes to ensure that the bare magnets are uniformly dispersed in cyclohexane, forming suspension C; 4) Take another 1000ml of cyclohexane and add 12ml of emulsifier CO-520; 5) Sonicate for 30 minutes to completely dissolve and disperse CO-520 in cyclohexane, forming solution D; 6) Slowly pour solution D into suspension C; 7) Stir ultrasonically for 30 minutes to form a preliminary oil-in-magnetic-core emulsion system; 8) Add 200 ml of an aqueous solution containing 0.5% (w / v) SDBS (sodium dodecylbenzenesulfonate) to the above system; 9) Add 10 ml of 28% concentrated ammonia (NH3·H2O) to the above system; 10) Ultrasonic stirring for 30 minutes. At this time, under the action of composite emulsifier (CO-520+SDBS) and ammonia, a stable water core / oil (W / O) reverse microemulsion system containing magnetic core is formed. 11) Take 40 ml of tetraethyl orthosilicate (TEOS) and dilute it with 160 ml of cyclohexane to obtain mixed solution E; 12) Add the mixed solution E slowly dropwise to the reaction system of step 10 using a constant flow pump (or with precise control of the dropping rate) at a rate of approximately 1 ml / min; (during the dropping process, only perform gentle magnetic or mechanical stirring, stop sonication, and avoid damaging the microemulsion structure.) 13) After the mixed solution E is added, continue to stir gently for 4 hours to allow TEOS to fully hydrolyze and condense at the water-core interface and form a uniform silanol shell on the surface of the magnetic core. 14) After the reaction is complete, transfer the product system out; 15) Washing: First wash 3 times with anhydrous ethanol (collect magnetic beads by magnetic separation, discard the supernatant, add fresh ethanol to disperse), then wash 3 times with purified water (same as above). 16) Finally, disperse the cleaned silanol magnetic beads in an appropriate amount of purified water to obtain the final product. The concentration can be adjusted as needed.

[0022] In the above steps, as a foundation, the bare magnets were well pre-dispersed in cyclohexane, and the silanol coating was optimized through a reverse microemulsion method. Then, an innovative emulsification system was constructed: a composite emulsifier (a combination of CO-520 and SDBS) combined with ammonia (NH3·H2O) catalysis was used to form a stable water-core microemulsion at the cyclohexane / water interface. CO-520 is an oil-soluble emulsifier, SDBS is a co-emulsifier, and sulfonate groups were introduced to assist in stabilization. Precise feeding control was achieved: a slow dropwise addition of a TEOS cyclohexane solution (1 ml / min) ensured that the TEOS precursor fully diffused into the water core, hydrolyzing and condensing on the surface of the water core to form a uniform and dense silica shell. Sufficient reaction time was provided: prolonged magnetic stirring after coating (4 hours) ensured complete reaction. The silica coating step can process batches of up to 20 g of bare magnets, far exceeding conventional small-scale trials (typically milligrams to grams).

[0023] like Figure 1-3 As shown, TEM / SEM reveals that the magnetic core size is uniform at approximately 500 nm, and the silicon shell coating is complete and of uniform thickness. This demonstrates that the products from a single batch exhibit uniform morphology and size. It has been successfully applied to the extraction of genomic DNA or total RNA from various biological samples, such as whole blood, serum, plasma, cultured cells, animal tissues, plant tissues, and swabs.

[0024] The silanol magnetic beads prepared in Example 2 were compared with commercially available silanol magnetic beads used as a comparative example. Nucleic acid was extracted from different samples and analyzed for influenza A virus and hepatitis B virus. The efficiency was compared, and the results are as follows: 1. Influenza A virus:

[0025] 2. Hepatitis B virus:

[0026] Based on the above results, regardless of whether the concentration is high or low, the CT value of the siloxane magnetic beads in Example 2 is superior to that of the commercially available siloxane magnetic beads in the comparative example, and the batch-to-batch variation of the siloxane magnetic beads in Example 2 is small, indicating good repeatability.

[0027] The above description is only a preferred embodiment of the present invention. It should be noted that those skilled in the art can make several modifications and improvements without departing from the inventive concept of the present invention, and these all fall within the protection scope of the present invention.

Claims

1. A method for preparing a silicon hydroxyl magnetic bead, characterized by, The method comprises the following steps: I. Preparation of ferrite bare magnetic particles: 1) Take 100 g of PEG-2000 and 300 g of anhydrous sodium acetate and mix them; 2) Add the powder of step 1) to 1500 ml of ethylene glycol and stir until completely dissolved to obtain solution A; 3) Take 150 g of ferric chloride hexahydrate and add it to 1500 ml of ethylene glycol and stir until completely dissolved to obtain solution B; 4) Slowly pour solution B into solution A and continue stirring for 30 minutes to ensure uniform mixing; 5) Divide the mixed solution into several polytetrafluoroethylene-lined hydrothermal synthesis kettles; 6) Place the synthesis kettles in an oven for heating reaction; 7) After the reaction is completed, naturally cool to room temperature; 8) Open the synthesis kettle and collect the black product, wash it with anhydrous ethanol by centrifugation at least three times to remove residual reactants, solvents and byproducts; 9) The obtained product is ferrite bare magnetic particles, which are ready for use or dried for storage; II. Preparation of silicon hydroxyl magnetic beads.

2. The method for preparing silanol magnetic beads as described in claim 1, characterized in that, In step I-6, the heating reaction is carried out at 150°C for 8 hours.

3. A method for preparing silanol magnetic beads as described in claim 1 or 2, characterized in that, The step II comprises: 1) Take 20 g of ferrite bare magnetic particles prepared in step I; 2) Add 20 g of bare magnetic particles to 1000 ml of cyclohexane; 3) Ultrasonically stir and disperse for 30 minutes to uniformly disperse the bare magnetic particles in the cyclohexane to form a suspension C; 4) Take another 1000 ml of cyclohexane and add 12 ml of emulsifier CO-520; 5) Ultrasonically treat for 30 minutes to completely dissolve and disperse CO-520 in cyclohexane to form solution D; 6) Slowly pour solution D into suspension C; 7) Ultrasonically stir for 30 minutes to form a preliminary emulsion system of magnetic core in oil; 8) Add 200 ml of an aqueous solution containing 0.5% (w / v) sodium dodecylbenzenesulfonate to the above system; 9) Add 10 ml of 28% concentrated ammonia water to the above system; 10) Ultrasonically stir for 30 minutes to form a stable water core / oil reverse microemulsion system containing magnetic cores; 11) Take 40 ml of tetraethyl orthosilicate and dilute it with 160 ml of cyclohexane to obtain a mixed solution E; 12) Slowly add the mixed solution E to the reaction system of step 10) at a rate of 1 ml / min; 13) After the addition of the mixed solution E is completed, continue to gently stir for 4 hours to form a uniform silicon hydroxyl shell on the surface of the magnetic cores; 14) After the reaction is completed, transfer the product system out; 15) Washing: first wash with anhydrous ethanol for 3 times, and then wash with purified water for 3 times; 16) Finally, disperse the washed silicon hydroxyl magnetic beads in purified water to obtain the final product, and adjust the concentration as needed.

4. Silicon hydroxyl magnetic beads, characterized in that, Prepared by the method of claim 1-3. Prepared by the method of claim 1-3.

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