A method for controlling the size of nanosilica and a preparation method of DNA tracer

Abstract

The application discloses a size control method of nano-silicon dioxide and a preparation method of a DNA tracer, and comprises the following steps: mixing anhydrous ethanol, triethylamine and ultrapure water to obtain an initial solution; adding tetraethyl orthosilicate into the initial solution, stirring and standing for a period of time to obtain a silica reaction stock solution; mixing the silica reaction stock solution with anhydrous ethanol and stirring; adding tetraethyl orthosilicate into the mixed solution after stirring and reacting for a period of time, repeating the operation until the silica particles in the reactants reach the required size, and finally obtaining the end product. The application has a greater time advantage in obtaining a specific size of the silica core without complex reaction conditions, does not need to carry out the reaction from the beginning to the end to obtain the specific size of the core, has a better industrial production prospect, and improves the adaptability of the preparation process of the DNA tracer to different scenes.

Description

Technical Field

[0001] This invention relates to the field of nanomaterials technology, and in particular to a method for controlling the size of nano-silica and a method for preparing DNA tracers. Background Technology

[0002] DNA tracers of different sizes exhibit varying permeability in different scenarios. Selecting a size appropriate for the application scenario ensures more reliable data. The choice of DNA tracer size depends on the availability of nano-silica of different sizes. Current methods for obtaining nano-silica require a complete reaction from start to finish to obtain a core of a specific particle size. This results in a long time required to obtain silica cores of a specific size, causing DNA tracers to lack adaptability to different size requirements.

[0003] Therefore, the applicant has developed a method for controlling the size of nano-silica and a method for preparing DNA tracers to solve the above problems. Summary of the Invention

[0004] This invention proposes a method for controlling the size of nano-silica and a method for preparing DNA tracers, in order to solve the problems of long time required to obtain silica cores of specific sizes in existing technologies and the lack of adaptability of DNA tracers to different size requirements.

[0005] The present invention achieves the above objectives through the following technical solutions:

[0006] This invention provides a method for controlling the size of nano-silica, comprising the following steps:

[0007] Anhydrous ethanol, triethylamine and ultrapure water were mixed and stirred to obtain the initial solution;

[0008] Tetraethyl orthosilicate was added to the initial solution, stirred and allowed to stand for a period of time to obtain the silica reaction stock solution.

[0009] Determine whether the size of the silica particles in the silica reaction stock solution has reached the required size. If yes, the final reactant is obtained. If not, the silica reaction stock solution is mixed with anhydrous ethanol and stirred. Tetraethyl orthosilicate is added to the stirred mixture and reacted for a period of time. The operation is repeated until the silica particles in the reactant reach the required size, and the final reactant is obtained.

[0010] In this reaction, various raw materials are first uniformly mixed by stirring. Tetraethyl orthosilicate hydrolyzes to generate silanol Si-OH. As the hydrolysis proceeds, the silanol gradually forms a Si-O-Si network structure under the catalysis of triethylamine. This condensation reaction causes particle formation and growth until a three-dimensional network structure of silica nuclei is formed. The silica nucleates, and the hydroxyl groups on the surface are negatively charged. Under static conditions, the positively charged triethylamine is adsorbed. As the encapsulation of triethylamine increases, the triethylamine exerts a steric hindrance effect, preventing the silica surface from continuing to undergo condensation reactions with free silanols or oligomers, thus inhibiting the particle size growth of the silica nuclei. The equilibrium point between the amount of triethylamine and tetraethyl orthosilicate and the silica size is around 50 nm. The silica synthesized in this way has a large number of reactive sites on its surface. Outside of triethylamine, a large amount of silanols and oligomers that have not reached the nucleation concentration are retained. When the triethylamine detaches from adsorption, the silica can continue to condense with silanols to increase the particle size. Furthermore, the most significant advantage of this method for synthesizing silicon dioxide is that it does not involve complex reaction control steps, can be stored for a long time under static conditions, is convenient to react, and has the conditions for industrial-scale preparation.

[0011] The silica reaction stock solution is diluted with anhydrous ethanol to reduce the concentration of silica, making it less likely for the cores to agglomerate during stirring.

[0012] By stirring, the adsorption equilibrium between triethylamine and silica is broken, exposing the hydroxyl groups on the silica surface. At this time, free silanols and oligomers will condense on the silica surface, increasing the silica particle size.

[0013] Adding tetraethyl orthosilicate is to replenish the silanols in the solution through hydrolysis. Adding tetraethyl orthosilicate in small amounts and multiple times is to control the concentration of free silanols produced by hydrolysis, avoid secondary nucleation causing uneven silica size, and steadily increase the existing silica particle size.

[0014] Further, anhydrous ethanol, triethylamine, and ultrapure water in a volume ratio of 30:1:10 were mixed and stirred to obtain the initial solution.

[0015] Further, anhydrous ethanol, triethylamine, and ultrapure water were mixed and stirred at 900 r / min at 25°C for 20 min to obtain the initial solution.

[0016] Furthermore, the volume ratio of the initial solution to the added tetraethyl orthosilicate is 41:3.

[0017] Further, tetraethyl orthosilicate is added to the initial solution, stirred at 900 r / min for 5 min, and allowed to stand at 25°C for 20-30 h to obtain the silica reaction stock solution.

[0018] Further, the silica reaction stock solution with a volume ratio of 1:1 is mixed with anhydrous ethanol and stirred.

[0019] Furthermore, the silica particles in the silica reaction solution have a diameter of 50 nm, and the silica particles in the final reactant have a diameter of 50 nm to 507 nm.

[0020] Adjusting the particle size between 50-507 nm is to meet the usage requirements of the final synthesized DNA nanoparticles in different environments. For example, DNA nanoparticles smaller than 200 nm can meet the requirements for circulation in vivo and have good in vivo circulation performance. DNA nanoparticles smaller than 100 nm are more uniformly distributed in fluids, have higher synchronization effects, and also have higher permeability. Moreover, DNA nanoparticles of different sizes can be used to determine the connectivity and dominant channels. Silica exceeding 500 nm in size simply requires more stacking of silicon sources, extending the overall nucleation time. Furthermore, beyond 500 nm, the silica concentration in the original solution can be further diluted by increasing the amount of silicon source added, or the silica size can be finely controlled by micro-stacking with larger cores.

[0021] Further, the silica reaction stock solution is mixed with anhydrous ethanol and stirred at a speed of 500 r / min.

[0022] Furthermore, the volume ratio of the mixture to the tetraethyl orthosilicate added therein is 4000:1-400:1.

[0023] Furthermore, tetraethyl orthosilicate is added to the stirred mixture and the reaction is carried out for 20-50 minutes.

[0024] This invention also provides a method for preparing a DNA nanotracer, comprising the following steps:

[0025] After centrifuging the final reactant obtained by the nano-silica size control method, the supernatant was discarded. The centrifuged precipitate was resuspended, washed, and centrifuged again. The washed and centrifuged solid was then dried.

[0026] Disperse dried silica with isopropanol, and repeat the operation until the silica is completely dispersed to obtain a silica isopropanol dispersion.

[0027] Add APTES to the silica isopropanol dispersion and stir. Then centrifuge and discard the supernatant. Suspend the precipitate with isopropanol, vortex and sonicate in an ultrasonic bath. Repeat the stirring and centrifugation operations in this step until unreacted APTES is completely removed. Finally, dry the precipitate to obtain aminated silica.

[0028] Aminated silica was dispersed in ultrapure water, vortexed and sonicated in an ultrasonic bath, then DNA solution was added, vortexed and sonicated in an ultrasonic bath, and the vortexing operation was repeated until the solution was homogeneous to obtain a dispersion, which was then allowed to stand.

[0029] The dispersion was centrifuged and the supernatant was discarded. The precipitate was then suspended in ultrapure water, vortexed, and sonicated in an ultrasonic bath until a homogeneous solution was formed. The dispersion was then centrifuged and the supernatant was discarded. The precipitate was then suspended in ultrapure water, vortexed, and sonicated in an ultrasonic bath until a homogeneous solution was formed. TMAPS methanol solution or APTES was then added to the solution and vortexed. Tetraethyl orthosilicate was then added and reacted at room temperature under vortexing. Tetraethyl orthosilicate was then added to the solution and the mixture was stirred. The stirred mixture was then centrifuged and the supernatant was discarded to obtain the DNA tracer.

[0030] The beneficial effects of this invention are as follows:

[0031] The present invention proposes a method for controlling the size of nano-silica and a method for preparing DNA tracers. This method does not require complex reaction conditions and has a greater time advantage in obtaining silica cores of a specific size. It does not require a complete reaction from start to finish to obtain a core of a specific particle size, and has better prospects for industrial production. It also improves the adaptability of the DNA tracer preparation process to different scenarios. Attached Figure Description

[0032] Figure 1 This is a scanning electron microscope image of aminated silicon dioxide at 50 nm in an embodiment of this application;

[0033] Figure 2 This is a scanning electron microscope image of 80 nm DNA nanoparticles in an embodiment of this application. Detailed Implementation

[0034] 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 with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. The components of the embodiments of the present invention described and shown in the accompanying drawings can generally be arranged and designed in various different configurations.

[0035] Therefore, the following detailed description of the embodiments of the invention provided in the accompanying drawings is not intended to limit the scope of the claimed invention, but merely to illustrate selected embodiments of the invention. All other embodiments obtained by those skilled in the art based on the embodiments of the invention without inventive effort are within the scope of protection of the invention.

[0036] It should be noted that similar labels and letters in the following figures indicate similar items. Therefore, once an item is defined in one figure, it does not need to be further defined and explained in subsequent figures.

[0037] In the description of this invention, it should be understood that the terms "upper," "lower," "inner," "outer," "left," "right," etc., indicate the orientation or positional relationship based on the orientation or positional relationship shown in the accompanying drawings, or the orientation or positional relationship commonly used when the product of this invention is in use, or the orientation or positional relationship commonly understood by those skilled in the art. They are only used to facilitate the description of this invention and to simplify the description, and are not intended to indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation. Therefore, they should not be construed as limitations on this invention.

[0038] Furthermore, the terms "first," "second," etc., are used only to distinguish descriptions and should not be interpreted as indicating or implying relative importance.

[0039] In the description of this invention, it should also be noted that, unless otherwise explicitly specified and limited, terms such as "set" and "connection" should be interpreted broadly. For example, "connection" can be a fixed connection, a detachable connection, or an integral connection; it can be a mechanical connection or an electrical connection; it can be a direct connection or an indirect connection through an intermediate medium; it can be a connection within two components. Those skilled in the art can understand the specific meaning of the above terms in this invention according to the specific circumstances.

[0040] The specific embodiments of the present invention will now be described in detail with reference to the accompanying drawings.

[0041] The anhydrous ethanol, triethylamine, tetraethyl orthosilicate, isopropanol, and APTES (γ-aminopropyltriethoxysilane) used in this application are all commercially available known products. The TMAPS methanol solution is a commercially available trimethyl[3-(trimethoxysilyl)propyl]ammonium chloride (50% in methanol) solution. The DNA dry powder is a commercially available product or a DNA dry powder designed by the user according to their needs.

[0042] The method for preparing the DNA solution in this application is as follows:

[0043] Two sample tubes containing 50 nmol / tube of DNA powder were placed at room temperature for slow thawing for 10-15 minutes. They were then centrifuged at 3000 rpm for 3 minutes to bring the powder to the bottom of the tubes. 1 mL of ultrapure water was added to each tube to disperse the DNA powder. The tubes were centrifuged at 3000 rpm for 3 minutes. The centrifuged solution was then transferred to a 15 mL centrifuge tube. The sample tubes were washed twice with 1 mL of ultrapure water and the water was added to the 15 mL centrifuge tube. 3.86 mL of ultrapure water was then added to the 15 mL centrifuge tube (total volume 9.86 mL). At this point, the DNA concentration was 50 μg / mL, and the DNA solution was obtained.

[0044] The present invention will be further illustrated by the following specific embodiments:

[0045] Example 1: Preparation of 50nm silicon dioxide.

[0046] In a 25 mL round-bottom flask, 15 mL of anhydrous ethanol, 0.5 mL of triethylamine, and 5 mL of ultrapure water were mixed. The mixture was stirred at 900 rpm at 25 °C for 20 min, and then 1.5 mL of tetraethyl orthosilicate (TEOS) was added. The mixture was then stirred at 900 rpm for 5 min and allowed to stand at 25 °C for 24 h. The diameter of the silica was monitored to obtain a silica stock solution with a diameter of 50 nm.

[0047] The diameter of silica can be monitored using existing particle size monitoring methods.

[0048] Example 2: Preparation of 187nm silicon dioxide

[0049] In a 25 mL round-bottom flask, mix 15 mL anhydrous ethanol, 0.5 mL triethylamine, and 5 mL ultrapure water. Stir the mixture at 900 rpm for 20 min at 25 °C. Then add 1.5 mL tetraethyl orthosilicate (TEOS). Stir the mixture at 900 rpm for 5 min and allow it to stand at 25 °C for 24 h. Mix 10 mL of the silica stock solution with 10 mL of anhydrous ethanol at 500 rpm. Add 25 μL of tetraethyl orthosilicate and react for 25 min. Monitor the diameter of the silica. Repeat this operation twice to obtain a silica stock solution with a diameter of 187 nm.

[0050] Example 3: Preparation of 206nm silicon dioxide

[0051] In a 25 mL round-bottom flask, mix 15 mL anhydrous ethanol, 0.5 mL triethylamine, and 5 mL ultrapure water. Stir the mixture at 900 rpm for 20 min at 25 °C. Then add 1.5 mL tetraethyl orthosilicate (TEOS). Stir the mixture at 900 rpm for 5 min and allow it to stand at 25 °C for 24 h. Mix 10 mL of the silica stock solution with 10 mL of anhydrous ethanol at 500 rpm. Add 25 μL of tetraethyl orthosilicate and react for 25 min. Monitor the diameter of the silica. Repeat this operation 4 times to obtain a silica stock solution with a diameter of 206 nm.

[0052] Example 4: Preparation of 213nm silicon dioxide

[0053] In a 25 mL round-bottom flask, mix 15 mL anhydrous ethanol, 0.5 mL triethylamine, and 5 mL ultrapure water. Stir the mixture at 900 rpm for 20 min at 25 °C. Then add 1.5 mL tetraethyl orthosilicate (TEOS). Stir the mixture at 900 rpm for 5 min and allow it to stand at 25 °C for 24 h. Mix 10 mL of the silica stock solution with 10 mL of anhydrous ethanol at 500 rpm. Add 25 μL of tetraethyl orthosilicate and react for 25 min. Monitor the diameter of the silica. Repeat this operation 5 times to obtain a silica stock solution with a diameter of 213 nm.

[0054] Example 5: Fabrication of a 507nm silicon dioxide core

[0055] In a 25 mL round-bottom flask, mix 15 mL anhydrous ethanol, 0.5 mL triethylamine, and 5 mL ultrapure water. Stir the mixture at 900 rpm for 20 min at 25 °C. Then add 1.5 mL tetraethyl orthosilicate (TEOS). Stir the mixture at 900 rpm for 5 min and allow it to stand at 25 °C for 24 h. Mix 10 mL of the silica stock solution with 10 mL of anhydrous ethanol at 500 rpm. Add 50 μL of tetraethyl orthosilicate and react for 20 min. Monitor the diameter of the silica. Repeat this operation 13 times to obtain a silica stock solution with a diameter of 507 nm.

[0056] In some embodiments, anhydrous ethanol, triethylamine and ultrapure water in a volume ratio of 30:1:10 are mixed and stirred to obtain the initial solution.

[0057] In some embodiments, anhydrous ethanol, triethylamine and ultrapure water are mixed and stirred at 900 r / min at 25°C for 20 min to obtain the initial solution.

[0058] In some embodiments, the volume ratio of the initial solution to the added tetraethyl orthosilicate is 41:3.

[0059] In some embodiments, tetraethyl orthosilicate is added to the initial solution, stirred at 900 r / min for 5 min, and allowed to stand at 25°C for 20-30 h to obtain the silica reaction stock solution.

[0060] In some embodiments, the silica reaction stock solution with anhydrous ethanol in a volume ratio of 1:1 is mixed and stirred.

[0061] In some embodiments, the diameter of the silica particles in the silica reaction stock solution is 50 nm, and the diameter of the silica particles in the final reactant is 50 nm-507 nm.

[0062] Adjusting the particle size between 50-507 nm is to meet the usage requirements of the final synthesized DNA nanoparticles in different environments. For example, DNA nanoparticles smaller than 200 nm can meet the requirements for circulation in vivo and have good in vivo circulation performance. DNA nanoparticles smaller than 100 nm are more uniformly distributed in fluids, have higher synchronization effects, and also have higher permeability. Moreover, DNA nanoparticles of different sizes can be used to determine the connectivity and dominant channels. Silica exceeding 500 nm in size simply requires more stacking of silicon sources, extending the overall nucleation time. Furthermore, beyond 500 nm, the silica concentration in the original solution can be further diluted by increasing the amount of silicon source added, or the silica size can be finely controlled by micro-stacking with larger cores.

[0063] In some embodiments, the silica reaction stock solution is mixed with anhydrous ethanol and then stirred at a speed of 500 r / min.

[0064] In some embodiments, the volume ratio of the mixture to the tetraethyl orthosilicate added therein is 4000:1-400:1.

[0065] In some embodiments, tetraethyl orthosilicate is added to the stirred mixture and the reaction is carried out for 20-50 minutes.

[0066] The following example, using the preparation of DNA nanotracers with a 50nm silica core, further illustrates this invention:

[0067] Preparation of silica isopropanol dispersion

[0068] The 50 nm silica core was centrifuged at 12000 rpm for 3 min, and the supernatant was discarded to remove triethylamine and free silanol. The solid was resuspended in anhydrous ethanol, washed, and centrifuged again. This washing process was repeated twice to further remove tetraethyl orthosilicate and triethylamine. Finally, the solid was dried completely in a 60 °C desiccator for 4 h.

[0069] Silica was dispersed in isopropanol (vortexed for 30 seconds and sonicated in an ultrasonic bath for 15 minutes). This process was repeated until the silica was completely dispersed and the liquid showed no stratification or precipitation. A silica isopropanol dispersion with a concentration of 10 mg / mL was prepared.

[0070] Silica amylation modification

[0071] Take 20 mL of the silica isopropanol dispersion. Add 100 μl of APTES. Stir at 900 rpm for 6 h at 60 °C. Centrifuge at 12000 rpm for 3 min and discard the supernatant. Resuspend the precipitate in isopropanol (vortex for 30 s and sonicate in an ultrasonic bath for 5 min). Repeat the stirring and centrifugation steps until unreacted APTES is completely removed. Finally, dry the precipitate in a 60 °C desiccator for 4 h. 50 mg of 50 nm aminated silica (SiO2-NH2) is obtained.

[0072] DNA encapsulated in silica

[0073] Prepare a 50 nmol / mL DNA working solution using ultrapure water. Disperse 50 mg of 50 nm aminated silica in 1 mL of ultrapure water (vortex for 30 s and sonicate in an ultrasonic bath for 5 min). Add 1 mL of DNA solution (vortex for 30 s and sonicate in an ultrasonic bath for 2 min). Repeat the dispersion process until the solution is homogeneous. Let stand for 25 min to allow the DNA to fully adsorb onto the silica surface. Centrifuge the mixture at 16000 rpm for 5 min and discard the supernatant. Resuspend the precipitate in 1 mL of ultrapure water, vortex for 30 s and sonicate in an ultrasonic bath for 2-5 min until a homogeneous solution is formed. Then centrifuge at 16000 rpm for 5 min and discard the supernatant. Resuspend the precipitate in 8 mL of ultrapure water, vortex for 30 s and sonicate in an ultrasonic bath for 2 min until a homogeneous solution is formed. Add 10 μL of APTES and vortex for 30 s, then add 20 μL of TEOS. The mixture was reacted at 25°C and 900 rpm for 4 hours at room temperature, followed by the addition of 160 μL of TEOS (tetraethyl orthosilicate). The mixture was then stirred at 25°C and 900 rpm for 48 hours. The mixture was centrifuged at 16000 rpm for 2-5 minutes, and the supernatant was discarded to obtain 80 nm DNA nanoparticles (SiO2-DNA-SiO2), i.e., DNA nanotracers.

[0074] The scanning electron microscope image of 50nm aminated silicon dioxide in the embodiments of this application is shown below. Figure 1 As shown, the scanning electron microscope image of the 80nm DNA nanoparticles prepared from the 50nm silica core in this embodiment of the application is as follows. Figure 2 As shown.

[0075] The advantages of this invention compared to the prior art are:

[0076] 1. No complex reaction conditions are required to prepare a large number of silica cores of about 50 nanometers in one go. In this invention, triethylamine is both a catalyst and can be mutually adsorbed with the negatively charged silica after nucleation to prevent the silica from continuing to grow. This is beneficial for the synthesis of small-diameter silica cores. Moreover, the reaction process is not affected by the reaction volume, which is conducive to large-scale production.

[0077] 2. Mix the 50nm silica core stock solution with anhydrous ethanol. Add tetraethyl orthosilicate and react for 20-50 minutes. Repeat this process. This allows for the adjustment of silica particle size from 50nm to 507nm. The adjustable silica particle size enables application in various scenarios, and the nanoparticles can move synchronously with the fluid. This improves data reliability.

[0078] 3. By repeatedly adding silicon sources in small amounts, the time required to prepare silica cores of a specific size is shortened. By adding silicon sources in small amounts multiple times, the market's demand for control over parameters such as particle size can be met more quickly without the need to readjust the core synthesis parameters.

[0079] 4. Multiple micro-additions avoid the scaling-up effect of nuclei in large-scale production, offering better prospects for industrial production. The hydrolysis and condensation processes of silicon source occur sequentially and simultaneously; the changing reaction rates of these two processes make the number of nuclei and particle size uncontrollable. Multiple micro-additions of the silicon source after dilution of the nucleus stock solution avoid conflicts between particle size control and nucleation control, which is beneficial for condition control in industrial production. It also avoids the impact of scaling-up effects caused by factors such as stirring on experimental results after the experimental parameters have been scaled up.

[0080] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the technical principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for controlling the size of nano-silica, characterized in that, Includes the following steps: Anhydrous ethanol, triethylamine and ultrapure water were mixed and stirred to obtain the initial solution; Tetraethyl orthosilicate is added to the initial solution, stirred, and allowed to stand for a period of time to obtain a silica reaction stock solution. During this process, tetraethyl orthosilicate hydrolyzes to generate silanol. As the hydrolysis reaction proceeds, the silanol gradually forms a Si-O-Si network structure under the catalysis of triethylamine, until a three-dimensional network structure of silica core is formed. The hydroxyl groups on the surface of silica nucleation are negatively charged and adsorb positively charged triethylamine under static conditions. As the encapsulation of triethylamine increases, triethylamine produces a steric hindrance effect, preventing the silica surface from continuing to undergo condensation reaction with free silanol or oligomers, thus inhibiting the particle size growth of the silica core. When triethylamine is desorbed, silica can continue to condense with silanol to increase particle size. Determine whether the size of the silica particles in the silica reaction stock solution has reached the required size. If yes, the final reactant is obtained. If not, the silica reaction stock solution is mixed with anhydrous ethanol and stirred. The adsorption equilibrium between triethylamine and silica is broken by stirring, exposing the hydroxyl groups on the silica surface. Tetraethyl orthosilicate is added to the stirred mixture and reacted for a period of time. The operation is repeated until the silica particles in the reactant reach the required size, and the final reactant is obtained.

2. The method for controlling the size of nano-silica according to claim 1, characterized in that, The initial solution was obtained by mixing anhydrous ethanol, triethylamine and ultrapure water in a volume ratio of 30:1:10 and stirring.

3. The method for controlling the size of nano-silica according to claim 1 or 2, characterized in that, The initial solution was obtained by mixing anhydrous ethanol, triethylamine and ultrapure water and stirring at 900 r / min at 25 °C for 20 min.

4. The method for controlling the size of nano-silica according to claim 1, characterized in that, The volume ratio of the initial solution to the added tetraethyl orthosilicate is 41:

3.

5. A method for controlling the size of nano-silica according to claim 1 or 4, characterized in that, Tetraethyl orthosilicate was added to the initial solution, and the mixture was stirred at 900 r / min for 5 min and allowed to stand at 25°C for 20-30 h to obtain the silica reaction stock solution.

6. The method for controlling the size of nano-silica according to claim 1, characterized in that, The silica reaction stock solution with a volume ratio of 1:1 was mixed with anhydrous ethanol and then stirred.

7. The method for controlling the size of nano-silica according to claim 1, characterized in that, The silica particles in the silica reaction stock solution have a diameter of 50 nm, and the silica particles in the final reactant have a diameter of 50 nm to 507 nm.

8. The method for controlling the size of nano-silica according to claim 7, characterized in that, The volume ratio of the mixture to the tetraethyl orthosilicate added therein is 4000:1-400:

1.

9. A method for controlling the size of nano-silica according to claim 1 or 8, characterized in that, After adding tetraethyl orthosilicate to the stirred mixture, react for 20-50 minutes.

10. A method for preparing a DNA tracer, characterized in that, Includes the following steps: After centrifuging the final reactant obtained by the nano-silica size control method according to any one of claims 1-9, the supernatant is discarded, the centrifuged precipitate is resuspended, then washed and centrifuged, and the washed and centrifuged solid is dried. Disperse dried silica with isopropanol, and repeat the operation until the silica is completely dispersed to obtain a silica isopropanol dispersion. Add APTES to the silica isopropanol dispersion and stir. Then centrifuge and discard the supernatant. Suspend the precipitate with isopropanol, vortex and sonicate in an ultrasonic bath. Repeat the stirring and centrifugation operations in this step until unreacted APTES is completely removed. Finally, dry the precipitate to obtain aminated silica. Aminated silica was dispersed in ultrapure water, vortexed and sonicated in an ultrasonic bath, then DNA working solution was added, vortexed and sonicated in an ultrasonic bath, and the dispersion operation was repeated until the solution was homogeneous to obtain a dispersion, which was then allowed to stand. The dispersion was centrifuged and the supernatant was discarded. The precipitate was then suspended in ultrapure water, vortexed, and sonicated in an ultrasonic bath until a homogeneous solution was formed. The dispersion was then centrifuged and the supernatant was discarded. The precipitate was then suspended in ultrapure water, vortexed, and sonicated in an ultrasonic bath until a homogeneous solution was formed. TMAPS methanol solution or APTES was then added to the solution and vortexed. Tetraethyl orthosilicate was then added and reacted at room temperature under vortexing. Tetraethyl orthosilicate was then added to the solution and the mixture was stirred. The stirred mixture was then centrifuged and the supernatant was discarded to obtain the DNA tracer.

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