Ultra-high density spherical nucleic acid and method of making

Abstract

The application provides a kind of ultrahigh density spherical nucleic acid and preparation method, its preparation method is: after nucleic acid solution and metal nanometer glue solution are mixed, it is placed in acoustic levitation reactor and is reacted, after acoustic levitation is formed into suspension drop, nucleic acid molecule is coupled densely on the surface of nano particle and forms nano particle;Nanoparticle is resuspended, centrifugal washing, remove the nucleic acid molecule that is not coupled, and spherical nucleic acid is obtained.Using acoustic levitation standing wave, the mixed solution of nucleic acid and metal nanometer sol is acoustic levitated into suspension drop, based on the synergistic effect of nondestructive enrichment evaporation and colloid surface reconstruction generated by acoustic wave, nucleic acid molecules and metal nanoparticles are compressed in physical space, local concentration increases, while ligand molecules on metal nanoparticles are stripped under the action of ultrasound, metal nanoparticles form stable covalent bond with sulfydryl on nucleic acid, and a layer of high-density nucleic acid molecules is coupled on the surface, so that the prepared spherical nucleic acid has higher coupling density and better coupling uniformity.

Description

Technical Field

[0001] This invention relates to the field of bio-nanomaterial synthesis technology, and in particular to an ultra-high density spherical nucleic acid and its preparation method. Background Technology

[0002] Spherical nucleic acids (SNAs) are multivalent nucleic acid-nanoparticle conjugates, initially composed of a gold core and a DNA shell, exhibiting properties distinct from those derived from nanoparticles and DNA. These well-defined nanostructures can serve as novel tags for bio / chemical sensing, therapy, molecular diagnostics, imaging, and programmable DNA assembly. These promising applications present significant challenges to the simplicity, efficiency, versatility, and scalability of SNA fabrication processes.

[0003] The electrostatic bilayer and dense surface packing formed by ligands on nanoparticles often pose significant challenges to DNA coupling. Current methods primarily employ salt aging to shield the electrostatic repulsion between gold nanoparticles (AuNPs) and DNA. However, this method is time-consuming, requiring more than two days to prevent salt-induced AuNP aggregation.

[0004] To overcome this limitation, some advanced synthetic methods have been developed. For example, a method for rapid and efficient synthesis of SNA using low pH and freezing has been reported (ZHANG X, SERVOS MR, LIU J. Instantaneous and Quantitative Functionalization of Gold Nanoparticles with Thiolated DNA Using a pH-Assisted and Surfactant-Free Route[J]. JOURNAL OF THE AMERICAN CHEMICAL SOCIETY, 2012, 134(17): 7266-9. LIU B, LIU J. Freezing Directed Construction of Bio / Nano Interfaces: Reagentless Conjugation, Denser Spherical Nucleic Acids, and Better Nanoflares[J]. JOURNAL OF THE AMERICAN CHEMICAL SOCIETY, 2017, 139(28): 9471-4.). This method achieves rapid modification of AuNPs surface DNA under pH=3 and low-temperature freeze-thaw conditions, respectively. Another method for the rapid synthesis of SNAs based on the instantaneous dehydration of n-butanol has been developed (HAO Y, LI Y, SONG L, et al. Flash Synthesis of Spherical Nucleic Acids with Record DNA Density[J]. JOURNAL OF THE AMERICAN CHEMICAL SOCIETY, 2021, 143(8):3065-9.), which utilizes the instantaneous dehydration effect induced by n-butanol extraction to produce a uniformly mixed and highly concentrated dehydrated DNA / AuNPs "solid solution", thereby achieving high-density modification of DNA.

[0005] Here, there are also studies that utilize microwave-assisted heating drying (HUANG M, XIONG E, WANG Y, et al. Fastmicrowave heating-based one-step synthesis of DNA and RNA modified goldnanoparticles[J]. NATURE COMMUNICATIONS, 2022, 13(1)) and evaporative drying (YE Y, HAO Y, YEM, et al. Evaporative Drying: A General and Readily Scalable Route to Spherical Nucleic Acids with Quantitative, Fully Tunable, and Record-High DNA Loading[J]. SMALL, 2022, 18(24).) to achieve the synthesis of SNAs with high DNA or RNA loading. The above-mentioned SNA synthesis methods are all based on physical space compression and increase their local concentration through dehydration to achieve rapid and high-density coupling. However, these methods require organic reagents, high temperature, low temperature or acidic conditions, and the coupling density of DNA is low and the uniformity is poor. Therefore, it is necessary to propose a high-density, high-uniformity, and green method for preparing SNA. Summary of the Invention

[0006] In view of the shortcomings of the prior art, the purpose of this invention is to provide an ultra-high density spherical nucleic acid and its preparation method, so as to solve the problems of the prior art.

[0007] To achieve the above objectives, the present invention provides the following technical solution:

[0008] A method for preparing ultra-high density spherical nucleic acids, comprising the following steps:

[0009] S1. Prepare nucleic acid solution and metal nanogel solution;

[0010] S2. After mixing the nucleic acid solution with the metal nanoparticle solution, the mixture is placed in an acoustic suspension reactor for reaction. After acoustic suspension into suspended droplets, the nucleic acid molecules are densely coupled on the surface of the nanoparticles to form nanoparticles.

[0011] S3. The nanoparticles obtained in step S2 are resuspended, centrifuged and washed to remove uncoupled nucleic acid molecules, resulting in spherical nucleic acids.

[0012] In a further embodiment, the nucleic acid solution in step S1 is a solution with a concentration of 10-100 μM formed by dissolving nucleic acid in deionized water.

[0013] In a further embodiment, the 5' end of the nucleotide base sequence of the nucleic acid is modified with a coupling group;

[0014] The base is one or more of adenine, guanine, thymine, or cytosine; the coupling group is one of thiol, carboxyl, amino, or silane.

[0015] Preferably, the length of the nucleotide base sequence is 10-100 bp.

[0016] In a further embodiment, the metal nanoparticle gel solution is formed by dispersing metal nanoparticles in an aqueous phase, and the concentration of the metal nanoparticle gel solution is 1-150 nM.

[0017] In a further embodiment, the metal nanoparticles are one of gold, silver, or copper;

[0018] The morphology of metal nanoparticles includes one or more of the following: nanospheres, nanorods, nanocubes, and nanooctahedrons.

[0019] The particle size of the metal nanoparticles is 5-60 nm.

[0020] Preferably, the gold nanoparticle adhesive solution is prepared as follows:

[0021] HAuCl4 was synthesized into gold nanoparticles using a reduction method. After repeated reduction, gold nanoparticles of various particle sizes were obtained. The synthesis solution was concentrated by centrifugation and then dispersed in the aqueous phase to form a gold nanoparticle gel solution. The reduction method included citrate reduction, citrate / tannic acid reduction, and hydroxylamine hydrochloride reduction.

[0022] In a further embodiment, the molar ratio of nucleic acid to metal nanoparticles in step S2 is 200:1-5000:1;

[0023] The acoustic suspension reactor has a power of 2-100mW and a reaction time of 5-60min; the number of suspended droplets is 1-20 drops.

[0024] In a further embodiment, the centrifugal washing in step S3 is performed at a speed of 3000-20000 rpm for 50-60 min.

[0025] The washing process involves centrifugation 2-4 times, using deionized water or PBS buffer solution as the washing solvent.

[0026] Another objective of this invention is to provide a spherical nucleic acid prepared by the above-described preparation method.

[0027] Acoustic levitation reactors are commercially available devices that suspend fluids and solid particles in an acoustic field. Acoustic levitation utilizes the interaction between standing waves in an acoustic field and an object to generate a vertical levitation force that overcomes the object's weight, allowing droplets to suspend in space. Without solid-liquid or solid-gas interfaces, it completely overcomes boundary effects and minimizes the physicochemical effects on solid surfaces, showing great potential for application in the synthesis of multifunctional suspended assemblies. By suspending droplets of a mixed solution containing nucleic acids and metal nanocolloids using acoustic levitation standing waves, the ligands on the metal nanoparticles detach under the synergistic effect of colloidal surface reconstruction and non-destructive enrichment evaporation generated by the acoustic waves, simultaneously coupling dense nucleic acid molecules to obtain spherical nucleic acids with ultra-high density modification.

[0028] This invention uses an acoustic suspension reactor to react nucleic acid molecule solution with metal nanoparticle gel solution to form a core-shell structure with metal nanoparticles as the core and nucleic acid molecules as the shell, with nucleic acid molecules densely coupled on the surface of metal nanoparticles.

[0029] This invention utilizes standing waves in an acoustic levitation reactor to acoustically suspend a mixture of nucleic acid and metal nanosol into suspended droplets. Furthermore, based on the synergistic effect of non-destructive enrichment evaporation and colloidal surface reconstruction generated by acoustic waves, boundary effects are avoided, and the electrostatic repulsion between metal nanoparticles and nucleic acid molecules is overcome. This physically compresses the nucleic acid molecules and metal nanoparticles, continuously increasing their local concentration. Simultaneously, ligand molecules on the metal nanoparticles are stripped away under ultrasonic action, allowing stable covalent bonds to form between the metal nanoparticles and the thiol groups on the nucleic acids. This results in a high-density layer of nucleic acid molecules coupled to the surface, giving the prepared spherical nucleic acids higher coupling density and better coupling uniformity.

[0030] Compared with existing freezing and n-butanol dehydration methods, the spherical nucleic acids prepared by this invention have higher modification density and better uniformity. Furthermore, the particle size distribution of the coupled spherical nucleic acids shifts towards larger sizes, with the average size increasing from 15.7 nm to 37.8 nm. This expands the application range of spherical nucleic acids in fields such as bio / chemical sensing, therapy, molecular diagnostics, and imaging.

[0031] The preparation method of the present invention does not require organic reagents, high or low temperature or other harsh conditions, and realizes green and simple preparation. Attached Figure Description

[0032] Figure 1 This is a schematic diagram of the acoustic suspension droplet concentration process;

[0033] Figure 2 The UV-vis spectra before and after the formation of spherical nucleic acids in Example 2;

[0034] Figure 3 The images show the DLS patterns before and after the formation of spherical nucleic acids in Example 2.

[0035] Figure 4 A quantitative fluorescence diagram showing the formation of spherical nucleic acids by coupling AuNPs of different sizes with DNA of different sequence lengths;

[0036] Figure 5 Gel electrophoresis diagram of the coupling of AuNPs of different sizes with DNA of different sequence lengths to form spherical nucleic acids. Detailed Implementation

[0037] The following specific examples illustrate the implementation of this patent application. Those skilled in the art can easily understand other advantages and effects of this patent application from the content disclosed in this specification. This patent application can also be implemented or applied through other different specific embodiments, and the details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of this patent application. It should be noted that, unless otherwise specified, the embodiments and features in the embodiments of this application can be combined with each other.

[0038] Please see Figure 1-5 The present invention provides the following technical solution:

[0039] A method for preparing high-density spherical nucleic acids, such as Figure 1 As shown, a solution of gold nanoparticles and a solution of nucleic acid molecules are mixed and placed in an acoustic levitation reactor, where they are acoustically levied into droplets. Over time, the droplets gradually concentrate, and the coupling density gradually increases. The preparation method includes the following steps:

[0040] S1. Prepare nucleic acid solution and metal nanogel solution;

[0041] S2. After mixing the nucleic acid solution with the metal nanoparticle solution, the mixture is placed in an acoustic suspension reactor for reaction. After acoustic suspension into suspended droplets, the nucleic acid molecules are densely coupled on the surface of the nanoparticles to form nanoparticles.

[0042] S3. The nanoparticles obtained in step S2 are resuspended, centrifuged and washed to remove uncoupled nucleic acid molecules, resulting in spherical nucleic acids.

[0043] Among them, nucleic acid solution is a solution with a concentration of 10-100 μM formed by dissolving nucleic acid in deionized water.

[0044] The 5' end of the nucleic acid base sequence is modified with a coupling group, the base sequence being one or more of adenine, guanine, thymine, or cytosine; the coupling group structure is one of thiol, carboxyl, amino, or silane groups; the length of the nucleic acid base sequence is 10-100 bp.

[0045] The following examples illustrate the selection of nucleic acids using DNA10, DNA21, and DNA59 with 5' end modified with thiol groups. Their base sequences are as follows:

[0046]

[0047] Example 1:

[0048] 1. 5nm gold nanoparticles were synthesized from HAuCl4 via a citrate / tannic acid reduction method. The synthesis steps are as follows:

[0049] Citrate / tannic acid reduction method: Add 1 ml of 1% HAuCl4 and 79 ml of deionized water to an Erlenmeyer flask and heat to 60°C in a constant temperature water bath (solution A). Separately, add 4 ml of 1% trisodium citrate, 3 ml of 1% tannic acid, 3 ml of 25 mol / L potassium carbonate, and 10 ml of deionized water to a constant temperature water bath and heat to 60°C (solution B). Add solution B to solution A and maintain the temperature at 60°C with stirring and heating for 15 min. After the solution turns dark red, heat the mixed solution to boiling and maintain boiling for 5 min to obtain a 5 nm gold nanoparticle solution.

[0050] 2. Preparation of gold nanoparticle gel solution: The gold nanoparticle solution is concentrated by centrifugation and then dispersed in the aqueous phase to form a gold nanoparticle gel solution; the centrifugation concentration process conditions are: centrifugation speed of 20,000 rpm and centrifugation time of 60 min.

[0051] 3. Prepare 100 μM nucleic acid solutions of DNA10, DNA21, or DNA59 respectively; add 0.3 μl of each of the above three nucleic acid solutions to 10 μl and 15 nM gold nanoparticle gel solutions respectively, and place them in an acoustic suspension reactor for 30-60 min. After acoustic suspension into suspended droplets, the nucleic acid molecules are densely coupled on the surface of the nanoparticles to form nanoparticles; then add deionized water to resuspend, centrifuge at 20,000 rpm to remove the supernatant, add deionized water to resuspend, centrifuge and wash, repeat the above operation twice to obtain three kinds of spherical nucleic acids.

[0052] Example 2:

[0053] 1. 15nm gold nanoparticles were synthesized from HAuCl4 via citrate reduction. The synthesis steps are as follows:

[0054] Citrate reduction method: Add 98.9 ml of deionized water and 1 ml of 3% sodium citrate to an Erlenmeyer flask, heat to boiling at 400 rpm, then add 0.1 ml of 250 mM HAuCl4, keep boiling and stirring for 7 min, and cool in an ice bath to obtain a solution of 15 nm gold particles.

[0055] 2. Preparation of gold nanoparticle gel solution: The gold particle solution is concentrated by centrifugation and then dispersed in the aqueous phase to form a gold nanoparticle gel solution; the centrifugation concentration process conditions are: centrifugation speed of 12000 rpm and centrifugation time of 15 min.

[0056] 3. Prepare 100 μM DNA10, DNA21 or DNA59 solutions. Take 0.3 μl of each of the above three nucleic acid solutions and add them to 10 μl of 5 nM gold nanoparticle gel solution respectively. Place them in an acoustic suspension reactor for 30-60 min. After acoustic suspension into suspension droplets, the nucleic acid molecules are densely coupled on the surface of the nanoparticles to form nanoparticles. Then add deionized water to resuspend, centrifuge at 12000 rpm and remove the supernatant. Add deionized water to resuspend, centrifuge and wash. Repeat the above operation twice to obtain three kinds of spherical nucleic acids.

[0057] Example 3:

[0058] 1. 25nm gold nanoparticles were synthesized from HAuCl4 via citrate reduction and hydroxylamine hydrochloride reduction. The synthesis steps are as follows:

[0059] Citrate reduction method: Add 98.9 ml of deionized water and 1 ml of 3% sodium citrate to an Erlenmeyer flask, heat to boiling at 400 rpm, then add 0.1 ml of 250 mM HAuCl4, keep boiling and stirring for 7 min, and cool in an ice bath to obtain a solution of 15 nm gold particles.

[0060] Hydroxylamine hydrochloride reduction method: Take 10 ml of 15 nm gold particle solution, add 9.8 ml of deionized water, 100 mM hydroxylamine hydrochloride and 0.2 ml of 1% sodium citrate, stir at 400 rpm for 5 min, then add 47.1 μl of 250 mM HAuCl4 and continue stirring for 1 h to obtain a solution containing 25 nm gold particles;

[0061] 2. Preparation of gold nanoparticle gel solution: The solution of gold particles is concentrated by centrifugation and then dispersed in the aqueous phase to form a gold nanoparticle gel solution; wherein, the process conditions for centrifugation concentration are: centrifugation speed of 8000 rpm and centrifugation time of 10 min.

[0062] 3. Prepare 100 μM DNA10, DNA21, or DNA59 solutions. Take 0.3 μl of each of the above three nucleic acid solutions and add them to 10 μl and 1 nM gold nanoparticle solutions, respectively. Place them in an acoustic suspension reactor for 30-60 min. After acoustic suspension into droplets, the nucleic acid molecules are densely coupled on the surface of the nanoparticles to form nanoparticles. Then, add deionized water to resuspend, centrifuge at 8000 rpm, remove the supernatant, add deionized water to resuspend, centrifuge and wash. Repeat the above operation twice to obtain three kinds of spherical nucleic acids.

[0063] Example 4:

[0064] 1. 45nm gold nanoparticles were synthesized from HAuCl4 via citrate reduction and hydroxylamine hydrochloride reduction. The synthesis steps are as follows:

[0065] Citrate reduction method: Add 98.9 ml of deionized water and 1 ml of 3% sodium citrate to an Erlenmeyer flask, heat to boiling at 400 rpm, then add 0.1 ml of 250 mM HAuCl4, keep boiling and stirring for 7 min, and cool in an ice bath to obtain a solution of 15 nm gold particles.

[0066] Hydroxylamine hydrochloride reduction method: Take 10 ml of 15 nm gold particle solution, add 9.8 ml of deionized water, 100 mM hydroxylamine hydrochloride and 0.2 ml of 1% sodium citrate, stir at 400 rpm for 5 min, then add 47.1 μl of 250 mM HAuCl4 and continue stirring for 1 h to obtain 25 nm gold particle solution.

[0067] Hydroxylamine hydrochloride reduction method: Take 10 ml of 25 nm gold particle solution, add 9.8 ml of deionized water, 100 mM hydroxylamine hydrochloride and 0.2 ml of 1% sodium citrate, stir at 400 rpm for 5 min, then add 47.1 μl of 250 mM HAuCl4 and continue stirring for 1 h to obtain a solution of 45 nm gold particles;

[0068] 2. Preparation of gold nanoparticle gel solution: The gold particle solution is concentrated by centrifugation and then dispersed in the aqueous phase to form a gold nanoparticle gel solution; the centrifugation concentration process conditions are: centrifugation speed of 6000 rpm and centrifugation time of 5 min.

[0069] 3. Prepare a 100 μM DNA10, DNA21, or DNA59 solution. Take 0.3 μl of each of the above three nucleic acid solutions and add them to 10 μl of 0.6 nM gold nanoparticle gel solution, respectively. Place them in an acoustic suspension reactor for 30-60 min. After acoustic suspension into droplets, the nucleic acid molecules are densely coupled on the surface of the nanoparticles to form nanoparticles. Then, add deionized water to resuspend, centrifuge at 6000 rpm, remove the supernatant, add deionized water to resuspend, centrifuge and wash. Repeat the above operation twice to obtain spherical nucleic acid.

[0070] Example 5:

[0071] like Figure 1 The top row shows a schematic diagram of the reaction. Nucleic acid molecules suspended in the droplets are densely coupled to the surface of gold nanoparticles under the action of the acoustic standing wave to form a nanoparticle structure. Figure 1The bottom row shows micrographs of the suspended droplets, demonstrating that the suspension reaction of this invention has a non-destructive enrichment effect. The volume of the suspended droplets gradually decreases over time. As shown in the figure, after 30 minutes, the volume of the suspended droplets shrinks from 1 mm to 110 micrometers, a reduction of nearly 10 times, which is significant. This is mainly because as the reaction proceeds, the solvent evaporates, causing the nucleic acid molecules and gold nanoparticles to be compressed in physical space. The concentration of nucleic acid molecules in the droplets continuously increases, and at the same time, under the action of ultrasound, the ligands on the surface of the gold nanoparticles are stripped, which helps the rapid and dense coupling of nucleic acid molecules to form spherical nucleic acids with ultra-high density modification.

[0072] The wavelengths of the maximum absorption peaks of the DNA59 nucleic acid molecules in Example 2 before and after coupling with 15 nm gold nanoparticles were determined using a UV-Vis spectrometer. Figure 2 As shown, the wavelength of the maximum absorption peak of the coupled spherical nucleic acid gradually increases with the progress of the acoustic suspension reaction. Specifically, after 30 minutes, the wavelength of the maximum absorption peak of the spherical nucleic acid changes from 520 nm to 530 nm.

[0073] The particle size distribution of the DNA21 nucleic acid molecules before and after coupling with 15 nm gold nanoparticles in Example 2 was determined using a DLS spectrometer. Figure 3 As shown, the particle size distribution of the coupled spherical nucleic acids shifts towards larger sizes, with the average size increasing from 15.7 nm to 37.8 nm.

[0074] This will further expand the application scope of the prepared spherical nucleic acids in fields such as bio / chemical sensing, therapy, molecular diagnostics, and imaging.

[0075] Example 6:

[0076] To demonstrate the high-density and high-uniformity modification of the spherical nucleic acids prepared in this invention, the following experiments were conducted:

[0077] Experiment 1:

[0078] The modified density of the spherical nucleic acids prepared in Examples 1-4 above was analyzed by fluorescence quantitative analysis. Fluorescent labeling (FAM) of the DNA was required in this experiment. The spherical nucleic acid solution was incubated with dithreitol at 60°C for 10 hours, followed by fluorescence quantitative analysis (excitation 495 nm, emission 500-600 nm) to obtain the results as follows. Figure 4 The coupling densities of gold nanoparticles with diameters of 5 nm, 15 nm, 25 nm, or 45 nm, respectively, with DNA10, DNA21, and DNA59 during the acoustic suspension reaction are shown.

[0079] Experiment 2:

[0080] A nano-gold solution of the same concentration and volume as in Experiment 1 was used to compare the spherical nucleic acids prepared by the freezing method and the n-butanol dehydration method reported in the background art with the nucleic acid molecular solution.

[0081] A bar chart comparing the coupling densities of spherical nucleic acids prepared by the three methods is shown below. Figure 4 As shown, the number of DNA molecules (unit: cells) coupled to the surface of gold nanoparticles in the spherical nucleic acids prepared by the present invention is significantly higher than that prepared by the freezing method and the n-butanol dehydration method. This indicates that the spherical nucleic acids prepared by the acoustic suspension method of the present invention have the highest coupling density.

[0082] Among them, 5nm gold nanoparticles and DNA10 formed spherical nucleic acids during the acoustic suspension reaction, with 45 DNA molecules coupled to the surface of the gold nanoparticles. This is comparable to the 46 spherical nucleic acids prepared by n-butanol dehydration and significantly higher than the 26 spherical nucleic acids prepared by the freezing method. 15nm gold nanoparticles and DNA10 formed spherical nucleic acids during the acoustic suspension reaction, with 621 DNA molecules coupled to the surface of the gold nanoparticles. This is significantly higher than the 266 and 461 spherical nucleic acids prepared by the freezing method and n-butanol dehydration method, respectively. During the acoustic levitation reaction, 25nm gold nanoparticles and DNA10 form spherical nucleic acids, with 1710 DNA molecules coupled to the surface of the gold nanoparticles, significantly higher than the 980 and 1350 spherical nucleic acids prepared by freezing and n-butanol dehydration methods, respectively. During the acoustic levitation reaction, 45nm gold nanoparticles and DNA10 form spherical nucleic acids, with 3555 DNA molecules coupled to the surface of the gold nanoparticles, significantly higher than the 2965 spherical nucleic acids prepared by n-butanol dehydration method, where freezing is not applicable.

[0083] Experiment 3:

[0084] Spherical nucleic acids prepared by the three methods in Experiment 2 and pure gold particle solutions were compared using agarose gel electrophoresis. The agarose concentration was 2 wt%, the electrophoresis buffer was 1xTBE, and the voltage was 13 V / cm. The results were as follows: Figure 5 The image shows a gel electrophoresis photograph. It can be seen from the figure that the spherical nucleic acid prepared in this invention has the narrowest bands, indicating better uniformity of its coupling density.

[0085] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for preparing ultra-high density spherical nucleic acids, characterized in that, Includes the following steps: S1. Prepare a nucleic acid solution and a metal nanogel solution; the 5' end of the nucleotide base sequence of the nucleic acid is modified with a coupling group, wherein the base is one or more of adenine, guanine, thymine or cytosine, and the coupling group is one of thiol, carboxyl, amino or silane. The length of the nucleotide base sequence is 10-100 bp; S2. The nucleic acid solution and the metal nanoparticle gel solution are mixed and placed in an acoustic levitation reactor for reaction. After acoustic levitation into suspended droplets, the nucleic acid molecules are densely coupled to the surface of the metal nanoparticles to form nanoparticles. The power of the acoustic levitation reactor is 2-100mW, the reaction time is 5-60min, and the number of suspended droplets is 1-20. S3. The nanoparticles obtained in step S2 are resuspended, centrifuged and washed to remove uncoupled nucleic acid molecules, resulting in spherical nucleic acids.

2. The preparation method according to claim 1, characterized in that: In step S2, the molar ratio of nucleic acid to metal nanoparticles is 200:1-5000:

1.

3. The preparation method according to claim 1, characterized in that: In step S1, the nucleic acid solution is a solution with a concentration of 10-100 μM formed by dissolving nucleic acid in deionized water.

4. The preparation method according to claim 1, characterized in that: The metal nanoparticle gel solution is formed by dispersing metal nanoparticles in an aqueous phase, and the concentration of the metal nanoparticle gel solution is 1-150 nM.

5. The preparation method according to claim 4, characterized in that: The metal nanoparticles are one of gold, silver, or copper.

6. The preparation method according to claim 4, characterized in that: The morphology of the metal nanoparticles includes one or more of the following: nanospheres, nanorods, nanocubes, and nanooctahedrons. The particle size of the metal nanoparticles is 5-60 nm.

7. The preparation method according to claim 5, characterized in that: The preparation method of the metal nanogel solution is as follows: HAuCl4 was synthesized into gold nanoparticles using a reduction method. After repeated reduction, gold nanoparticles of various particle sizes were obtained. The synthesis solution was concentrated by centrifugation and then dispersed in the aqueous phase to form a gold nanoparticle gel solution. The reduction method included citrate reduction, citrate / tannic acid reduction, and hydroxylamine hydrochloride reduction.

8. The preparation method according to claim 1, characterized in that: The centrifugal speed for centrifugal washing in step S3 is 3000. 20,000 rpm, time is 50 60min; The number of centrifugal washes was 2. Wash 4 times with deionized water or PBS buffer solution as the washing solvent.

9. A spherical nucleic acid prepared by any one of claims 1-8.

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