Preparation method of nucleic acid molecule rapidly and quantitatively coupled with colloidal gold protected by ascorbic acid

Abstract

The invention relates to the technical field of synthesis of biological nano-materials, and provides a preparation method of nucleic acid molecules rapidly and quantitatively coupled with colloidal gold protected by ascorbic acid, ascorbic acid modified gold nanoparticles AA-AuNPs are adopted as a core carrier, strong electrostatic repulsion between the gold nanoparticles AuNPs and the nucleic acid molecules is effectively overcome, and the nucleic acid molecules are rapidly and quantitatively coupled to the colloidal gold nanoparticles AA-AuNPs. The nucleic acid loading efficiency and the coupling layer compactness are improved. In a sulfhydrylation terminal nucleic acid molecule system, AA-anion mediated electrostatic shielding effect and Au-S covalent bonds have a synergistic effect, so that sulfhydrylation terminal nucleic acid molecules and AA-AuNPs are instantly self-assembled and only mixed, and stable coupling is completed within 30 seconds. In a nucleic acid molecule system at the end of adenine, a concentration strategy is adopted, coupling of a single adenine base A1 to AA-AuNPs is utilized, dependence of a traditional method on a polyadenine sequence is broken through, universal coupling of nucleic acid molecules is achieved, and the density and stability of SNA are improved.

Description

Technical Field

[0001] The present invention relates to the technical field of biological nanomaterial synthesis, and more specifically, to a preparation method for rapidly and quantitatively coupling nucleic acid molecules with ascorbic acid-protected colloidal gold. Background Art

[0002] Spherical Nucleic Acid (SNA) is a core-shell type nanocomposite formed by the assembly of nucleic acids and nanoparticles. Its typical structure is a rigid hybrid system composed of a gold nanocore and a densely modified DNA shell layer on the surface. SNA not only combines the plasmonic effect of nanoparticles and the molecular recognition function of nucleic acids, but also generates unique physicochemical properties (such as enhanced cell penetrability, anti-enzymatic stability, etc.) through the synergistic effect of the two, making it show significant advantages in the fields of biosensing, theranostics, high-throughput molecular diagnosis, in vivo imaging, and dynamic nanoassembly. However, the existing SNA preparation technologies are limited by problems such as low nucleic acid loading efficiency, non-uniform surface modification, and process complexity, and it is difficult to meet the requirements of large-scale production and multi-scenario applications. Developing a universal synthesis method with high-efficiency coupling, controllable structure, and simplified operation has become the key challenge to promote its practical application.

[0003] In the construction of Spherical Nucleic Acid (SNA), the binding methods between DNA and gold nanoparticles (AuNPs) are divided into two major systems: thiolation modification and non-thiolation adsorption, which rely on Au-S covalent bonds and weak interactions such as adenine, respectively. Since the salt aging method based on Au-S bonds was proposed in 1996, the core process of thiolated DNA modification of gold nanoparticles (AuNPs) depends on gradually increasing the salt concentration to shield the electrostatic repulsion of DNA and achieve high-density modification. However, this method has significant defects: the preparation process takes more than 48 hours, and the high-salt environment is prone to inducing nanoparticle aggregation, affecting the stability of the final product. To improve the coupling efficiency of thiolated DNA, researchers have proposed various optimization strategies, such as low pH assistance, freeze-thaw, n-butanol dehydration, microwave, and acoustic levitation technologies. Although these methods can accelerate the coupling process to a certain extent, each still has limitations. Low pH conditions (pH = 3.0) will cause DNA depurination damage and increase operation complexity; the freeze-thaw method is not suitable for long-chain DNA due to the energy consumption during the phase change process and the ice crystal shearing effect; the n-butanol dehydration method requires organic solvents, which may affect biocompatibility; microwave and acoustic levitation technologies can improve the binding rate, but have strong equipment dependence and are accompanied by potential risks of thermal degradation or nanoparticle deformation. Therefore, the existing thiolated DNA modification methods still face multiple challenges in terms of efficiency, stability, and operability.

[0004] In the non-thiolated DNA system, the current coupling strategy mainly relies on the coordination adsorption of adenine (A) and AuNPs, and typical methods include salt aging and freeze-thaw methods. However, this strategy requires the introduction of polyadenine (≥2 A, commonly A) at the 3' or 5' end of the probe sequence. 10 ) as an anchoring module, thereby reducing the length of the effective sequence and limiting the flexibility of probe design. In addition, the preparation cycle is still more than 48 hours, and the DNA density of the final SNA is low, affecting its stability and biological function. Although the low pH method is also applicable to the immobilization of non-thiolated DNA, its complicated operation limits its wide application. In recent years, the microwave-assisted method has improved the coupling efficiency by additionally introducing a poly-T weak adsorption module, and the evaporation drying method can complete DNA modification within 12 minutes, but its broad-spectrum adaptability to monoadenine (A1) DNA is still limited. Therefore, how to improve the preparation efficiency of non-thiolated DNA-SNA while ensuring stability and break through the limitations of existing methods on sequence design is still a core scientific problem that needs to be solved in this field. Summary of the invention

[0005] The present invention provides a method for preparing a colloidal gold rapid quantitative coupling nucleic acid molecule protected by ascorbic acid, which can not only significantly improve the construction efficiency of SNA, but also take into account high sequence versatility, high DNA density and stability.

[0006] According to the invention, a method for preparing ascorbic acid protected colloidal gold for rapid quantitative coupling of nucleic acid molecules comprises directly mixing a colloidal solution of gold nanoparticles AA-AuNPs protected by ascorbic acid anions AA- as ligands with a nucleic acid molecule solution in a certain proportion to achieve rapid, quantitative and high-density coupling; wherein the nucleic acid molecules include nucleic acid molecules with adenine ends and nucleic acid molecules with thiolated ends; the nucleic acid molecules with thiolated ends achieve instant self-assembly through the electrostatic shielding effect mediated by AA and the synergistic effect of Au-S bonds; the nucleic acid molecules with adenine ends are coupled with AA-AuP through a single adenine base A.

[0007] Preferably, the number of adenine bases required for successful coupling of adenine-terminated nucleic acid molecules to AA-AuNPs is as low as 1 and the density is as high as 0.10 / nm. 2 The time required is less than 60 seconds; the time for the thiol-terminated nucleic acid molecules to successfully couple to AA-AuNPs is less than 30 seconds, and the density reaches 0.18 / nm 2 , and remains stable in high salt concentration environments.

[0008] Preferably, AA-AuNPs are gold nanoparticles with a diameter of 5-60 nanometers, and their surface ligand is ascorbate anion AA-; AA-AuNPs are directly hydrothermally synthesized using ascorbic acid as a reducing agent; or prepared by ligand exchange reaction of gold nanoparticles protected by other ligands; the other ligands include citric acid, CTAB, and PVP.

[0009] Preferably, only 1 adenine is contained at the 5' end or 3' end of the nucleic acid molecule with an adenine terminus; the chain length is 11-60 nt; the 5' end of the nucleic acid molecule with a thiolated terminus is modified with a C6-SH group, and the chain length is 21-60 nt.

[0010] Preferably, the coupling method of the nucleic acid molecule with an adenine terminus is to mix the nucleic acid molecule with an adenine terminus at a molar ratio of 150:1-500:1 with AA-AuNPs, add 9 times the volume of n-butanol for dehydration and concentration, and then add 0.5×TBE buffer for resuspension to form spherical nucleic acid; among them, the concentration method includes but is not limited to dehydration with n-butanol.

[0011] Preferably, the coupling method of the nucleic acid molecule with a thiolated terminus is to mix a 100 μM solution of the thiolated nucleic acid molecule with 1-10 nM AA-AuNPs at a molar ratio of 1000:1-10000:1, complete the coupling within 30 seconds by vortex oscillation at room temperature, and obtain high-purity spherical nucleic acid by centrifugal washing.

[0012] Preferably, by regulating the number of adenine A bases at the end of the nucleic acid molecule, the control of the surface modification density of gold nanoparticles is achieved; when the number of adenines at the 5' end or 3' end of the nucleic acid sequence is controlled within 1-30, the surface coupling density regulation of 0.08-0.13 per nm is achieved through the coordination of the terminal A with the surface of the gold nanoparticles; the coupling process is completed within 30-60 seconds at room temperature; the prepared spherical nucleic acid maintains monodisperse stability in a 1 M concentration NaCl solution, and no obvious aggregation phenomenon is observed after storage at 4°C for 7 days. 2 Preferably, the nucleic acid molecule coupling reaction occurs under neutral pH conditions.

[0013] Preferably, the coupling density of nucleic acid molecules on the surface of each gold nanoparticle is regulated within the range of 0.06-0.18 per nm; the change in density is achieved by adjusting the molar ratio of DNA to gold nanoparticles of 1000:1 to 10000:1; the coupling process completes nucleic acid modification within 15-30 seconds at room temperature by mixing; the prepared spherical nucleic acid shows a stable particle size distribution with PDI < 0.2 by DLS measurement in a 1 M NaCl solution, and remains monodisperse even after being placed in a 1 M NaCl solution for 7 days.

[0014] Preferably, the coupling density of nucleic acid molecules on the surface of each gold nanoparticle is regulated within the range of 0.06-0.18 per nm; the change in density is achieved by adjusting the molar ratio of DNA to gold nanoparticles of 1000:1 to 10000:1; the coupling process completes nucleic acid modification within 15-30 seconds at room temperature by mixing; the prepared spherical nucleic acid shows a stable particle size distribution with PDI < 0.2 by DLS measurement in a 1 M NaCl solution, and remains monodisperse even after being placed in a 1 M NaCl solution for 7 days. 2 Preferably, the coupling density of nucleic acid molecules on the surface of each gold nanoparticle is regulated within the range of 0.06-0.18 per nm; the change in density is achieved by adjusting the molar ratio of DNA to gold nanoparticles of 1000:1 to 10000:1; the coupling process completes nucleic acid modification within 15-30 seconds at room temperature by mixing; the prepared spherical nucleic acid shows a stable particle size distribution with PDI < 0.2 by DLS measurement in a 1 M NaCl solution, and remains monodisperse even after being placed in a 1 M NaCl solution for 7 days.

[0015] Aiming at the DNA assembly limitation caused by the strongly ionized surface of traditional citrate ligand gold nanoparticles (Cit-AuNPs) under near-neutral conditions, the present invention innovatively constructs a system of ascorbic acid-modified gold nanoparticles (AA-AuNPs). Mechanism studies have shown that: in a neutral pH environment, the strongly ionized nanointerface formed by Cit3- ligands will exacerbate the electrostatic repulsion with nucleic acid molecules; while the AA ligand forms a weakly ionized surface layer by converting to the single-charged AA- state, effectively reducing this electrostatic barrier. When a thiolated / non-thiolated nucleic acid and a metal nano-sol with ascorbic acid as a ligand are mixed, the thiol group forms an Au-S covalent bond with the Au atoms on the surface of the gold nanoparticles, and spherical nucleic acids are obtained by mixing; the non-thiolated nucleic acid coordinates and adsorbs with the Au atoms on the surface of the gold nanoparticles to realize the coupling of nucleic acid molecules on the surface of the nanoparticles, and spherical nucleic acids are obtained.

[0016] In the present invention, a nucleic acid molecule solution and a metal nano-colloid solution are directly mixed to form a core-shell structure with metal nanoparticles as the core and nucleic acid molecules as the shell, and the nucleic acid molecules are densely coupled on the surface of the metal nanoparticles.

[0017] The spherical nucleic acids prepared by the present invention have breakthrough advantages compared with existing synthesis technologies: existing methods rely on complex processes such as salt aging, low pH, freeze dehydration or organic solvent concentration, and have defects such as harsh reaction conditions and long time consumption (from several hours to several days); for non-thiolated DNA systems, although current technologies can avoid the need for thiol modification, they require that the DNA sequence must contain at least two consecutive adenines to achieve effective adsorption, and their strict sequence dependence leads to insufficient technical versatility and difficulty in adapting to the design of conventional DNA probes with a single adenine (A1) structure. The present invention proposes a method for the instant self-assembly of AuNPs-thiolated DNA and the universal coupling of non-thiolated DNA with single adenine based on the regulation of ascorbic acid ligands. Through the reconstruction of the ligand layer on the surface of AuNPs by ascorbic acid molecules, the double electric layer and the steric hindrance are simultaneously reduced, enabling the thiolated DNA to complete efficient self-assembly in seconds without salt aging. More breakthroughly, by establishing an adenine-ascorbic acid composite adsorption interface, the present method first realizes the universal coupling of non-thiolated DNA under single adenine conditions, successfully breaking through the sequence limitation of continuous double adenines in traditional technologies, and providing a solution for the customized design and industrial production of SNAs. Its "mix and get" process characteristics greatly reduce production costs and equipment dependence, and support kilogram-scale large-scale production. Description of the Drawings

[0018] Figure 1 Schematic diagram of the process for the one-step self-assembly of ascorbic acid ligand gold nanoparticles and thiolated nucleic acids to prepare spherical nucleic acids;

[0019] Figure 2Schematic diagram of the process for preparing spherical nucleic acids from ascorbic acid ligand gold nanoparticles and non-thiolated nucleic acids;

[0020] Figure 3 Transmission electron micrograph of ascorbic acid ligand gold nanoparticles;

[0021] Figure 4 Particle size distribution diagram of ascorbic acid ligand gold nanoparticles;

[0022] Figure 5 UV-vis diagrams before and after the formation of spherical nucleic acids;

[0023] Figure 6 Schematic diagram of the principle for the formation of spherical nucleic acids;

[0024] Figure 7 DLS diagrams before and after the formation of spherical nucleic acids;

[0025] Figure 8 Zeta potential diagrams before and after the formation of spherical nucleic acids;

[0026] Figure 9 Gel electrophoresis diagrams before and after the formation of spherical nucleic acids;

[0027] Figure 10 Salt tolerance stability diagram of spherical nucleic acids;

[0028] Figure 11 Fluorescence quantification diagram of spherical nucleic acids formed by coupling DNA with different sequence lengths. Detailed implementation manners

[0029] To further understand the content of the present invention, the present invention will be described in detail in combination with the accompanying drawings and embodiments. It should be understood that the embodiments are only for explaining the present invention rather than limiting it.

[0030] Example 1

[0031] As Figure 1 and Figure 2 shown, this example provides a method for preparing ascorbic acid-protected colloidal gold for rapid quantitative coupling of nucleic acid molecules, which directly mixes the colloidal solution of ascorbic acid anion AA--protected gold nanoparticles AA-AuNPs with the nucleic acid molecule solution in a certain proportion to achieve rapid, quantitative, and high-density coupling, effectively overcoming the strong electrostatic repulsion between gold nanoparticle bottles AuPs and nucleic acid molecules; wherein, the nucleic acid molecules include nucleic acid molecules with adenine terminals and nucleic acid molecules with thiolated terminals; the nucleic acid molecules with thiolated terminals achieve immediate self-assembly through the synergistic effect of the electrostatic shielding effect mediated by AA and the Au-S bond; the nucleic acid molecules with adenine terminals are coupled with AA-AuP through a single adenine base A.

[0032] The number of adenine bases required for the successful coupling of nucleic acid molecules with adenine termini to AA-AuNPs is as low as 1, and the density reaches 0.10 per nm. 2 The time required for the successful coupling of nucleic acid molecules with thiolated termini to AA-AuNPs is less than 30 seconds, and the density reaches 0.18 per nm. 2 And it remains stable in a high-salt concentration environment.

[0033] AA-AuNPs are gold nanoparticles with a diameter of 5-60 nanometers, and their surface ligand is ascorbate anion AA-; AA-AuNPs are directly hydrothermally synthesized using ascorbic acid as a reducing agent; or prepared by ligand exchange reaction of other ligand-protected gold nanoparticles with ascorbic acid; the other ligands include citric acid, CTAB, and PVP.

[0034] The 5' or 3' end of the nucleic acid molecule with an adenine terminus contains only 1 adenine; the chain length is 11-60 nt; the 5' end of the nucleic acid molecule with a thiolated terminus is modified with a C6-SH group, and the chain length is 21-60 nt.

[0035] The coupling method of nucleic acid molecules with adenine termini is to mix nucleic acid molecules with adenine termini and AA-AuNPs at a molar ratio of 150:1-500:1, add 9 volumes of n-butanol for dehydration and concentration, and then resuspend with 0.5×TBE buffer to form spherical nucleic acids; among them, the concentration method includes but is not limited to n-butanol dehydration.

[0036] The coupling method of nucleic acid molecules with thiolated termini is to mix a 100 μM solution of thiolated nucleic acid molecules and 1-10 nM AA-AuNPs at a molar ratio of 1000:1-10000:1, complete the coupling within 30 seconds by vortex oscillation at room temperature, and obtain high-purity spherical nucleic acids by centrifugal washing.

[0037] By regulating the number of adenine A bases at the termini of nucleic acid molecules, the control of the surface modification density of gold nanoparticles is achieved; when the number of adenine at the 5' or 3' end of the nucleic acid sequence is controlled within 1-30, a surface coupling density regulation of 0.08-0.13 per nm is achieved through the coordination of terminal A with the surface of gold nanoparticles. 2 The surface coupling density regulation is completed within 30-60 seconds at room temperature; the prepared spherical nucleic acids maintain monodisperse stability in a 1 M concentration NaCl solution, and no obvious aggregation phenomenon is observed after storage at 4°C for 7 days.

[0038] The nucleic acid molecule coupling reaction occurs under neutral pH conditions.

[0039] The coupling density of nucleic acid molecules on the surface of each gold nanoparticle is 0.06-0.18 per nm. 2Regulation within a certain range; the change in density is achieved by adjusting the molar ratio of DNA to gold nanoparticles from 1000:1 to 10000:1; the coupling process completes nucleic acid modification in 15 - 30 seconds at room temperature, and it can be obtained by mixing; the prepared spherical nucleic acid is measured by DLS in 1M NaCl solution, showing a stable particle size distribution with PDI < 0.2, and it remains monodispersed even after being placed in 1M NaCl solution for 7 days.

[0040] This example provides a spherical nucleic acid prepared by the above preparation method. In the spherical nucleic acid, 200 DNA strands are coupled to the surface of each gold nanoparticle, and it maintains dispersion stability in a high - concentration NaCl solution; the red - shift amount of the surface plasmon resonance absorption peak is ≥ 3nm.

[0041] In this example, ascorbic acid molecules achieve double - layer compression and steric - hindrance reduction by reconstructing the ligand layer on the surface of AuNPs, enabling the thiolated DNA to complete efficient self - assembly within seconds without salt aging. More importantly, for the first time, this method utilizes the adenine - ascorbic acid composite adsorption interface to achieve the universal coupling of non - thiolated DNA under the condition of single adenine (A1), breaking through the sequence limitation of continuous double - adenine in traditional methods. This strategy not only significantly improves the construction efficiency of SNA, but also takes into account high sequence versatility, high DNA density, and stability, providing a new solution for the customized design and industrial production of SNA.

[0042] In the following examples, the nucleic acids are exemplified by the following sequences for illustration, and their base sequences are as follows:

[0043]

[0044] All the sequences used are synthesized by Sangon Biotech.

[0045] Example 2

[0046] This example provides a spherical nucleic acid, and its preparation method includes the following steps:

[0047] 2.1. Prepare gold particles with ascorbic acid as the ligand (AA - AuNP). It is synthesized by the ascorbic acid reduction method from HAuCl4, and its synthesis steps are as follows:

[0048] Ascorbic acid reduction method: The preparation of gold nanoparticles adopts the ascorbic acid reduction-ligand synergistic regulation method, which includes the following steps: In a two-necked flask protected by nitrogen, add 100 mL of an aqueous solution of chloroauric acid (HAuCl4·3H2O) with a concentration of 0.25 mM. Heat the reaction system to 80 ± 5 °C and maintain a constant temperature through a constant temperature oil bath. Subsequently, add 2 mL of a 100 mM ascorbic acid solution, and at the same time, continuously stir with a magnetic stirrer at 800 rpm for 10 min. During the reaction process, it is observed that the color of the solution gradually changes from light yellow to characteristic wine red, indicating that the nucleation and growth process of gold nanoparticles is completed. Immediately transfer the flask to an ice-water bath for rapid cooling treatment after the reaction to effectively terminate particle aggregation and lock the morphological characteristics.

[0049] The morphology of the product was characterized by transmission electron microscopy (TEM) ( Figure 3 ), and the particle size of 60 randomly selected particles was statistically analyzed by combining with ImageJ software. The results showed that the prepared gold nanoparticles with ascorbic acid as the surface ligand had a particle size distribution of (24.0 ± 8.1) nm ( Figure 4 ).

[0050] 2.2. Preparation of gold nanogold solution: The gold nanoparticle solution was centrifugally concentrated and then dispersed in the aqueous phase to form a gold nanogold solution; among them, the process conditions for centrifugal concentration were: the centrifugal speed was 9000 rpm, and the centrifugal time was 20 min.

[0051] 2.3. Prepare a 100 μM nucleic acid solution of DNA21; take 5 μl of the above nucleic acid solution and add it to 50 μL of a 10 nM gold nanogold solution. Mix with a vortex mixer, and the nucleic acid molecules are densely coupled on the surface of the nanoparticles to form nanoparticles; then add deionized water to resuspend, centrifuge at 9000 rpm, remove the supernatant, add deionized water to resuspend and centrifuge for washing, and repeat the above operation 2 times to obtain spherical nucleic acid.

[0052] Example 3

[0053] The difference between this example and Example 2 is that:

[0054] Prepare a 100 μM nucleic acid solution of DNA-A1; take 5 μL of the above nucleic acid solution and add it to 50 μL of a 10 nM gold nanogold solution containing 5 mM salt. After vortexing and centrifuging, add 450 μL of n-butanol, vortex, and then add 100 μL of 0.5×TBE to resuspend. Centrifuge at 9000 rpm, remove the supernatant, add deionized water to resuspend and centrifuge for washing, and repeat the above operation 2 times to obtain spherical nucleic acid.

[0055] Example 4

[0056] This example conducts experimental analysis, specifically as follows:

[0057] To evaluate the superiority of gold nanoparticles with ascorbic acid as ligand (AA-AuNP), gold nanoparticles with sodium citrate as ligand (Cit-AuNP) of the same concentration and size were synthesized. In the experiment of synthesizing SNA provided in this example, equal volumes of AA-AuNP and Cit-AuNP were taken for parallel experiments. Both groups of systems were divided into experimental groups and control groups with / without DNA added. As Figure 5 shown in part a, when DNA21 was not added, both AA-AuNP and Cit-AuNP aggregated under 100 mM salt; after adding DNA21, Cit-AuNP still aggregated under 100 mM salt, but AA-AuNP successfully maintained the colloidal dispersion state even after 7 days under 1000 mM salt; as Figure 5 shown in part b, when DNA-A1 was not added, both AA-AuNP and Cit-AuNP aggregated; after adding DNA-A1, Cit-AuNP still showed aggregation, but AA-AuNP successfully maintained the colloidal dispersion state. It is shown that in a neutral pH environment, the strongly ionized nanointerface formed by Cit3- ligand will exacerbate the electrostatic repulsion with nucleic acid molecules; while the AA ligand forms a weakly ionized surface layer by converting to the single-charged AA- state, effectively reducing this electrostatic barrier. When the thiolated / non-thiolated nucleic acid and metal nanosol with ascorbic acid as ligand are mixed, the thiol forms an Au-S covalent bond with the Au atoms on the surface of the gold nanoparticles, and spherical nucleic acid is obtained by mixing; the non-thiolated coordinates and adsorbs with the Au atoms on the surface of the gold nanoparticles to realize the coupling of nucleic acid molecules on the surface of the nanoparticles, and spherical nucleic acid ( Figure 6 ).

[0058] Spectroscopic Characterization of the Coupling Effect of Gold Nanoparticles

[0059] The DNA21 / DNA-A1 modified gold nanoparticles (SNA) prepared in Example 2 and Example 3 were optically characterized using a UV-visible spectrophotometer. The specific operation is as follows: An aqueous solution of the original gold nanoparticles (AA-AuNP) and the prepared spherical nucleic acid solution were placed in a quartz cuvette and scanned over the full wavelength range (400 - 800 nm) at room temperature with a scanning interval of 0.5 nm.

[0060] As Figure 7 shown in part a, the original AA-AuNP showed a characteristic surface plasmon resonance absorption peak at 522.5 nm, while the maximum absorption peak of the spherical nucleic acid solution modified with DNA21 showed a significant red shift and stabilized at 526.5 nm. The maximum absorption peak of the spherical nucleic acid solution modified with DNA-A1 showed a significant red shift and stabilized at 525.5 nm ( Figure 7Part b) The mechanism for the generation of the redshift phenomenon is as follows: When single-stranded DNA is directionally modified through Au-S covalent bonds and coupled to the surface of gold nanoparticles or adenine forms a coordination bond with gold particles, the following dual effects are caused: (1) The dielectric constant on the surface of the nanoparticles changes due to the coating of the DNA molecular layer; (2) The surface charge distribution of AuNP is affected by the negatively charged DNA phosphate backbone, resulting in charge recombination. The above experimental data confirm the successful coupling of DNA molecules with gold nanoparticles.

[0061] Dynamic light scattering (DLS) particle size analysis

[0062] The particle size distribution of the spherical nucleic acids (SNA) in Example 2 and Example 3 was characterized using a dynamic light scattering spectrometer. The specific method is as follows: The samples before and after coupling (AA-AuNP and SNA) were diluted to the same concentration with ultrapure water and measured under constant temperature conditions at 25°C.

[0063] As Figure 8 shown in Part a), the hydrodynamic diameter of the original AA-AuNP was 43.8 nm, while the average particle size of the spherical nucleic acid significantly increased to 58.7 nm after being modified with DNA21, and the average particle size of the spherical nucleic acid significantly increased to 50.2 nm after being modified with DNA-A1. As Figure 8 shown in Part b). The particle size distribution curve shifted towards the larger size direction (the peak position shifted from 43.8 nm to 58.7 nm), indicating that after DNA molecules were directionally modified through Au-S covalent bonds and coupled to the surface of gold nanoparticles or adenine formed a coordination bond with gold particles, a stable monolayer coating structure was formed.

[0064] Surface charge analysis based on Zeta potential

[0065] The surface charge of the spherical nucleic acid (SNA) modified with DNA21 in Example 2 or DNA-A1 in Example 3 was characterized using a nanoparticle size and Zeta potential analyzer. The specific method is as follows: The samples before and after coupling (AA-AuNP and SNA) were diluted to the same concentration (0.1 nM) with an aqueous solution and three independent measurements were performed using a Malvern potentiometric sample cell under constant temperature conditions at 25°C.

[0066] The experimental data show ( Figure 9 in Part a) that the Zeta potential of the original AA-AuNP was (-21.1 ± 2.2) mV (n = 3), while the surface charge of the spherical nucleic acid significantly decreased to (-32.7 ± 3.7) mV (n = 3) after being modified with DNA21; and the surface charge of the spherical nucleic acid significantly decreased to (-31.4 ± 3.8) mV (n = 3) after being modified with DNA-A1 ( Figure 9Part b) This charge inversion phenomenon stems from the negative charge coverage of the phosphate backbone in DNA molecules: When single-stranded DNA is directionally coupled to the nanoparticle surface through Au-S bonds or adenine forms a coordination bond with gold nanoparticles for coupling, its phosphate groups form a stable charge recombination layer with the surface of AA-AuNP.

[0067] Verification of spherical nucleic acid coupling by gel electrophoresis migration behavior

[0068] The coupling effects of the spherical nucleic acids (SNA) prepared in Example 2 and Example 3 with unmodified gold nanoparticles (AA-AuNP) were verified and analyzed by agarose gel electrophoresis technology. Specifically, a 2 wt% agarose gel was used as the separation medium, and a 1×TBE buffer solution containing 89 mM Tris-borate and 2 mM EDTA (pH 8.0) was used as the electrophoresis solution system. During the sample pretreatment process, a solution containing 0.1% sodium dodecyl sulfate (SDS) was particularly used to re-concentrate and purify the SNA and AA-AuNP, and then it was mixed with an equal volume of 0.6 M sucrose solution to form a density gradient to ensure the effective sedimentation of the nanoparticles in the electrophoresis loading wells. Electrophoresis separation was carried out for 5 minutes under the condition of a constant voltage of 200 V, and the results are as Figure 10 shown with significant difference characteristics.

[0069] The electrophoresis pattern clearly shows that the spherical nucleic acid (SNA) prepared in the present invention exhibits an obvious retarded migration behavior in the gel matrix, and the migration distance of its band is significantly shorter than that of the unmodified gold nanoparticles (AA-AuNP). This phenomenon is attributed to the fact that the high-density nucleic acid molecules covalently coupled and modified on the SNA surface significantly increase the molecular weight of the complex, resulting in a decrease in the electrophoresis mobility; it proves the successful assembly of nucleic acid molecules on the gold nanoparticle surface.

[0070] Quantify the number of DNAs on each gold particle

[0071] To further verify the universality of sequence design of the spherical nucleic acids prepared by the present invention, DNA21 and DNA59 with significant length differences and secondary structure characteristics were selected as model sequences for the loading analysis. Specifically, carboxyfluorescein (FAM, excitation / emission wavelength 488 nm / 520 nm) was covalently modified at the 3'-end of the two oligonucleotide chains, and spherical nucleic acids were prepared according to the method described in Example 2. To accurately quantify the nucleic acid loading on the surface of single particles, the obtained SNA solution was incubated with 10 mM dithiothreitol (DTT, pH 8.0) at 60 °C with constant shaking for 10 hours, and the complete dissociation of the DNA-AuNP complex was achieved through a thiol competition displacement reaction. Fluorescence quantitative analysis of the modification density was performed on the spherical nucleic acids prepared according to the method described in Example 3. Fluorescent labeling (FAM) of DNA was required in this experiment. The spherical nucleic acid solution was incubated with mercaptoethanol at 60 °C for 10 hours, followed by fluorescence quantitative analysis. After centrifugation of the dissociation solution, it was scanned using a fluorescence spectrophotometer in the range of excitation wavelength 488 nm and emission wavelength 500 - 600 nm, and quantitative calculations were performed based on the pre-established FAM standard curve. As Figure 11 shown, the average loadings of DNA21-SNA and DNA59-SNA were 248 ± 15 strands / particle and 235 ± 18 strands / particle, respectively. This result indicates that the thiol-DNA directed coupling technology developed in this example is not affected by sequence secondary structure or steric hindrance effects. Among them, DNA-A1 could form spherical nucleic acids according to the method described in Example 3, and the number of DNA coupled to the surface of AA-AuNP was (194 ± 24), which indicates that the ideal sequence universality of synthesizing non-thiol DNA spherical nucleic acids relying only on one adenine has been achieved.

[0072] The above schematically describes the present invention and its implementation manners. This description is not restrictive, and what is shown in the drawings is only one of the implementation manners of the present invention. The actual structure is not limited thereto. Therefore, if those of ordinary skill in the art are inspired by it and, without departing from the gist of the present invention, creatively design structural manners and embodiments similar to the technical solution, they shall fall within the protection scope of the present invention.

Claims

1. A method for preparing ascorbic acid-protected colloidal gold rapidly and quantitatively conjugated with nucleic acid molecules, characterized in that: The colloidal solution of ascorbate anion AA⁻-protected gold nanoparticles AA-AuNPs is directly mixed with the nucleic acid molecule solution in a certain ratio to achieve rapid, quantitative, and high-density coupling; among them, the nucleic acid molecules include nucleic acid molecules with adenine termini and nucleic acid molecules with thiolated termini; the nucleic acid molecules with thiolated termini achieve immediate self-assembly through the synergistic effect of the electrostatic shielding effect mediated by AA and the Au-S bond; the nucleic acid molecules with adenine termini are coupled with AA-AuP through a single adenine base A.

2. The preparation method of an ascorbic acid-protected colloidal gold for rapid quantitative coupling of nucleic acid molecules according to claim 1, wherein: The number of adenine bases required for the successful coupling of nucleic acid molecules with adenine termini to AA-AuNPs is as low as 1, and the density reaches 0.10 per nm 2 ; the time required for the successful coupling of nucleic acid molecules with thiolated termini to AA-AuNPs is less than 30 seconds, and the density reaches 0.18 per nm 2 , and it remains stable in a high-salt concentration environment.

3. The preparation method of an ascorbic acid-protected colloidal gold for rapid quantitative coupling of nucleic acid molecules according to claim 1, wherein: AA-AuNPs are gold nanoparticles with a diameter of 5-60 nanometers, and their surface ligand is ascorbate anion AA⁻; AA-AuNPs are directly hydrothermally synthesized using ascorbic acid as a reducing agent; or prepared by ligand exchange reaction of other ligand-protected gold nanoparticles with ascorbic acid; the other ligands include citric acid, CTAB, and PVP.

4. The preparation method of ascorbic acid-protected colloidal gold for rapid quantitative coupling of nucleic acid molecules according to claim 1, characterized in that: The 5' end or 3' end of the nucleic acid molecule with an adenine terminus contains only 1 adenine; the chain length is 11-60 nt; the 5' end of the nucleic acid molecule with a thiolated terminus is modified with a C6-SH group, and the chain length is 21-60 nt.

5. The preparation method of an ascorbic acid-protected colloidal gold for rapid quantitative coupling of nucleic acid molecules according to claim 1, characterized in that: The coupling method of the nucleic acid molecule with an adenine terminus is to mix the nucleic acid molecule with an adenine terminus and AA-AuNPs at a molar ratio of 150:1-500:1, add 9 times the volume of n-butanol for dehydration and concentration, and then resuspend with 0.5×TBE buffer to form spherical nucleic acids; among them, the concentration method includes but is not limited to dehydration with n-butanol.

6. The preparation method of ascorbic acid-protected colloidal gold for rapid quantitative coupling of nucleic acid molecules according to claim 1, wherein: The coupling method of the nucleic acid molecule with a thiolated terminus is to mix the 100 μM thiolated nucleic acid molecule solution and 1-10 nM AA-AuNPs at a molar ratio of 1000:1-10000:1, complete the coupling within 30 seconds by vortex oscillation at room temperature, and obtain high-purity spherical nucleic acids by centrifugal washing.

7. A method for preparing ascorbic acid-protected colloidal gold for rapid quantitative coupling of nucleic acid molecules according to claim 5, characterized in that: By regulating the number of adenine (A) bases at the ends of nucleic acid molecules, the surface modification density of gold nanoparticles is controlled; when the number of adenine bases at the 5' or 3' end of the nucleic acid sequence is controlled between 1 and 30, a surface coupling density regulation of 0.08 - 0.13 per nm is achieved through the coordination of the terminal A with the surface of the gold nanoparticles; the coupling process is completed within 30 - 60 seconds at room temperature; the prepared spherical nucleic acid maintains monodisperse stability in a 1 M concentration NaCl solution, and no obvious aggregation phenomenon is observed after storage at 4°C for 7 days. 2 The surface coupling density regulation of...; the coupling process is completed within 30 - 60 seconds at room temperature; the prepared spherical nucleic acid maintains monodisperse stability in a 1 M concentration NaCl solution, and no obvious aggregation phenomenon is observed after storage at 4°C for 7 days. It should be noted that there seems to be some incomplete or unclear information in the original text, especially in the part " 2 " and the beginning of "", which may affect the full understanding and translation quality. You may want to check and clarify the content for more accurate translation.

8. The preparation method of an ascorbic acid-protected colloidal gold for rapid quantitative coupling of nucleic acid molecules according to claim 1, characterized in that: The nucleic acid molecule coupling reaction occurs under neutral pH conditions.

9. The preparation method of an ascorbic acid-protected colloidal gold for rapid quantitative coupling of nucleic acid molecules according to claim 7, characterized in that: The coupling density of nucleic acid molecules on the surface of each gold nanoparticle is regulated within the range of 0.06 - 0.18 molecules / nm 2 The density is adjusted by regulating the molar ratio of DNA to gold nanoparticles from 1000:1 to 10000:1; the nucleic acid modification is completed within 15 - 30 seconds under room temperature conditions during the coupling process by mixing; the prepared spherical nucleic acid is measured by DLS in 1M NaCl solution, showing a stable particle size distribution with PDI < 0.2, and remaining monodispersed even after being placed in 1M NaCl solution for 7 days.

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