Stable gold nanoparticles and methods of making and using the same

Abstract

The application provides a gold nanoparticle with enhanced stability and a preparation method and application thereof, and belongs to the technical field of gold nanoparticles. The gold nanoparticle with enhanced stability provided by the application is modified on the surface with a hairpin DNA; the nucleotide sequence of the hairpin DNA is shown as SEQ ID NO. 1. The gold nanoparticle of the application is modified by the hairpin DNA, and the stability is significantly improved, specifically including tolerance to a strong acid environment, stable existence in an environment with a pH of 2 or more, and a substantial improvement in salt tolerance, tolerance to 100 mM Mg 2+ of the above ion strength.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of gold nanoparticles, and particularly relates to a gold nanoparticle with enhanced stability and a preparation method and application thereof. BACKGROUND

[0002] Gold nanoparticles (AuNPs) have unique optical properties such as surface plasmon resonance (SPR), which have become one of the preferred materials for constructing rapid visual detection methods. DNA, as a natural biological molecule, is often used as a ligand to combine with AuNPs to form recognition elements (DNA-AuNPs), which are used to prepare rapid test strips, kits and the like, greatly improving the efficiency of food safety monitoring. However, gold nanoparticles face the challenge of poor stability in practical applications. Stability is crucial for gold nanoparticles, because unstable gold nanoparticles may agglomerate, oxidize or change their surface properties, resulting in a decrease in their performance or even loss of their original functions. For example, in biomedical applications, the agglomeration of gold nanoparticles may affect their distribution and metabolism in the body, and even cause toxicity problems; in catalytic applications, changes in surface properties may reduce their catalytic activity and selectivity.

[0003] The stability of AuNPs can be effectively improved by reasonable design of the surface ligand of AuNPs, selection of appropriate dispersants and solvent environments, and the like. The commonly used method is to add or modify high molecular substances such as polyethylene glycol (PEG), DNA, and the like. By adding high-concentration PEG to keep the colloid stable, although the modifiability of the surface of AuNPs is retained, this method greatly limits its application in actual samples considering the compatibility of different liquid food matrices and detection systems. Moreover, PEG is modified on the surface of AuNPs to improve stability by increasing steric hindrance, but due to the large molecular weight of PEG wrapped on the surface of AuNPs, it is difficult to modify other target recognition elements and cannot be applied. With the research on the topological structure of polymers, a new way to improve the stability of AuNPs is provided. The cyclic PEG is modified on the AuNPs to adjust the stability of AuNPs by changing the conformation of the ligand on the surface of the nanoparticles. The results show that the dispersion stability is good under extreme conditions such as freezing or heating, greatly improving the stability of AuNPs. However, the PEG cyclization step is complicated and the conditions are harsh, which is not conducive to large-scale production.

[0004] Hairpin DNA (HpDNA), a special DNA structure, forms through base complementarity or mismatch, enabling it to form quasi-circular DNA while retaining its 5' end for modification onto AuNPs surfaces. However, hairpin DNA, formed from only a few bases, is susceptible to melting or rearrangement of its stem double strand due to temperature fluctuations, pH shifts, and changes in ionic strength. This structural instability directly weakens the anti-aggregation ability of the DNA-AuNPs complex. Therefore, it is urgent to screen for hairpin DNA with highly stable conformations to construct gold nanoparticles that are salt- and acid-resistant with significantly improved stability. Summary of the Invention

[0005] In view of this, the purpose of the present invention is to provide a gold nanoparticle with enhanced stability, its preparation method and application.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0007] The present invention provides a gold nanoparticle with enhanced stability, wherein the surface of the gold nanoparticle is modified with hairpin DNA; the nucleotide sequence of the hairpin DNA is shown in SEQ ID NO.1.

[0008] Preferably, the 5' end of the hairpin DNA is connected with 1 to 10 T bases.

[0009] Preferably, the nucleotide sequence of the hairpin DNA includes any one of SEQ ID NO.2 to SEQ ID NO.6.

[0010] Preferably, the 5' end of the hairpin DNA is modified with a thiol group.

[0011] The present invention also provides a method for preparing the above-mentioned gold nanoparticles, comprising the following steps: mixing hairpin DNA with a gold nanoparticle solution, gradually adding a salt solution, increasing the salt concentration in the reaction solution by 50 mM at each step, with an interval of 1 h between each step, until reaching 300 mM, and centrifuging to obtain gold nanoparticles with hairpin DNA modified on the surface.

[0012] Preferably, the preparation method of the nano-gold solution includes the following steps: mixing water with a 1% (w / w) HAuCl4 solution, heating to boiling, then mixing with a 1% (w / w) sodium citrate solution, maintaining a gentle boil, and obtaining the nano-gold solution after the solution color stabilizes and no longer changes.

[0013] Preferably, the volume ratio of HAuCl4 solution to sodium citrate solution is 1:4.

[0014] Preferably, the salt solution is a NaCl solution with a concentration of 5M.

[0015] Preferably, the centrifugation conditions are 12,000 rpm for 15 minutes.

[0016] This invention also provides the application of the above-described gold nanoparticles or the above-described preparation method in biological detection or drug delivery.

[0017] The beneficial effects of this invention are:

[0018] The gold nanoparticles of this invention, modified with hairpin DNA, exhibit significantly improved stability, specifically including tolerance to strong acid environments (stable at pH 2 and above) and greatly enhanced salt tolerance (able to withstand 100 mM Mg). 2+ The above ionic strength. Attached Figure Description

[0019] Figure 1 The images show the spectral and electron micrographs of gold nanoparticles from different groups, where (a) is a structural simulation of hairpin DNA, (b) is a UV-vis spectrum, and (c) to (i) are TEM images of gold nanoparticles from different groups, respectively.

[0020] Figure 2 Potentiograms and FTIR spectra of gold nanoparticles from different groups are shown, where (a) is the potentiogram and (b) is the FTIR spectrum.

[0021] Figure 3 The results show the salt tolerance stability of different groups. (a) shows the results when the NaCl concentration in the system is 1M. The top image shows the photographic results of different groups, corresponding to D1, T0, T1, T3, T5, T7, and T10 from left to right. The bottom image shows the spectral measurement results. (b) shows the results when the MgCl2 concentration in the system is 50mM. The top image shows the photographic results of different groups, corresponding to D-1, T0, T1, T3, T5, T7, and T10 from left to right. The bottom image shows the spectral measurement results. (c) shows the results when the MgCl2 concentration in the system is 100mM. The top image shows the photographic results of different groups, corresponding to D-1, T0, T1, T3, T5, T7, and T10 from left to right. The bottom image shows the spectral measurement results.

[0022] Figure 4 The results show the acid resistance stability of different groups, where (a) is the result at pH 1, (b) is the result at pH 2, (c) is the result at pH 3, and (d) is the result at pH 4. The upper figures in (a) to (d) are the photographic results of different groups, corresponding to D1, T0, T1, T3, T5, T7, and T10 from left to right, respectively. The lower figures are the spectral measurement results. Detailed Implementation

[0023] The present invention provides a gold nanoparticle with enhanced stability, wherein the surface of the gold nanoparticle is modified with hairpin DNA; the nucleotide sequence of the hairpin DNA is 5'-TCTGCGAAGCAGA-3' (SEQ ID NO.1).

[0024] In this invention, the structural simulation diagram of the hairpin DNA with the nucleotide sequence shown in SEQ ID NO.1 is as follows: Figure 1 As shown in (a), the hairpin DNA with the nucleotide sequence shown in SEQ ID NO.1 has a strong intramolecular binding force and a Tm value of 85℃. It is not easily affected by high temperature or complex matrix and has strong conformational stability. Using it to modify gold nanoparticles can greatly improve the stability of gold nanoparticles.

[0025] In this invention, the 5' end of the hairpin DNA preferably has 1 to 10 T bases attached, and the nucleotide sequence of the 5' end of the hairpin DNA with 1 to 10 T bases attached preferably includes any one of 5'-TTCTGCGAAGCAGA-3' (SEQ ID NO. 2), 5'-TTTTCTGCGAAGCAGA-3' (SEQ ID NO. 3), 5'-TTTTTTCTGCGAAGCAGA-3' (SEQ ID NO. 4), 5'-TTTTTTTTCTGCGAAGCAGA-3' (SEQ ID NO. 5), and 5'-TTTTTTTTTTTCTGCGAAGCAGA-3' (SEQ ID NO. 6). In this invention, the 5' end of the hairpin DNA preferably has a thiol (-SH) modification.

[0026] The present invention also provides a method for preparing the above-mentioned gold nanoparticles, comprising the following steps: mixing hairpin DNA with a gold nanoparticle solution, gradually adding a salt solution, increasing the salt concentration in the reaction solution by 50 mM at each step, with an interval of 1 h between each step, until reaching 300 mM, and centrifuging to obtain gold nanoparticles with hairpin DNA modified on the surface.

[0027] In this invention, the preferred method for preparing the nano-gold solution includes the following steps: mixing water with a 1% (w / w) HAuCl4 solution, heating to boiling, then mixing with a 1% (w / w) sodium citrate solution, maintaining a gentle boil, and obtaining the nano-gold solution after the solution color stabilizes and no longer changes. In this invention, the preferred volume ratio of HAuCl4 solution to sodium citrate solution is 1:4; the preferred salt solution is NaCl solution, and the preferred concentration of the NaCl solution is 5M; the preferred centrifugation conditions are 12000 rpm for 15 min.

[0028] This invention also provides the application of the above-described gold nanoparticles or the above-described preparation method in biological detection or drug delivery.

[0029] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0030] Unless otherwise specified, the following embodiments are all conventional methods.

[0031] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0032] Example 1

[0033] A gold nanoparticle with enhanced stability, wherein the surface of the gold nanoparticle is modified with hairpin DNA containing thiol groups, the nucleotide sequence of the hairpin DNA containing thiol groups being 5'-SH-TCTGCGAAGCAGA-3'.

[0034] The preparation method is as follows:

[0035] (1) Synthesis of nano gold: Add 95ml of ultrapure water to an Erlenmeyer flask, add 1ml of 1% HAuCl4 solution, stir and heat to boiling, then add 4mL of 1% sodium citrate solution, continue stirring and keep boiling gently. After the solution color stabilizes and no longer changes, continue stirring until the solution cools to room temperature to obtain nano gold solution, which is stored at 4℃.

[0036] (2) An excess of hairpin DNA sequence (5'-SH-TCTGCGAAGCAGA-3') with thiol groups was added to the gold nanoparticle solution for reaction. The mixture was continuously treated with 5M NaCl solution, which was added dropwise. After the first drop, the salt concentration was 50mM. In each subsequent step, the salt concentration in the reaction solution was increased by 50mM, with an interval of 1h between each step, until it reached 300mM. The mixture was then centrifuged at 12000rpm for 15min to obtain gold nanoparticles.

[0037] Example 2

[0038] A gold nanoparticle with enhanced stability is provided, wherein the surface of the gold nanoparticle is modified with hairpin DNA containing thiol groups, and the nucleotide sequence of the hairpin DNA containing thiol groups is 5'-SH-TTCTGCGAAGCAGA-3'. The preparation method is the same as in Example 1.

[0039] Example 3

[0040] A gold nanoparticle with enhanced stability is provided, wherein the surface of the gold nanoparticle is modified with hairpin DNA containing thiol groups, and the nucleotide sequence of the hairpin DNA containing thiol groups is 5'-SH-TTTTCTGCGAAGCAGA-3'. The preparation method is the same as in Example 1.

[0041] Example 4

[0042] A stability-enhanced gold nanoparticle, the surface of which is modified with hairpin DNA containing thiol groups, the nucleotide sequence of which is 5'.

[0043] -SH-TTTTTTCTGCGAAGCAGA-3'. The preparation method is the same as in Example 1.

[0044] Example 5

[0045] A stability-enhanced gold nanoparticle, the surface of which is modified with hairpin DNA containing thiol groups, the nucleotide sequence of which is 5'.

[0046] -SH-TTTTTTTTCTGCGAAGCAGA-3'. The preparation method is the same as in Example 1.

[0047] Example 6

[0048] A stability-enhanced gold nanoparticle, the surface of which is modified with hairpin DNA containing thiol groups, the nucleotide sequence of which is 5'.

[0049] -SH-TTTTTTTTTTTCTGCGAAGCAGA-3'. The preparation method is the same as in Example 1.

[0050] Comparative Example 1

[0051] The difference from Example 1 is that the hairpin DNA in Example 1 is replaced with linear DNA, the nucleotide sequence of which is 5'-GGAACCCATAACAAAT-3' (SEQ ID NO. 7). All other aspects are the same as in Example 1. When modifying gold nanoparticles with the linear DNA, thiol modification is also required at the 5' end of the linear DNA. The nucleotide sequence of the linear DNA with the thiol group is 5'-GGAACCCATAACAAAT-3' (SEQ ID NO. 7).

[0052] -SH-GGAACCCATAACAAAT-3'.

[0053] Example 7

[0054] The gold nanoparticles obtained in Example 1 were designated as group T0 (also known as T0-AuNPs), the gold nanoparticles obtained in Example 2 were designated as group T1 (also known as T1-AuNPs), the gold nanoparticles obtained in Example 3 were designated as group T3 (also known as T3-AuNPs), the gold nanoparticles obtained in Example 4 were designated as group T5 (also known as T5-AuNPs), the gold nanoparticles obtained in Example 5 were designated as group T7 (also known as T7-AuNPs), the gold nanoparticles obtained in Example 6 were designated as group T10 (also known as T10-AuNPs), and the gold nanoparticles obtained in Comparative Example 1 were designated as group DNA1 (also known as DNA1-AuNPs, AuNP, D1, or D-1). The following tests were performed on each group:

[0055] (1) The UV-vis of the gold nanoparticles obtained in Examples 1-6 and Comparative Example 1 were measured respectively:

[0056] Each of the gold nanoparticles obtained in Examples 1-6 and Comparative Example 1 was prepared into a 10 μL system with water, wherein the concentration of each gold nanoparticle was 4 nM. The spectra of the different groups of gold nanoparticles were measured using Nano Drop, and the results are as follows: Figure 1 As shown in (b) of the diagram.

[0057] Further TEM characterization was performed on different groups, and the results are as follows: Figure 1 As shown in (c) to (i), it can be seen that the gold nanoparticles modified with different hairpin DNA are of the same size and have a uniform morphology, and there is no difference in morphology compared with the gold nanoparticles modified with linear DNA, indicating that the hairpin DNA modification of the present invention has no effect on the gold nanoparticles themselves.

[0058] (2) The potentials and FTIR spectra of gold nanoparticles from different groups were measured respectively, and the results are as follows: Figure 2 As shown. By Figure 2 As can be seen in (a), the zeta potential of gold nanoparticles modified with hairpin DNA is higher than that of gold nanoparticles modified with linear DNA, indicating that the charge stability of hairpin DNA is higher than that of linear DNA. Figure 2 As can be seen in (b), the characteristic peaks of hairpin DNA-modified gold nanoparticles show more significant intensity changes, indicating that the interaction between hairpin DNA and gold nanoparticles is stronger, which helps to improve the stability of gold nanoparticles.

[0059] (3) Salt tolerance stability test:

[0060] Each of the gold nanoparticles obtained in Examples 1-6 and Comparative Example 1 was used to prepare 10 μL systems with water, where the concentration of each gold nanoparticle was 4 nM. Different volumes of 5 M NaCl or MgCl2 solution were added, and the remaining volume was made up with water to prepare 1 M NaCl solution or 50 mM and 100 mM MgCl2 solutions, respectively. The color changes were observed and photographed after mixing, followed by spectral measurement. The results are as follows: Figure 3 As shown.

[0061] Depend on Figure 3 As shown in (a), when the NaCl concentration in the system is 1M, the nanoparticles modified with linear DNA sequences show no absorption peak, and the solution color turns purple. However, the nanoparticles modified with hairpin DNA all show a significant absorption peak at 525 nm, and the solution color remains red, indicating that hairpin DNA can maintain stable dispersion in a 1M NaCl system. To further verify the tolerable charge intensity, a stronger charge, Mg... 2+ To verify, from Figure 3 As can be seen from (b) in the figure, when Mg in the solution 2+ At a concentration of 50 mM, the nanoparticles modified with T3, T5, T7, and T10 all maintained stable dispersion, the solution appeared red, and the corresponding spectrum also showed a clear absorption peak at 525 nm without shift. Subsequently, further increases in Mg... 2+ Content up to 100mM, from Figure 3 As shown in (c), T3, T5, T7, and T10 still maintain stable absorption peaks. This indicates that the hairpin DNA-modified nanoparticles can withstand 100 mM Mg. 2+ The above ionic strength indicates that the ionic strength tolerance is significantly higher than that of linear DNA.

[0062] (4) Acid resistance stability test

[0063] Take the gold nanoparticles obtained in Examples 1-6 and Comparative Example 1, respectively, and prepare 10 μL systems with water, where the concentration of each gold nanoparticle is 4 nM. Add different volumes of HCl solution, and make up the remaining volume with water to prepare solutions with pH values ​​of 1, 2, 3, and 4, respectively. Observe the color changes after mixing and take pictures, then measure the spectra. The results are as follows: Figure 4 As shown.

[0064] Depend on Figure 4 As shown in (a), when pH is 1, both the hairpin DNA and linear DNA-modified nanoparticle solutions are blue, and their spectral peaks also shift. Figure 4As shown in (b), at pH 2, solutions of T7 and T10 appear red, and their spectra maintain an absorption peak at 525 nm, while other hairpin DNA and linear DNA cannot remain stable. Similarly, as pH increases, the stability of hairpin DNA and linear DNA gradually improves, reaching a point at pH 3 (see...). Figure 4 In (c) of the above, almost all hairpin DNA remains stable; at pH 4 (see [reference]). Figure 4 In (d) of the diagram, linear DNA also maintains stable dispersion. This indicates that hairpin DNA-modified gold nanoparticles can withstand strongly acidic environments and remain stable in environments above pH 2, exhibiting significantly higher stability than linear DNA.

[0065] 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 principle 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 stability-enhanced gold nanoparticle, characterized in that, The surface of the gold nanoparticles is modified with a hairpin DNA; the nucleotide sequence of the hairpin DNA is shown as SEQ ID NO. 1; The 5' end of the hairpin DNA is connected with 1-10 bases of T; The nucleotide sequence of the hairpin DNA comprises any one of SEQ ID NO. 2-SEQ ID NO.

6.

2. The gold nanoparticle of claim 1, wherein, The 5' end of the hairpin DNA is modified with a thiol group.

3. The method of claim 1 or 2, wherein the gold nanoparticles are prepared by the method comprising the steps of: The method comprises the following steps: mixing the hairpin DNA with a gold nanoparticle solution, gradually adding a salt solution, increasing the salt concentration in the reaction solution by 50mM at each step, with an interval of 1h, and finally to 300mM, and centrifuging to obtain gold nanoparticles modified with the hairpin DNA on the surface.

4. The production method according to claim 3, characterized by, The preparation method of the gold nanoparticle solution comprises the following steps: mixing water with a 1% HAuCl4 solution by mass fraction, heating to boiling, mixing with a 1% sodium citrate solution by mass fraction, keeping it slightly boiling, and obtaining the gold nanoparticle solution after the color of the solution is stable and no longer changes.

5. The preparation method according to claim 4, characterized in that, The volume ratio of the HAuCl4 solution to the sodium citrate solution is 1:

4.

6. The preparation method according to claim 3, characterized in that, The salt solution is a NaCl solution, and the concentration of the NaCl solution is 5M.

7. The preparation method according to claim 3, characterized in that, The centrifugation condition is 12000rpm for 15min.

8. The gold nanoparticles of any one of claims 1-2 or the preparation method of any one of claims 3-7 are used in biological detection or drug delivery.

Images