Gold nanoparticles with enhanced stability as well as preparation method and application of gold nanoparticles

Abstract

The invention provides gold nanoparticles with enhanced stability and a preparation method and application thereof, and belongs to the technical field of gold nanoparticles. The surface of the gold nanoparticle with enhanced stability provided by the invention is modified with hairpin DNA (Deoxyribose Nucleic Acid); the nucleotide sequence of the hairpin DNA is as shown in SEQ ID NO. 1. The gold nanoparticles are modified by hairpin DNA, the stability is remarkably improved, particularly, the gold nanoparticles can tolerate a strong acid environment, can stably exist in an environment with the pH value being 2 or above, and can tolerate the ionic strength of 100 mM Mg < 2 + > or above, and the salt tolerance is greatly improved.

Description

Technical Field

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

[0002] Gold nanoparticles (AuNPs) have become one of the preferred materials for constructing rapid visualization detection methods due to their unique optical properties such as surface plasmon resonance (SPR). As a natural biomolecule, DNA is often used as a ligand to bind with AuNPs to form a recognition element (DNA-AuNPs), which is used to prepare rapid detection test strips, kits, etc., greatly improving the efficiency of food safety monitoring. However, gold nanoparticles face challenges in poor stability in practical applications. Stability is crucial for gold nanoparticles because unstable gold nanoparticles may aggregate, oxidize, or change surface properties, resulting in a decline in their performance or even loss of their original functions. For example, in biomedical applications, the aggregation of gold nanoparticles may affect their distribution and metabolism in vivo, and even cause toxicity problems; in catalytic applications, the change in surface properties may reduce their catalytic activity and selectivity.

[0003] By means of reasonably designing the surface ligands of AuNPs, selecting appropriate dispersants and solvent environments, etc., the stability of AuNPs can be effectively improved. The currently commonly used method is to add or modify polymer substances, such as polyethylene glycol (PEG), DNA, etc. By adding a high concentration of PEG to keep the colloid stable, although the modifiability on the surface of AuNPs is retained, considering the compatibility of different liquid food matrices with the detection system, this method is greatly limited in the application of actual samples. Moreover, when PEG is modified on the surface of AuNPs to enhance stability by increasing steric hindrance, since the PEG molecule has a large molecular weight and is wrapped on the surface of AuNPs, it is difficult to modify other target recognition elements and thus cannot be applied. With the research on the topological structure of polymers, a new idea is provided for improving the stability of AuNPs. By modifying cyclic PEG on AuNPs, the stability of AuNPs is regulated by changing the conformation of the surface ligands of the nanoparticles. The results show that good dispersion stability is maintained under extreme conditions such as freezing or heating, greatly improving the stability of AuNPs. However, the PEG cyclization step is cumbersome and the conditions are harsh, which is not conducive to large-scale production.

[0004] Hairpin DNA (HpDNA), as a special DNA structure, forms HpDNA through base complementarity or mismatch. It can form circular-like DNA and retain the 5'-end that can be modified on the surface of AuNPs. However, the double-stranded stem of the hairpin DNA formed by only a few bases is vulnerable to temperature fluctuations, pH shifts, and changes in ionic strength, resulting in strand separation or rearrangement. This structural instability directly weakens the anti-aggregation ability of the DNA-AuNP complex. Therefore, it is urgent to screen hairpin DNAs with highly stable conformations to construct gold nanoparticles with salt tolerance, acid tolerance, and 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 invention purpose, the present invention provides the following technical solutions:

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

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

[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 preparation method of the above-mentioned gold nanoparticle, including the following steps: mixing the hairpin DNA with a nano-gold solution, gradually dropping a salt solution, increasing the salt concentration in the reaction solution by 50 mM each step, with an interval of 1 h between each step, and finally reaching 300 mM, and centrifuging to obtain a gold nanoparticle with hairpin DNA modified on its surface.

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

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

[0014] Preferably, the salt solution is an NaCl solution, and the concentration of the NaCl solution is 5 M.

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

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

[0017] Advantages of the present invention:

[0018] The gold nanoparticles of the present invention are modified with hairpin DNA, and the stability is significantly improved. Specifically, they can tolerate a strong acid environment, can stably exist in an environment with a pH above 2, and the salt tolerance is greatly improved, and they can tolerate an ionic strength of 100 mM Mg 2+ or higher. Description of the drawings

[0019] Figure 1 are the spectra and electron microscopy images of gold nanoparticles in different groups, where (a) is the structural simulation diagram of hairpin DNA, (b) is the UV-vis spectrum diagram, and (c) to (i) are the TEM images of gold nanoparticles in different groups respectively;

[0020] Figure 2 are the potential diagrams and FTIR spectrum diagrams of gold nanoparticles in different groups, where (a) is the potential diagram and (b) is the FTIR spectrum diagram;

[0021] Figure 3 are the salt tolerance stability results of different groups, where (a) is the result when the NaCl concentration in the system is 1 M. The upper figure is the photographing results of different groups, corresponding to D1, T0, T1, T3, T5, T7, T10 from left to right respectively, and the lower figure is the spectrum measurement result; (b) is the result when the MgCl2 concentration in the system is 50 mM. The upper figure is the photographing results of different groups, corresponding to D-1, T0, T1, T3, T5, T7, T10 from left to right respectively, and the lower figure is the spectrum measurement result; (c) is the result when the MgCl2 concentration in the system is 100 mM. The upper figure is the photographing results of different groups, corresponding to D-1, T0, T1, T3, T5, T7, T10 from left to right respectively, and the lower figure is the spectrum measurement result;

[0022] Figure 4 are the acid tolerance stability results 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, (d) is the result at pH 4. The upper figures in (a) to (d) are the photographing results of different groups, corresponding to D1, T0, T1, T3, T5, T7, T10 from left to right respectively, and the lower figures are the spectrum measurement results. Detailed implementation manners

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

[0024] In the present invention, the structural simulation diagram of the hairpin DNA with the nucleotide sequence shown in SEQ ID NO.1 is as shown in Figure 1 (a) in. The hairpin DNA with the nucleotide sequence shown in SEQ ID NO.1 has strong intramolecular binding force, its Tm value reaches 85 °C, is not easily interfered by high temperature or complex matrix, has strong conformational stability, and using it to modify gold nanoparticles can greatly improve the stability of gold nanoparticles.

[0025] In the present invention, preferably 1 to 10 bases T are connected to the 5'-end of the hairpin DNA, and the nucleotide sequences in which 1 to 10 bases T are connected to the 5'-end of the hairpin DNA preferably include 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), 5'-TTTTTTTTTTTCTGCGAAGCAGA-3' (SEQ ID NO.6). In the present invention, the 5'-end of the hairpin DNA is preferably modified with a thiol group (-SH).

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

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

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

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

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

[0031] In the following embodiments, the materials, reagents, etc. used, unless otherwise specified, can all be obtained from commercial channels.

[0032] Example 1

[0033] A gold nanoparticle with enhanced stability, the surface of the gold nanoparticle is modified with a hairpin DNA with a thiol group, and the nucleotide sequence of the hairpin DNA with a thiol group is 5'-SH-TCTGCGAAGCAGA-3'.

[0034] The preparation method is as follows:

[0035] (1) Synthesis of gold nanoparticles: Add 95 ml of ultrapure water to a conical flask, add 1 ml of a 1% HAuCl4 solution, stir and heat to boiling, then add 4 mL of a 1% sodium citrate solution, continue to stir and keep slightly boiling. After the solution color is stable and no longer changes, continue to stir. Wait for the solution to cool to room temperature to obtain a gold nanoparticle solution, and store it at 4°C.

[0036] (2) Add an excessive amount of the hairpin DNA sequence with a thiol group (5'-SH-TCTGCGAAGCAGA-3') to the gold nanoparticle solution for reaction, continuously treat the mixture with a 5M NaCl solution, and add dropwise gradually. The salt concentration is 50 mM after the first dropwise addition, and the salt concentration in the reaction solution is increased by 50 mM in each step. Each step is separated by 1 h, and finally reaches 300 mM. Then centrifuge the mixture at 12000 rpm for 15 min to obtain the gold nanoparticles.

[0037] Example 2

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

[0039] Example 3

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

[0041] Example 4

[0042] A gold nanoparticle with enhanced stability, the surface of the gold nanoparticle is modified with a hairpin DNA with a thiol group, and the nucleotide sequence of the hairpin DNA with a thiol group is 5’

[0043] -SH-TTTTTTCTGCGAAGCAGA-3’. The preparation method is the same as that of Example 1.

[0044] Example 5

[0045] A gold nanoparticle with enhanced stability, the surface of the gold nanoparticle is modified with a hairpin DNA with a thiol group, and the nucleotide sequence of the hairpin DNA with a thiol group is 5’

[0046] -SH-TTTTTTTTCTGCGAAGCAGA-3’. The preparation method is the same as that of Example 1.

[0047] Example 6

[0048] A gold nanoparticle with enhanced stability, the surface of the gold nanoparticle is modified with a hairpin DNA with a thiol group, and the nucleotide sequence of the hairpin DNA with a thiol group is 5’

[0049] -SH-TTTTTTTTTTTCTGCGAAGCAGA-3’. The preparation method is the same as that of 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, and the nucleotide sequence of the linear DNA is 5’-GGAACCCATAACAAAT-3’ (SEQ ID NO.7). The rest are the same as Example 1. When using the linear DNA to modify the gold nanoparticles, it is also necessary to modify the 5’ end of the linear DNA with a thiol group. The nucleotide sequence of the linear DNA with a thiol group is 5’

[0052] -SH-GGAACCCATAACAAAT-3’.

[0053] Example 7

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

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

[0056] 10 μL systems of the gold nanoparticles obtained in Examples 1 to 6 and Comparative Example 1 were respectively prepared with water, and the concentration of each gold nanoparticle was 4 nM. The spectra of the gold nanoparticles in different groups were measured with a Nano Drop, and the results are shown in Figure 1 as shown in (b) of.

[0057] Furthermore, TEM characterization was carried out on different groups, and the results are shown in Figure 1 as shown in (c) to (i) of. It can be seen that the sizes of the gold nanoparticles modified with different hairpin DNAs are consistent, the morphologies are uniform, and there are no morphological differences from 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 the gold nanoparticles in different groups were measured respectively, and the results are shown in Figure 2 as shown. From Figure 2 (a) of, it can be seen that the Zeta potentials of the gold nanoparticles modified with hairpin DNA are all higher than those of the gold nanoparticles modified with linear DNA, and the charge stability of the hairpin DNA is higher than that of the linear DNA. From Figure 2 (b) of, it can be seen that the characteristic peaks of the gold nanoparticles modified with hairpin DNA show more significant intensity changes, indicating that the interaction between the hairpin DNA and the gold nanoparticles is stronger, which helps to improve the stability of the gold nanoparticles.

[0059] (3) Salt tolerance stability determination:

[0060] 10 μL systems were respectively prepared with the gold nanoparticles obtained in Examples 1 - 6 and Comparative Example 1 using water, where the concentration of each gold nanoparticle was 4 nM. Different volumes of 5 M NaCl or MgCl₂ solution were respectively added, and the remaining volume was made up with water to prepare NaCl solutions with a concentration of 1 M or MgCl₂ solutions with concentrations of 50 mM and 100 mM. After mixing, the color change was observed and photographed, and then the spectrum was measured. The results are as Figure 3 shown.

[0061] As can be seen from (a) in Figure 3 , when the NaCl concentration in the system was 1 M, there was no absorption peak in the spectrum of the nanoparticles modified with linear DNA sequences, and the solution color changed to purple. However, the nanoparticles modified with hairpin DNA all had obvious absorption peaks at 525 nm, and the solution colors all remained red, indicating that the hairpin DNA could remain stably dispersed in a 1 M NaCl system. To further verify the tolerable charge intensity, Mg 2+ was used for verification. As can be seen from (b) in Figure 3 , when the Mg 2+ content in the solution was 50 mM, the nanoparticles modified with T3, T5, T7, and T10 all remained stably dispersed, the solution showed red, and the corresponding spectra also showed obvious absorption peaks at 525 nm without shifting. Subsequently, the Mg 2+ content was further increased to 100 mM. As can be seen from (c) in Figure 3 , T3, T5, T7, and T10 still maintained stable absorption peaks. This shows that the nanoparticles modified with hairpin DNA can tolerate an ionic strength of more than 100 mM Mg 2+ , and the ionic strength tolerance is significantly higher than that of linear DNA.

[0062] (4) Acid resistance stability determination

[0063] 10 μL systems were respectively prepared with the gold nanoparticles obtained in Examples 1 - 6 and Comparative Example 1 using water, where the concentration of each gold nanoparticle was 4 nM. Different volumes of HCl solution were respectively added, and the remaining volume was made up with water to prepare solutions with pH values of 1, 2, 3, and 4. After mixing, the color change was observed and photographed, and then the spectrum was measured. The results are as Figure 4 shown.

[0064] As can be seen from (a) in Figure 4 , when the pH was 1, the solutions of the nanoparticles modified with hairpin DNA and linear DNA were both blue, and at the same time, the spectral peaks also shifted. As can be seen from Figure 4As can be seen from (b) in [reference], when the pH is 2, the solution colors of T7 and T10 show red, and the spectrum still maintains the absorption peak at 525 nm, while other hairpin DNAs and linear DNAs cannot exist stably. Similarly, as the pH increases, the stability of hairpin DNAs and linear DNAs gradually improves. When the pH is 3 (see Figure 4 in (c)), almost all hairpin DNAs can be kept stable; when the pH is 4 (see Figure 4 in (d)), the linear DNAs can also be kept stably dispersed. This shows that the gold nanoparticles modified with hairpin DNAs can tolerate a strong acid environment and exist stably in an environment with a pH above 2, and their stability is significantly higher than that of linear DNAs.

[0065] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can still be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. A gold nanoparticle with enhanced stability, characterized in that, The surface of the gold nanoparticles is modified with hairpin DNA; the nucleotide sequence of the hairpin DNA is as shown in SEQ ID NO.

1.

2. The gold nanoparticles according to claim 1, characterized in that, 1 to 10 bases T are connected to the 5'-end of the hairpin DNA.

3. The gold nanoparticles according to claim 2, characterized in that, The nucleotide sequence of the hairpin DNA includes any one of SEQ ID NO.2 to SEQ ID NO.

6.

4. The gold nanoparticles according to any one of claims 1 to 3, characterized in that, The 5'-end of the hairpin DNA is modified with a thiol group.

5. The method for preparing the gold nanoparticles according to any one of claims 1 to 4, characterized in that, It includes the following steps: mixing the hairpin DNA with a nano-gold solution, gradually dropping a salt solution, increasing the salt concentration in the reaction solution by 50 mM each step, with an interval of 1 h between each step, and finally reaching 300 mM, and centrifuging to obtain gold nanoparticles with hairpin DNA modified on the surface.

6. The preparation method according to claim 5, characterized in that, The preparation method of the nano-gold solution includes the following steps: mixing water with a 1% HAuCl4 solution, heating to boiling, then mixing with a 1% sodium citrate solution, maintaining gentle boiling, and obtaining the nano-gold solution after the solution color is stable and no longer changes.

7. The preparation method according to claim 6, wherein The volume ratio of the HAuCl4 solution to the sodium citrate solution is 1:

4.

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

9. The preparation method according to claim 5, characterized in that, The centrifugation conditions are centrifugation at 12000 rpm for 15 min.

10. The application of the gold nanoparticles according to any one of claims 1 to 4 or the preparation method according to any one of claims 5 to 9 in biological detection or drug delivery.

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