A high-density modified oligoA-tailed DNA-gold nanomaterial and its preparation method

Abstract

This invention relates to the field of biotechnology, and more particularly to a high-density oligoA-tailed DNA-gold nanomaterial and its preparation method. The oligoA-tailed DNA-gold nanomaterial comprises AuNPs, and / or AuNRs, and modified oligoA-tailed DNA; the oligoA-tailed DNA sequence is shown in SEQ ID No. 1. This preparation method is low-cost, easy to operate, and can achieve high-density oligoA-tailed DNA modification on the surface of gold nanoparticles with different morphologies, while exhibiting high stability.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and more particularly to a high-density modified oligoA-tailed DNA-gold nanomaterial and its preparation method. Background Technology

[0002] DNA-functionalized gold nanoparticles (DNA-AuNPs), proposed by Mirkin et al. in 1996, have attracted widespread attention and are widely used in biosensors, materials fabrication, and nanomedicine. For the coupling of DNA with AuNPs, thiolated DNA is currently widely used. Thiolated DNA self-assembles on the surface of AuNPs through strong Au-S interactions. However, the synthesis of thiolated DNA is complex and costly. On the other hand, studies have shown that four DNA bases can adsorb onto the surface of AuNPs, with adenine (A) having the strongest affinity and thymine (T) the weakest. Therefore, oligoA-tailed DNA modification methods based on polyA tags have been developed, such as salt aging, low pH methods, and freeze-thaw methods. These methods are mainly used for oligoA-tailed DNA functionalization of gold nanospheres, but have not yet been applied to oligoA-tailed DNA functionalization of gold nanorods (AuNRs). The recently developed butanol dehydration method (DEBT) removes water between thiol-modified DNA and AuNPs, forcing the DNA to contact and adsorb onto the AuNP surface. Conversely, DEBT removes the hexadecyltrimethylammonium bromide (CTAB) surfactant from the AuNR surface during AuNR modification, making DNA easier to attach to the AuNR surface. While DEBT has enabled oligoA-tailed DNA modification on the AuNR surface, successful modification requires at least a 50 nt poly A tag. The presence of long poly A tags increases the cost of DNA synthesis and limits the modification density of oligoA-tailed DNA on the AuNR surface.

[0003] For the modification of oligoA-tailed DNA on the surface of AuNPs, transient fluctuations in the DNA structure cause its base orientation to align with AuNPs, leading to adsorption onto the AuNP surface. Therefore, the presence of DNA secondary structure significantly influences the modification of non-thiol DNA on the AuNP surface. Previous studies on the interaction between DNA and AuNPs have shown that adding a polyT tag to DNA can effectively alter the DNA secondary structure and enhance the anchoring effect. However, currently, at least a 5nt polyA tag is generally required to prepare oligoA-tailed DNA-AuNPs, which limits the modification density of oligoA-tailed DNA. Currently, there is no cost-effective, efficient, and universal method for high-density oligoA-tailed DNA-AuNPs / AuNRs modification.

[0004] Based on this, this application proposes a high-density oligoA-tailed DNA-gold nanomaterial and its preparation method. Based on the influence of polyT tags and temperature on DEBT, high-density oligoA-tailed DNA modification was achieved on AuNPs surfaces with different morphologies. This method is rapid, simple, cost-effective, efficient, and versatile. It can be extended to the conjugation of almost all types of DNA sequences, which will expand the applications of oligoA-tailed DNA-AuNPs / AuNRs in biomedicine and nanotechnology. Summary of the Invention

[0005] This invention provides a high-density oligoA-tailed DNA-gold nanomaterial and its preparation method, which is low in cost, easy to operate, and can achieve high-density oligoA-tailed DNA modification on the surface of gold nanomaterials with different morphologies, and has high stability, thus solving the problems existing in the prior art.

[0006] One of the technical solutions adopted in this invention is:

[0007] A high-density modified oligoA-tailed DNA-gold nanomaterial is provided, comprising AuNPs, and / or AuNRs, and modified oligoA-tailed DNA; the oligoA-tailed DNA sequence is shown in SEQ ID No. 1.

[0008] The second technical solution adopted in this invention is:

[0009] The preparation method of the above-mentioned high-density modified oligoA-tailed DNA-gold nanomaterial includes the following steps:

[0010] 1) Mix oligoA-tailed DNA with gold nanoparticles and vortex to obtain a mixture;

[0011] 2) Add NaNO3 to the above mixture and vortex mix until homogeneous;

[0012] 3) Add n-butanol to the mixed solution in step (2) and vortex rapidly to mix the two phases evenly;

[0013] 4) Heat the mixed solution from step (3) for a certain period of time;

[0014] 5) Add TBE buffer to the heated mixture to separate the two phases;

[0015] 6) Reheat the mixed solution from step (5) for a certain period of time;

[0016] 7) Centrifuge to separate the two phases and remove the lower layer of oligoA-tailed DNA-modified gold nanomaterial solution;

[0017] 8) After adding an appropriate amount of ultrapure water, centrifuge and remove the supernatant to obtain high-density oligoA-tailed DNA-gold nanomaterials.

[0018] Furthermore, the temperature range for steps 4) and 6) is 25-90℃, and the total heating time is 0-60min.

[0019] Further, in step 1), the molar ratio of oligoA-tailed DNA to gold nanomaterials is greater than 200:1; in step 2), the concentration of NaNO3 is 5-50 mM; and in step 3), the volume ratio of n-butanol to the mixed solution is greater than 5:1.

[0020] Furthermore, the molar ratio of oligoA-tailed DNA to AuNPs is greater than 200:1 (20nm AuNPs) or 20:1 (5nm AuNPs); the molar ratio of oligoA-tailed DNA to AuNRs is greater than 5000:1.

[0021] Further, in step 2), the NaNO3 concentration is 15-20 mM.

[0022] Furthermore, the gold nanomaterial is AuNRs modified with oligoA-tailed DNA, and its preparation steps are as follows:

[0023] 1) Mix oligoA-tailed DNA and AuNRs at a molar ratio greater than 5000:1 and vortex to obtain a mixture;

[0024] 2) Add NaNO3 to the above mixture and vortex mix until homogeneous;

[0025] 3) Add n-butanol to the mixed solution and vortex rapidly to ensure the two phases are mixed evenly;

[0026] 4) Heat the mixed solution in a water bath at a temperature of 80-90℃ for 30 minutes;

[0027] 5) Add TBE buffer to the heated mixture to separate the two phases;

[0028] 6) Reheat the mixed solution in a water bath at a temperature of 80-90℃ for 30 minutes;

[0029] 7) Centrifuge to separate the two phases and remove the lower layer of oligoA-tailed DNA-AuNRs solution;

[0030] 8) After adding an appropriate amount of ultrapure water, centrifuge and remove the supernatant to obtain high-density oligoA-tailed DNA-AuNRs.

[0031] The third technical solution adopted in this invention is:

[0032] An oligoA-tailed DNA-mediated core-satellite nanostructure was obtained by hybridization of oligoA-tailed DNA-AuNRs prepared above with an excess of oligoA-tailed DNA-AuNPs, incubating overnight at room temperature, and centrifuging to remove the supernatant.

[0033] Furthermore, the two modified oligoA-tailed DNA nanogold materials were hybridized in 50 mM NaCl; the final concentration of oligoA-tailed DNA-AuNRs was 0.5 nM.

[0034] The beneficial effects of this invention are:

[0035] 1. The DEBT-based labeling strategy of this invention is a rapid, simple, cost-effective and universal method for modifying oligoA-tailed DNA-AuNPs / AuNRs. It is the first to achieve high-density functionalization of oligoA-tailed DNA on the surface of AuNRs, and can be extended to the coupling of almost all types of DNA sequences. This will expand the application of oligoA-tailed DNA-AuNPs / AuNRs in biomedicine and nanotechnology.

[0036] 2. This invention enables rapid oligoA-tailed DNA modification of AuNPs at room temperature by introducing a polyT tag into the DNA sequence. The prepared high-density oligoA-tailed DNA-AuNPs exhibit excellent stability, effectively solving the problem of easy aggregation of oligoA-tailed DNA-AuNPs under high salt concentration conditions. When AuNPs are modified at high temperatures, the secondary structure of the DNA is destroyed by the high temperature, thus eliminating dependence on the polyT tag and further increasing the modification density while maintaining good stability. Regarding the influence of the dense CTAB bilayer on the AuNRs surface, this invention achieves high-density modification of oligoA-tailed DNA on the AuNRs surface at high temperatures by using a polyT tag and heating.

[0037] 3. The present invention uses a core-satellite nanostructure developed with oligoA-tailed DNA, which can significantly reduce costs compared to thiol-modified DNA. Attached Figure Description

[0038] Figure 1 A schematic diagram of the protocol for preparing oligoA-tailed DNA-AuNPs via DEBT;

[0039] Figure 2 Characterization of oligoA-tailed DNA-AuNPs of the present invention;

[0040] Figure 3 The results of the sequence dependence study on the modification of oligoA-tailed DNA-AuNPs in this invention;

[0041] Figure 4 The results show the stability of oligoA-tailed DNA-AuNPs in this invention;

[0042] Figure 5 The results show the stability of oligoA-tailed DNA-AuNRs in this invention;

[0043] Figure 6 This is a schematic diagram of the core-satellite nanostructure assembled according to the present invention;

[0044] in, Figure 2In the figure, a represents the normalized extinction spectra of AuNPs before and after functionalization of oligoA-tailed DNA, and the inset shows the particle size data of AuNPs before and after functionalization of oligoA-tailed DNA; b represents the number of oligoA-tailed DNA roots modified by salt aging, low pH, freeze-thaw and DEBT methods, respectively; c represents the number of oligoA-tailed DNA roots modified by DEBT at room temperature and high temperature.

[0045] Figure 3 In the image, 'a' shows photos of AuNPs after different combinations of poly tags; 'b' shows AuNPs and photos modified using T... 12 A2, T 12 C2 and T 12 Agarose gel electrophoresis image of G2-modified AuNPs; c is a photograph of AuNPs modified with different poly A tag lengths;

[0046] Figure 4 In Figure a, the extinction spectra of AuNPs, oligoA-tailed DNA-AuNPs, and a mixture of AuNPs and oligoA-tailed DNA in 167 mM NaNO3 are shown; the inset in the upper right corner is an actual photograph; Figure b shows the change of normalized UV-Vis absorption intensity of oligoA-tailed DNA-AuNPs at 520 nm over time at different NaNO3 concentrations; Figure c shows the change of normalized UV-Vis absorption intensity of oligoA-tailed DNA-AuNPs at 520 nm over time at different pH values.

[0047] Figure 5 In Figure a, the extinction spectra of AuNRs, oligoA-tailed DNA-AuNRs, and a mixture of AuNRs and oligoA-tailed DNA in 167 mM NaNO3 are shown; the inset in the upper right corner is an actual photograph; Figure b shows the change of normalized UV-Vis absorption intensity of oligoA-tailed DNA-AuNRs at 520 nm over time at different NaNO3 concentrations; Figure c shows the change of normalized UV-Vis absorption intensity of oligoA-tailed DNA-AuNRs at 520 nm over time at different pH values.

[0048] Figure 6Image a shows a schematic diagram of the core-satellite nanostructure assembled from oligoA-tailed DNA-AuNRs and oligoA-tailed DNA-AuNPs; image b shows the normalized extinction spectra of AuNRs before and after oligoA-tailed DNA functionalization, and the inset at the bottom shows the potential data of AuNPs before and after oligoA-tailed DNA functionalization; image c shows the surface oligoA-tailed DNA modification results of AuNRs; and image d shows a representative TEM image of the core-satellite nanostructure. Detailed Implementation

[0049] To clearly illustrate the technical features of this solution, the invention will be described in detail below through specific embodiments and in conjunction with the accompanying drawings.

[0050] In this invention, unless otherwise specified, the equipment and raw materials used are commercially available or commonly used in the field. The methods in the following embodiments, unless otherwise specified, are conventional methods in the field.

[0051] It should be noted that the preparation process of AuNPs and AuNRs is omitted in the following embodiments, and they can be prepared using existing general preparation methods.

[0052] The DNA sequences used in the following examples are shown in Table 1.

[0053] Table 1

[0054]

[0055] Example 1

[0056] A method for preparing high-modification-density oligoA-tailed DNA-AuNPs, comprising the following steps:

[0057] a. In the presence of 20mM NaNO3, oligoA-tailed DNA (T 12 A2) Add to citrate-terminated AuNPs (final concentration 10 nM) at the desired molar ratio to achieve a final volume of 100 μL.

[0058] b. Add n-butanol (700 μL) to the above mixed solution and vortex mix rapidly.

[0059] c. Add 200 μL of 0.5×TBE buffer (pH 7.4) to the above solution and vortex quickly to mix.

[0060] d. Separate the two-phase solution by centrifugation at 4000 rpm for a few seconds;

[0061] e. After adding an appropriate amount of ultrapure water, centrifuge and remove the supernatant to obtain modified oligoA-tailed DNA-AuNPs.

[0062] The oligoA-tailed DNA-AuNPs obtained above were characterized, see [reference needed]. Figure 2 The results showed that the extinction peak of oligoA-tailed DNA-AuNPs exhibited a 4 nm red shift, indicating the presence of oligoA-tailed DNA attached to the surface of AuNPs.

[0063] To further demonstrate the attachment of oligoA-tailed DNA to AuNPs, dynamic light scattering analysis was performed. The hydrated particle size of the oligoA-tailed DNA-AuNPs (30.1 nm) conjugate was larger than that of the naked AuNPs (22.7 nm).

[0064] Subsequently, based on FAM-labeled oligoA-tailed DNA (T 12 A2-FAM), this sequence in T 12 Based on A2 (SEQ ID No. 1), fluorescent groups were modified, and it is not listed separately in the sequence listing. The differences in the amount of oligoA-tailed DNA that could be modified by different methods were compared; see [link to relevant documentation]. Figure 2 b. The results showed that, based on DEBT, the number of oligoA-tailed DNA strands attached to each AuNP (20 nm) was 271, which is 1.7 times that of the freeze-thaw method and far higher than that of the salt aging method and the low pH method.

[0065] Following the aforementioned preparation steps, after step b, heating was performed at 75℃-85℃ for 30 minutes, during which the buffer solution from step c was added; the above-mentioned high-temperature heating modification can further increase the number of oligoA-tailed DNA modified roots, see [link to relevant documentation]. Figure 2 c.

[0066] Examine the decorative effects of combining two poly tags:

[0067] Following the above preparation steps, change the poly tag combination to T. 12 C2, T 12 G2; A 12 T2, A 12 C2, A 12 G2;C 12 A2, C 12 T2, C 12 G2; G 12 A2, G 12 T2, G 12C2;T 12 A3, T 12 A4, T 12 A5 (such as the DNA sequences corresponding to SEQ ID No2-SEQ ID No15 in Table 1) was used to prepare non-thiolized DNA-AuNPs.

[0068] Three combinations: T 12 A2, T 12 C2 and T 12 G2 can be used to prepare non-thiolated DNA-AuNPs, but when using T... 12 When AuNPs are combined with polybases (C, G) other than A2, they will irreversibly aggregate, such as... Figure 3 a. This indicates that the Poly T tag of the present invention is crucial for the successful modification of nonthiol-modified DNA-AuNPs.

[0069] Of the three combinations, T 12 The electrophoretic bands of A2-modified nonthiolized DNA-AuNPs showed greater lag. (See also...) Figure 3 In section b, it indicates that the density of its modified non-thiolized DNA is higher. Also, see... Figure 3 In the case of c, increasing the length of the poly A tag also causes irreversible aggregation of AuNPs.

[0070] The stability of the oligoA-tailed DNA-AuNPs prepared above was investigated:

[0071] In the presence of salt, the stability differences between AuNPs, oligoA-tailed DNA-AuNPs, and mixtures of AuNPs and oligoA-tailed DNA were compared. Figure 4 In step a, the stability of oligoA-tailed DNA-AuNPs was significantly improved, proving that oligoA-tailed DNA was successfully modified. Simultaneously, oligoA-tailed DNA-AuNPs, when monodispersed in 1M NaNO3 solution, maintained a stable UV-Vis absorption peak at 520 nm for 20 min. (See [link to documentation]). Figure 4 b.

[0072] See Figure 4 In addition, the stability of oligoA-tailed DNA-AuNPs was tested over a wide pH range (pH 4–12). The results showed that oligoA-tailed DNA-AuNPs exhibited excellent pH tolerance.

[0073] Example 2

[0074] A method for preparing high-density modified oligoA-tailed DNA-AuNRs, comprising the following steps:

[0075] a. In the presence of 15mM NaNO3, oligoA-tailed DNA was added to CTAB-capped AuNRs (final concentration 2nM) at the required molar ratio to reach a final volume of 100μL.

[0076] b. Add 700 μL of n-butanol to the mixed solution obtained in step (1) and vortex mix quickly;

[0077] c. Heat the mixed solution in a water bath at 80°C for 30 minutes;

[0078] d. Add 200 μL of 0.5×TBE buffer (pH 7.4) to the above solution and vortex quickly to mix;

[0079] e. Continue heating the above mixture in a water bath at 80°C for 30 minutes;

[0080] f. Centrifuge at 4000 rpm for a few seconds to separate the two phases; remove the lower oligoA-tailed DNA-AuNRs solution;

[0081] g. After adding an appropriate amount of ultrapure water, centrifuge and remove the supernatant to obtain high-density oligoA-tailed DNA-AuNRs.

[0082] Stability study of the oligoA-tailed DNA-AuNRs prepared above:

[0083] In the presence of salt, the stability differences among AuNRs, oligoA-tailed DNA-AuNRs, and mixtures of AuNRs and oligoA-tailed DNA were compared. The stability of oligoA-tailed DNA-AuNRs was significantly improved, demonstrating successful DNA modification; simultaneously, oligoA-tailed DNA-AuNRs, monodispersed in 1M NaNO3 solution, maintained a stable UV-Vis absorption peak at 635 nm for 20 min. (See attached figures). Figure 5 a and b.

[0084] See Figure 5 In addition, the stability of oligoA-tailed DNA-AuNRs was tested over a wide pH range (pH 4–12). The results showed that oligoA-tailed DNA-AuNRs exhibited excellent pH tolerance.

[0085] Characterization of the oligoA-tailed DNA-AuNRs prepared above:

[0086] The UV-Vis spectrum of oligoA-tailed DNA-AuNRs solution was measured, such as... Figure 6 Figure b. The results showed that the extinction peak of oligoA-tailed DNA-AuNRs exhibited a 5 nm blue shift, indicating the presence of oligoA-tailed DNA on the AuNRs surface. Zeta potential analysis further confirmed the oligoA-tailed DNA modification on the AuNRs surface.

[0087] Due to the presence of CTAB, AuNRs exhibit a positive potential of 42.6. However, after modification with oligoA-tailed DNA, the potential of oligoA-tailed DNA-AuNRs becomes -34.7, further demonstrating the functionalization of oligoA-tailed DNA on the surface of AuNRs.

[0088] Calculations based on fluorescence measurements showed that the number of oligoA-tailed DNA-modified roots on the surface of AuNRs was 1502. (See [link to relevant documentation]) Figure 6 c.

[0089] Example 3

[0090] The excess oligoA-tailed DNA-AuNPs (5nm)(N) prepared in Example 1 were used. 10 A 70 AuNPs (molar ratio of 20:1) and oligoA-tailed DNA-AuNRs (T) prepared in Example 2 12 N 10 A2:AuNRs (7500:1 molar ratio) were hybridized in 50 mM NaCl; the mixture was incubated overnight at room temperature.

[0091] The final concentration of oligoA-tailed DNA-AuNRs was 0.5 nM, and the sample was centrifuged at 6500 g for 5 min to obtain the nuclear-satellite sample solution.

[0092] A 4 μL core-satellite sample solution was deposited on the carbon film surface and dried at room temperature. See the results below. Figure 6 .

[0093] The T used in this embodiment 10 N 10 A2, N 10 A 70 The sequence information is shown in Table 1.

[0094] The above specific embodiments should not be construed as limiting the scope of protection of the present invention. For those skilled in the art, any alternative improvements or modifications made to the embodiments of the present invention shall fall within the scope of protection of the present invention.

[0095] Any aspects of this invention not described in detail are well-known to those skilled in the art.

Claims

1. A high-density modified oligoA-tailed DNA-gold nanomaterial, characterized in that, The oligoA-tailedDNA-gold nanomaterial is AuNPs and / or AuNRs and oligoA-tailed DNA modified on their surface; the oligoA-tailed DNA sequence is shown in SEQ ID No.

1.

2. A method for preparing high-density modified oligoA-tailed DNA-gold nanomaterials, characterized in that, The following steps are included: 1) Mix oligoA-tailed DNA with gold nanoparticles and vortex to obtain a mixture; 2) Add NaNO3 to the above mixture and vortex mix thoroughly; 3) Add n-butanol to the mixed solution in step (2) and vortex rapidly to mix the two phases evenly; 4) Heat the mixed solution from step (3) for a certain period of time; 5) Add TBE buffer to the heated mixture to separate the two phases; 6) Reheat the mixed solution from step (5) for a certain period of time; 7) Centrifuge to separate the two phases and remove the lower layer of oligoA-tailed DNA-modified gold nanomaterial solution; 8) After adding an appropriate amount of ultrapure water, centrifuge and remove the supernatant to obtain high-density oligoA-tailed DNA-gold nanomaterials; The temperature range for steps 4) and 6) is 25-80℃, and the total heating time is 0-30 min; the oligoA-tailed DNA sequence is shown in SEQ ID No.

1.

3. The method for preparing high-density modified oligoA-tailed DNA-gold nanomaterials according to claim 2, characterized in that, Step 1) The molar ratio of oligoA-tailed DNA to gold nanomaterials is greater than 200:1; Step 2) The concentration of NaNO3 is 5-50 mM; Step 3) The volume ratio of n-butanol to the mixed solution is greater than 5:

1.

4. The method for preparing high-density modified oligoA-tailed DNA-gold nanomaterials according to claim 2, characterized in that, The high-density modified oligoA-tailed DNA-gold nanomaterial is AuNRs or AuNPs modified with oligoA-tailed DNA, and its preparation steps are as follows: 1) Mix oligoA-tailed DNA with AuNRs / AuNPs at a molar ratio greater than 200:1 and vortex to obtain a mixture; 2) Add NaNO3 to the above mixture and vortex mix thoroughly; 3) Add n-butanol to the mixed solution and vortex rapidly to ensure the two phases are mixed evenly; 4) Heat the mixed solution in a water bath at a temperature of 50-80℃ for 20-30 minutes; 5) Add TBE buffer to the heated mixture to separate the two phases; 6) Reheat the mixed solution in a water bath at a temperature of 50-80℃ for 20-30 minutes; 7) Centrifuge to separate the two phases and remove the lower layer of oligoA-tailed DNA-AuNRs / AuNPs solution; 8) After adding an appropriate amount of ultrapure water, centrifuge and remove the supernatant to obtain high-density oligoA-tailed DNA-AuNRs / AuNPs.

5. An oligoA-tailed DNA-mediated core-satellite nanostructure, characterized in that, The oligoA-tailed DNA-AuNRs prepared according to claim 4 were mixed and hybridized with an excess of oligoA-tailed DNA-AuNPs, incubated overnight at room temperature, and the supernatant was removed by centrifugation to obtain the final product.

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