High-modification-density oligo A-tailed DNA-nanogold material and preparation method thereof

Abstract

The invention relates to the technical field of biology, in particular to an oligo A-tailed DNA-nanogold material with high modification density and a preparation method of the oligo A-tailed DNA-nanogold material. The oligo A-tag DNA-nanogold material is prepared from AuNPs (AuNPs), and / or AuNRs (AuNPs) and modified oligo A-tag DNA (Deoxyribose Nucleic Acid), wherein the AuNRs are AuNPs, AuNRs and the modified oligo A-tag DNA; and an oligo A-tag DNA (Deoxyribose Nucleic Acid) sequence is as shown in SEQ ID No 1. The preparation method is low in cost and easy to operate, high-density oligoA-tailed DNA modification on the surfaces of the nanogold with different morphologies can be achieved, and stability is high.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to a high-modification-density oligoA-tailed DNA-nano-gold material and a preparation method thereof. Background Art

[0002] DNA-functionalized gold nanoparticles (DNA-AuNPs), proposed by Mirkin et al. in 1996, have attracted widespread attention and are widely used in biosensors, material manufacturing, and nanomedicine. For the coupling of DNA to AuNPs, thiolated DNA is currently widely used. Thiolated DNA self-assembles on the surface of AuNPs through strong Au-S interactions. However, the synthesis process of thiolated DNA is complicated and costly. On the other hand, studies have shown that the four DNA bases can be adsorbed to the surface of AuNPs, among which adenine (A) has the strongest affinity for AuNPs, while thymine (T) has the weakest affinity. Therefore, oligoA-tailed DNA modification methods based on poly A tags have been developed, such as salt aging method, low pH method, freeze-thaw method, etc. 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 (ANRs). The recently developed butanol dehydration method (DEBT) can remove water between thiolated DNA and AuNPs, forcing DNA to contact and adsorb to the surface of AuNPs. On the other hand, when modifying AuNRs, DEBT can remove the cetyltrimethylammonium bromide (CTAB) surfactant on the surface of AuNRs, making it easier for DNA to be modified to the surface of AuNRs. Based on DEBT, oligoA-tailed DNA modification on the surface of AuNRs was achieved, but successful modification of oligoA-tailed DNA requires at least 50nt poly A tags. The presence of long poly A tags increases the cost of DNA synthesis and also limits the modification density of oligoA-tailed DNA on the surface of AuNRs.

[0003] For the modification of oligoA-tailed DNA on the surface of AuNPs, the fluctuation of the transient structure of DNA causes its bases to face AuNPs, thereby adsorbing to the surface of AuNPs. Therefore, the presence of DNA secondary structure has a significant effect on the non-thiol DNA modification on the surface of AuNPs. Previous studies on the interaction between DNA and AuNPs have shown that adding poly T tags to DNA can effectively change the secondary structure of DNA and enhance the anchoring effect. However, at present, at least 5nt of poly A tags are generally required to achieve the preparation of oligoA-tailed DNA-AuNPs, which limits the modification density of oligoA-tailed DNA. At present, there is no cost-effective and universal method for modifying high-density oligoA-tailed DNA-AuNPs / AuNRs.

[0004] Based on this, this application proposes a high-modification density oligoA-tailed DNA-nano-gold material and preparation method. Based on the influence of poly T tags and temperature on DEBT, high-density oligoA-tailed DNA modification is achieved on the surface of AuNPs with different morphologies. It is fast, simple, cost-effective and universal. It can be extended to the coupling of almost all types of DNA sequences, which will expand the application of oligoA-tailed DNA-AuNPs / AuNRs in biomedicine and nanotechnology. Summary of the invention

[0005] The invention provides a high-modification-density oligoA-tailed DNA-nano-gold material and a preparation method thereof, which are low in cost and easy to operate, can realize high-density oligoA-tailed DNA modification on the surface of nano-gold with different morphologies, and have high stability, thus solving the problems existing in the prior art.

[0006] One of the technical solutions adopted by the present invention is:

[0007] Provided is a high-modification-density oligoA-tailed DNA-nano-gold material, which comprises AuNPs, and / or AuNRs and modified oligoA-tailed DNA; the oligoA-tailed DNA sequence is shown in SEQ ID No1.

[0008] The second technical solution adopted by the present invention is:

[0009] The method for preparing the above-mentioned high-modification density oligoA-tailed DNA-nanogold material comprises the following steps:

[0010] 1) Mixing oligoA-tailed DNA and nanogold material and vortexing to obtain a mixed solution;

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

[0012] 3) adding n-butanol to the mixed solution of step (2) and vortexing rapidly to mix the two phases evenly;

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

[0014] 5) Adding TBE buffer to the heated mixed solution to separate the two phases;

[0015] 6) heating the mixed solution in step (5) again for a certain period of time;

[0016] 7) Separate the two phases by centrifugation and take out the lower layer of the oligoA-tailed DNA modified nanogold material solution;

[0017] 8) Add appropriate amount of ultrapure water and centrifuge, remove the supernatant, and obtain oligoA-tailed DNA-nanogold material with high modification density.

[0018] Furthermore, the temperature range of step 4) and step 6) is 25-90° C., and the total heating time is 0-60 min.

[0019] Furthermore, in step 1) the molar ratio of oligoA-tailed DNA to the nanogold material is greater than 200:1; in step 2) the concentration of NaNO3 is 5-50mM; 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 the mixture of oligoA-tailed DNA and AuNPs is greater than 200:1 (20nm AuNPs) or 20:1 (5nm AuNPs); the molar ratio of the mixture of oligoA-tailed DNA and AuNRs is greater than 5000:1.

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

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

[0023] 1) mixing oligoA-tailed DNA and AuNRs at a molar ratio greater than 5000:1 and vortexing to obtain a mixed solution;

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

[0025] 3) adding n-butanol to the mixed solution and vortexing rapidly to mix the two phases evenly;

[0026] 4) Heat the mixed solution in a water bath at 80-90°C for 30 min;

[0027] 5) Adding TBE buffer to the heated mixed solution to separate the two phases;

[0028] 6) Heat the mixed solution again in a water bath at 80-90°C for 30 min;

[0029] 7) Separate the two phases by centrifugation and take out the lower layer of oligoA-tailed DNA-AuNRs solution;

[0030] 8) Add appropriate amount of ultrapure water and centrifuge, remove the supernatant, and obtain oligoA-tailed DNA-AuNRs with high modification density.

[0031] The third technical solution adopted by the present invention is:

[0032] An oligoA-tailed DNA-mediated core-satellite nanostructure is obtained by hybridizing the oligoA-tailed DNA-AuNRs prepared as above with excess oligoA-tailed DNA-AuNPs, incubating them at room temperature overnight, and removing the supernatant by centrifugation.

[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] Beneficial effects of the present invention:

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

[0036] 2. The present invention can achieve rapid oligoA-tailed DNA modification of AuNPs at room temperature by introducing a poly T tag into the DNA sequence. The prepared high-density oligoA-tailed DNA-AuNPs have excellent stability and can effectively solve the problem that oligoA-tailed DNA-AuNPs are prone to agglomeration under high salt concentration conditions. When AuNPs are modified under high temperature conditions, the secondary structure of DNA will be destroyed with the high temperature, thereby getting rid of the dependence on the poly T tag, further improving the modification density, and having good stability. In order to reduce the influence of the dense CTAB double layer on the surface of AuNRs, the present invention achieves high-density modification of oligoA-tailed DNA on the surface of AuNRs at high temperature by heating while using the poly T tag.

[0037] 3. The core-satellite nanostructure developed by the present invention using oligoA-tailed DNA can significantly reduce costs compared to thiolated DNA. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 Schematic diagram of the preparation of oligoA-tailed DNA-AuNPs by DEBT;

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

[0040] Figure 3 The results of investigation on the sequence dependence of the modification of oligoA-tailed DNA-AuNPs of the present invention are as follows;

[0041] Figure 4 This is the stability result of oligoA-tailed DNA-AuNPs of the present invention;

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

[0043] Figure 6 A schematic diagram of the core-satellite nanostructure assembled by the present invention;

[0044] in, Figure 2a is the normalized extinction spectrum of AuNPs before and after oligoA-tailed DNA functionalization, and the inset is the particle size data of AuNPs before and after oligoA-tailed DNA functionalization; b is the number of oligoA-tailed DNA modified by salt aging method, low pH method, freeze-thaw method and DEBT; c is the number of modified oligoA-tailed DNA based on DEBT at room temperature and high temperature;

[0045] Figure 3 a is a picture of AuNPs modified with different poly tags; b is a picture of AuNPs and 12 A2, T 12 C2 and T 12 Agarose gel electrophoresis of G2-modified AuNPs; c is a photo of AuNPs modified with different poly A tag lengths;

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

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

[0048] Figure 6Figure a is a schematic diagram of the core-satellite nanostructure assembled by oligoA-tailed DNA-AuNRs and oligoA-tailed DNA-AuNPs; b is the normalized extinction spectrum of AuNRs before and after oligoA-tailed DNA functionalization, and the lower inset is the potential data of AuNPs before and after oligoA-tailed DNA functionalization; c is the surface oligoA-tailed DNA modification result of AuNRs; d is a representative TEM photo of the core-satellite nanostructure. DETAILED DESCRIPTION

[0049] In order to clearly illustrate the technical features of the present invention, the present invention is described in detail below through specific implementation methods in conjunction with the accompanying drawings.

[0050] In the present invention, unless otherwise specified, the equipment and raw materials used can be purchased from the market or are commonly used in the art. The methods in the following embodiments, unless otherwise specified, are all conventional methods in the art.

[0051] It should be noted that the following examples omit the preparation process of AuNPs and AuNRs, and the existing general preparation methods can be used to prepare them.

[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 oligoA-tailed DNA-AuNPs with high modification density, which is completed by the following steps:

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

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

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

[0060] d. Centrifuge at 4000 rpm for a few seconds to separate the two phases of the solution;

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

[0062] The oligoA-tailed DNA-AuNPs prepared above were characterized, see Figure 2 The results show that the extinction peak of oligoA-tailed DNA-AuNPs red-shifted by 4 nm, 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 oligoA-tailed DNA-AuNPs (30.1 nm) conjugates was larger than that of bare AuNPs (22.7 nm).

[0064] Subsequently, based on the FAM-labeled oligoA-tailed DNA (T 12 A2-FAM), the sequence in T 12 A2 (SEQ ID No. 1) was modified with a fluorescent group and is no longer listed separately in the sequence table. The difference in the amount of oligoA-tailed DNA that can be modified by different modification methods is compared, see Figure 2 The results showed that based on DEBT, the number of oligoA-tailed DNA attached to each AuNP (20 nm) was 271 chains, which was 1.7 times that of the freeze-thaw method and much higher than the salt aging method and the low pH method.

[0065] Referring to the above preparation steps, after step b, heating is performed at 75°C-85°C for 30 minutes, during which the buffer of step c is added; the modification mediated by the above high temperature heating can further increase the number of oligoA-tailed DNA modification roots, see Figure 2 Middle c.

[0066] Investigate the modification effects of two poly tag combinations:

[0067] According to the above preparation steps, the poly tag combinations were changed 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 (DNA sequences corresponding to SEQ ID No2-SEQ ID No15 in Table 1) was used to prepare non-thiolated DNA-AuNPs.

[0068] Three combinations: T 12 A2, T 12 C2 and T 12 G2 can achieve the preparation of non-thiolated DNA-AuNPs, but when T 12 When other poly base (C, G) tags are combined except A2, AuNPs will aggregate irreversibly, e.g. Figure 3 Figure a shows that the Poly T tag of the present invention is crucial for the successful modification of non-thiolated DNA-AuNPs.

[0069] Among the three combinations, T 12 The electrophoretic band of non-thiolated DNA-AuNPs modified with A2 is more delayed, see Figure 3 In b, it is shown that the density of non-thiolated DNA modified is higher. Figure 3 In middle c, after increasing the length of the poly A tag, AuNPs also undergo irreversible aggregation.

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

[0071] In the presence of salt, the stability differences among AuNPs, oligoA-tailed DNA-AuNPs, and the mixture of AuNPs and oligoA-tailed DNA were compared, e.g. Figure 4 In a, the stability of oligoA-tailed DNA-AuNPs is significantly improved, proving that oligoA-tailed DNA is successfully modified. At the same time, oligoA-tailed DNA-AuNPs are monodispersed in 1M NaNO3 solution, and the UV-visible absorption peak at 520nm remains stable within 20min, see Figure 4 Middle b.

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

[0073] Example 2

[0074] A method for preparing oligoA-tailed DNA-AuNRs with high modification density, which is completed by the following steps:

[0075] a. Add oligoA-tailed DNA to CTAB-capped AuNRs (final concentration 2 nM) at the desired molar ratio in the presence of 15 mM NaNO3 to a final volume of 100 μL;

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

[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 quickly vortex to mix;

[0079] e. Continue heating the mixed solution 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 of the solution; remove the lower layer of oligoA-tailed DNA-AuNRs solution;

[0081] g. Add appropriate amount of ultrapure water and centrifuge, remove the supernatant, and obtain oligoA-tailed DNA-AuNRs with high modification density.

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

[0083] In the presence of salt, the stability differences between AuNRs, oligoA-tailed DNA-AuNRs, and the mixture of AuNRs and oligoA-tailed DNA were compared. The stability of oligoA-tailed DNA-AuNRs was significantly improved, proving that DNA was successfully modified; at the same time, oligoA-tailed DNA-AuNRs were monodispersed in 1M NaNO3 solution, and the UV-visible absorption peak at 635nm remained stable within 20min. Figure 5 a and b.

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

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

[0086] The UV-visible spectra of oligoA-tailed DNA-AuNRs solutions were measured. Figure 6 Middle b. The results show that the extinction peak of oligoA-tailed DNA-AuNRs exhibits a 5nm blue shift, indicating the presence of oligoA-tailed DNA attached to the AuNRs surface. The oligoA-tailed DNA modification on the AuNRs surface was further verified by zeta potential.

[0087] Due to the presence of CTAB, AuNRs exhibited a positive potential of 42.6, but after oligoA-tailed DNA modification, the potential of oligoA-tailed DNA-AuNRs became -34.7, further proving the functionalization of oligoA-tailed DNA on the surface of AuNRs.

[0088] The number of oligoA-tailed DNA modified roots on the surface of AuNRs was calculated by fluorescence measurement to be 1502, see Figure 6 Middle c.

[0089] Example 3

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

[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 a nuclear-satellite sample solution.

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

[0093] This example uses T 10 N 10 A2、N 10 A 70 The sequences are shown in Table 1.

[0094] The above specific implementation manner cannot be used as a limitation on the protection scope of the present invention. For those skilled in the art, any substitution, improvement or change made to the implementation manner of the present invention falls within the protection scope of the present invention.

[0095] The matters not described in detail in the present invention are all known technologies to those skilled in the art.

Claims

1. A high-modification-density oligoA-tailed DNA-nano-gold material, characterized in that: The oligoA-tailed DNA-nanogold material is AuNPs and / or AuNRs and modified oligoA-tailed DNA; the oligoA-tailed DNA sequence is shown in SEQ ID No1.

2. A method for preparing a high-modification-density oligoA-tailed DNA-nano-gold material, characterized in that: The steps are as follows: 1) Mixing oligoA-tailed DNA and nanogold material and vortexing to obtain a mixed solution; 2) Add NaNO3 to the above mixture and vortex mix evenly; 3) adding n-butanol to the mixed solution of step (2) and vortexing rapidly to mix the two phases evenly; 4) heating the mixed solution of step (3) for a certain period of time; 5) Adding TBE buffer to the heated mixed solution to separate the two phases; 6) heating the mixed solution in step (5) again for a certain period of time; 7) Separate the two phases by centrifugation and take out the lower layer of the oligoA-tailed DNA modified nanogold material solution; 8) Add appropriate amount of ultrapure water and centrifuge, remove the supernatant, and obtain oligoA-tailed DNA-nanogold material with high modification density.

3. The method for preparing the high-modification-density oligoA-tailed DNA-nano-gold material according to claim 2, characterized in that: The temperature range of step 4) and step 6) is 25-90° C., and the total heating time is 0-60 min.

4. The method for preparing the high-modification density oligoA-tailed DNA-nano-gold material according to claim 2, characterized in that: Step 1) the molar ratio of oligoA-tailed DNA to the gold nanoparticles 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.

5. The method for preparing the high-modification density oligoA-tailed DNA-nano-gold material according to claim 2, characterized in that: The high-modification-density oligoA-tailed DNA-nano-gold material is AuNRs or AuNPs modified with oligoA-tailed DNA, and the preparation steps are as follows: 1) mixing oligoA-tailed DNA with AuNRs / AuNPs at a molar ratio greater than 200:1 and vortexing to obtain a mixed solution; 2) Add NaNO3 to the above mixture and vortex mix evenly; 3) adding n-butanol to the mixed solution and vortexing rapidly to mix the two phases evenly; 4) Heat the mixed solution in a water bath at a temperature of 50-90°C for 20-40 min; 5) Adding TBE buffer to the heated mixed solution to separate the two phases; 6) Heat the mixed solution again in a water bath at a temperature of 50-90°C for 20-40 min; 7) Separate the two phases by centrifugation and take out 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 oligoA-tailed DNA-AuNRs / AuNPs with high modification density.

6. An oligoA-tailed DNA-mediated core-satellite nanostructure, characterized in that: The oligoA-tailed DNA-AuNRs prepared according to claim 5 are mixed and hybridized with excess oligoA-tailed DNA-AuNPs, incubated at room temperature overnight, and centrifuged to remove the supernatant to obtain.

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