A method for rapidly preparing nucleic acid-modified nanogold probes mediated by halide ions

Abstract

The invention discloses a method for rapidly preparing nucleic acid-modified nano-gold probes mediated by halide ions, and relates to the technical field of gold nanomaterials. The method comprises the following steps: using a thiol reducing agent to react with thiol nucleic acid to eliminate disulfide bonds between nucleic acid chains to obtain treated thiol nucleic acid; mixing the treated thiol nucleic acid, a surfactant and gold nanoparticles, immediately adding a halide salt, performing a mixing reaction, and separating to obtain a nucleic acid-modified nano-gold probe; the halide salt is a metal bromide or a metal fluoride. The method of the invention is characterized in that a high concentration of halide salt is added at one time, and has the advantages of short preparation time, high nucleic acid loading capacity and universality, and the nucleic acid-modified nano-gold probe prepared according to the method of the invention has good colloidal stability.

Description

Technical Field

[0001] The invention relates to the technical field of gold nanomaterials, and in particular to a method for rapidly preparing nucleic acid-modified nanogold probes mediated by halide ions. Background Art

[0002] Gold nanoparticles (AuNPs) are the most typical colloidal nanoparticles. Their unique optical properties make them widely used in nanobiophotonics, making them a typical representative of the application of nanotechnology in biological systems.

[0003] In 1951, Turkevich et al. prepared AuNPs for the first time by trisodium citrate reduction method, and Frens et al. further improved it. AuNPs have attracted widespread attention due to their unique photoelectric and physicochemical properties and good biocompatibility.

[0004] With the advent of nanobiotechnology, AuNPs have been widely used in biomedicine, biosensing, drug delivery, and DNA-mediated assembly, thanks to the extensive work done on sulfhydryl DNA (HS-DNA) modified on the surface of AuNPs. Mirkin et al. used salt aging (NaCl) to avoid aggregation of AuNPs by slowly and repeatedly adding salt. The entire modification process usually takes 1 to 2 days, and an excess of DNA is required to ensure a good DNA loading capacity. In addition, the modification effect of this method is not ideal for large-sized AuNPs (>40nm), and obvious aggregation will occur. In the case of polyethylene glycol-spaced modified DNA, the research group achieved an ideal DNA loading capacity by aging with ~0.7M sodium chloride under ultrasonic treatment. Later, someone proposed the freezing method, which is considered to be a faster way to promote the salt aging process by increasing the local salt concentration through the gradual formation of ice crystals, but this method requires strict conditions (-20℃ or dry ice) and excess DNA.

[0005] In summary, there is currently no ideal DNA-modified AuNP method that can simultaneously achieve simplicity, rapidity, high loading capacity, and versatility. Summary of the invention

[0006] The purpose of the present invention is to provide a method for rapidly preparing nucleic acid-modified nanogold probes mediated by halide ions to solve the problems of the above-mentioned prior art. The method of the present invention is characterized by immediately adding a high concentration of halide salt, having the advantages of short preparation time, high loading capacity and universality, and the nucleic acid-modified nanogold probes prepared according to the method of the present invention have good colloidal stability.

[0007] To achieve the above object, the present invention provides the following solutions:

[0008] The present invention provides a method for rapidly preparing nucleic acid-modified nanogold probes mediated by halide ions, comprising the following steps:

[0009] Using a thiol reducing agent to react with thiol nucleic acid to eliminate the disulfide bonds between nucleic acid chains to obtain treated thiol nucleic acid;

[0010] After mixing the treated thiol nucleic acid, surfactant and gold nanoparticles, a halide salt is immediately added to carry out a mixing reaction, and then the nucleic acid-modified nanogold probe is separated;

[0011] The halide salt is a metal bromide or a metal fluoride.

[0012] Furthermore, the nucleic acid is DNA or RNA.

[0013] Furthermore, the metal bromide is NaBr.

[0014] Furthermore, the metal fluoride is NaF.

[0015] Furthermore, the thiol reducing agent is tris(2-carboxyethyl)phosphine.

[0016] Furthermore, the surfactant is Tween-20.

[0017] Furthermore, the particle size of the gold nanoparticles is 13-50 nm; the gold nanoparticles are prepared by trisodium citrate reduction method.

[0018] Furthermore, the molar ratio of the treated thiol nucleic acid to the gold nanoparticles is 150-1000:1.

[0019] Furthermore, the mixing reaction time is 0.5-2h.

[0020] Furthermore, the ratio of the amount of the thiol reducing agent to the amount of the thiol nucleic acid is 100:1.

[0021] The present invention discloses the following technical effects:

[0022] Compared with the prior art, the advantages of the present invention include:

[0023] (1) Compared with the most common salt aging modification method, the present invention gets rid of the cumbersome steps of multi-step salt addition, thereby greatly improving the reaction efficiency, and does not require the addition of additional buffer, which is very simple and easy to operate.

[0024] (2) Compared with the pH 3-mediated nanogold complex preparation method proposed by Liu's research group, which has a better effect on sequences rich in A bases, the method proposed in the present invention has no sequence dependence and is universal. It can be completed quickly without pH adjustment and the addition of additional reagents. The operation method is simple and has the advantages of being fast and economical.

[0025] (3) Compared with the freezing method for preparing gold nanocomposites proposed by Liu's research group, the method proposed in the present invention does not require a harsh low-temperature environment and can complete the reaction at room temperature.

[0026] (4) The present invention has the advantages of being rapid, efficient and economical, and the nucleic acid-modified nanogold probe prepared according to the method of the present invention has good colloidal stability and a high DNA loading capacity. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0028] Figure 1 Schematic diagram of the connection of poly DNA sequences to AuNPs mediated by different halide ions of the present invention; wherein A is a schematic diagram of the connection reaction; B is a comparison diagram of the products of each reaction system before and after centrifugation;

[0029] Figure 2 Schematic diagram of the connection of AuNPs with aptamer sequences mediated by different halide ions of the present invention; wherein A is a schematic diagram of the mechanism of halide ions in the DNA / AuNP system; B is the tolerance of AuNPs and different DNAs to halide ions (AuNP particle size and concentration: 13 nm, 20 nM; DNA / AuNP is 150:1 (molar ratio); reaction time: 1 h; halide ion concentration: 200 mM;

[0030] Figure 3The results of the loading test of AuNPs connected to aptamer sequences mediated by different halide ions of the present invention; wherein A is the effect of NaF concentration on the PS2M / -A / -T loading capacity; B is the effect of NaF, NaCl or NaBr concentration on the CEA / -A / -T loading capacity of AuNP (13nm); C is the effect of different NaF concentrations on the PS2M loading capacity of AuNP (13nm); D is the effect of NaBr concentration at different times on the CEA loading capacity of AuNP (13nm); EJ is the NaF, NaCl or NaBr modified on AuNP The amount of DNA (AuNP concentration: 20 nM for 13 nm, 10 nM for 20 nm, and 1 nM for 50 nm; DNA / AuNP of 13 nm AuNP is 150:1 (molar ratio), DNA / AuNP of 20 nm AuNP is 250:1, and DNA / AuNP of 50 nm AuNP is 1000:1; reaction time: 1 h for 13 and 20 nm AuNP, and 2 h for 50 nm AuNP; halide ion concentration: 200 mM); E is the concentration of 13 nm connected to PS2M, PS2M-A, and PS2M-T AuNP results; F is the result of PS2M, PS2M-A and PS2M-T connected to 20nm AuNP; G is the result of PS2M, PS2M-A and PS2M-T connected to 50nm AuNP; H is the result of CEA, CEA-A and CEA-T connected to 13nm AuNP; I is the result of CEA, CEA-A and CEA-T connected to 20nmAuNP; J is the result of CEA, CEA-A and CEA-T connected to 50nmAuNP;

[0031] Figure 4 The figure is a comparison of the gel electrophoresis results of the aptamer sequence connected to AuNP of the present invention and other methods; wherein pH 3 represents the method of comparative example 2; salt-aging represents the method of comparative example 1 (i.e., salt aging method); NaF or NaBr represents the method of Example 2;

[0032] Figure 5It is a comparison chart of the hydrated particle size and PDI of AuNPs connected to the aptamer sequences of the present invention and other methods; wherein, pH 3 represents the method of comparative example 2; salt-aging represents the method of comparative example 1; NaF or NaBr represents the method of Example 2; A is the result of PS2M, PS2M-A and PS2M-T connected to 13nmAuNP; B is the result of CEA, CEA-A and CEA-T connected to 13nmAuNP; C is the result of PS2M, PS2M-A and PS2M-T connected to 20nmAuNP; D is the result of CEA, CEA-A and CEA-T connected to 20nmAuNP; E is the result of PS2M, PS2M-A and PS2M-T connected to 50nmAuNP; F is the result of CEA, CEA-A and CEA-T connected to 50nmAuNP;

[0033] Figure 6 The effect of different surfactants on AuNP modification; AuNP size and concentration: 13nm, 20nm; the ratio of DNA to is 150:1; halide salt: 200mM NaF for PS2M / -A / -T, NaBr for CEA / -A / -T; surfactant concentration: 0.1%; reaction time: 1h. DETAILED DESCRIPTION

[0034] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0035] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0036] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0037] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0038] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0039] The DNA sequences used in the present invention are shown in Table 1.

[0040] Table 1

[0041]

[0042] Note: The nucleotide sequences in the table are labeled with SH-C6 at the 5' end; for nucleic acid loading assay, the 5' end is labeled with SH-C6 and the 3' end is labeled with 6-FAM.

[0043] Example 1 Synthesis of gold nanoparticles

[0044] 30 mL of chloroauric acid aqueous solution (0.01% w / v) was injected into a round-bottom flask equipped with a reflux condenser and heated to boiling. Then 900 μL of trisodium citrate solution (1% w / v) was added to the flask and refluxed under magnetic stirring. After the solution color gradually changed from colorless and black to dark red, heating was continued for 10 min, and then heating was stopped and cooled to room temperature to obtain a 13 nm AuNP seed solution, which was then separated to obtain 13 nm AuNP.

[0045] 100 mL of chloroauric acid aqueous solution (0.01% w / v) was injected into a round-bottom flask equipped with a reflux condenser and heated to boiling. Then 2 mL of trisodium citrate solution (1% w / v) was added to the flask and refluxed under magnetic stirring. After the color of the solution gradually changed from colorless and black to dark red, heating was continued for 15 min, and then heating was stopped. The solution was cooled to room temperature and separated to obtain 20 nm AuNPs.

[0046] 100 μL of chloroauric acid solution (1% w / v) and 9.378 mL of ultrapure water were added to a 20 mL serum bottle, and then 400 μL of 13 nm AuNP seed solution was added to the serum bottle and stirred rapidly at room temperature. After that, 22 μL of sodium citrate solution (1%) was added, followed by 100 μL of hydroquinone (0.03 M) and the reaction was completed for 10 min to obtain 50 nm AuNP.

[0047] Example 2 Halide ion-mediated rapid DNA modification and quantification of gold nanoparticles

[0048] 1. Methods

[0049] Halide ion-mediated rapid DNA modification of gold nanoparticles:

[0050] In order to quantify the amount of DNA adsorbed on AuNPs, carboxyfluorescein (3'FAM, 5'SH-C6) labeled thiol DNA was used to modify AuNPs as detailed below:

[0051] The thiol DNA (sequences shown in Table 1) solution was treated with tri(2-carboxyethyl)phosphine (TCEP) (the molar ratio of thiol DNA to TCEP was 1:100) at room temperature for 2 h to eliminate the disulfide bonds between DNA chains. The treated thiol DNA solution (concentration 100 μM), gold nanoparticle solution (13 nm AuNP 20 nmol / L, 20 nm AuNP 10 nmol / L and 50 nm AuNP 1 nmol / L, respectively) and Tween-20 were mixed to obtain a mixed solution, and a halide salt (200 mM NaF, NaBr, NaCl and NaI), react at room temperature for 0.5h (maximum time can reach 2h), then centrifuge at 4°C (13nm AuNP is centrifuged at 12000rpm for 15min; 20nm AuNP is centrifuged at 10000rpm for 15min; 50nm AuNP is centrifuged at 5000rpm for 10min), discard the supernatant to remove the unmodified thiol DNA, and obtain DNA-modified gold nanoparticles (DNA / AuNP). Among them, in the mixed solution, the molar ratio of 13nm AuNP: AuNP and treated thiol DNA is 1:150; the molar ratio of 20nm AuNP and treated thiol DNA is 1:250; the molar ratio of 50nm AuNP and treated thiol DNA is 1:1000. The final concentration of Tween-20 in the mixed solution is 0.1% v / v.

[0052] Method for detecting DNA loading: After centrifuging DNA / AuNP, carefully aspirate the supernatant, disperse 100μL DNA / AuNP samples in buffer (10mM Tris-HCl, pH 8.0, 100mM DTT) in each group, react for 12h, all thiol-DNA-FAM is replaced, then centrifuge at 12000rpm for 10min, take the supernatant and add it to the black ELISA plate to measure the FAM-DNA fluorescence intensity, where the excitation wavelength is 488nm and the emission wavelength is 520nm. Afterwards, the quantitative DNA concentration is calculated by the standard curve of FAM-DNA under the same buffer conditions.

[0053] 2. Results

[0054] like Figure 1 As shown, for A14 Sequence, DNA / AuNP conjugates in F - , Cl - and Br - Good dispersion is maintained under Cl - After centrifugation, significant aggregation occurred. This phenomenon was attributed to the F - It can help to "squeeze" DNA on AuNPs and maintain stability with lyophilicity, while Br with better affinity and unique packing - Occupies the A 14 and avoids potential bridging between polyA (polyA is prone to adhere to AuNP). - Without sufficient affinity and unique properties to support the formation of stable DNA / AuNP conjugates, the physical stacking generated by centrifugation caused each AuNP to strongly aggregate, resulting in a poor dispersion state.

[0055] For T 14 Sequence, DNA / AuNP conjugates before and after centrifugation in F - , Cl - and Br - This result may be due to the fact that T has the lowest affinity compared to other bases or halide ions. In this case, the sulfhydryl group plays a role in helping T 14 T plays a key role in achieving better modification on AuNPs. 14 The sequence also maintains a balance of dispersibility in the DNA / AuNP conjugate colloidal system. In addition, the thiol group helps to form a stable Au-S bond on the AuNP surface, making it resistant to 200 mM NaCl. However, due to the T and Cl - The affinity of Cl - The induced coupling product showed slight aggregation, resulting in - and Br - Compared with the above, the coupling effect is poor (chaotic state). In addition, T has the smallest molecular structure among the four bases, which is conducive to reducing the steric hindrance in the coupling process.

[0056] For G 14 Sequence, only F - The dispersion is good, but significant aggregation occurs under Cl- or Br-. This is also due to the fact that F -The affinity for AuNPs is the lowest and it promotes colloidal stability, resulting in well-dispersed DNA / AuNP conjugates. In addition, G-rich sequences tend to form G-quadruplexes through intra- or intermolecular means, regardless of the addition of any halide ions. This also stems from the fact that single-stranded DNA can better protect AuNPs during the modification process, while the protective effect weakens after forming more complex conformations.

[0057] For C 14 Sequence, DNA / AuNP conjugates in F - The dispersion of C- and Br- was good (also attributed to its excellent lyophilic properties), but Cl- and Br- did not disperse well after centrifugation (visible aggregates appeared). 14 Under weak acid conditions, a stable tetrahedral "i-motif" is formed. 14 The stacked negative charges increase and shield the negative charges on the AuNP surface, reducing the electrostatic repulsion of each AuNP.

[0058] In addition, I - As for the use of I - The strong affinity of I to AuNPs leads to a close arrangement of each AuNP and no space for polyDNA coupling. - The strong etching property of is also not conducive to coupling. Therefore, the following analysis does not discuss I - .

[0059] Figure 2 The effects of halide ions on the tolerance of two common 18nt DNA aptamer sequences, PS2M (G-rich) and CEA-binding aptamer (A- and T-rich), to AuNPs were investigated. Figure 2 As shown in Figure 2B, when AuNPs are modified with PS2M, only F- remains well dispersed due to its unique lyophilicity, while Cl- and Br - This result is consistent with the above results (G 14 sequence, Figure 1 ), it is concluded that the formation of G-quadruplexes failed to protect AuNPs, making them unable to tolerate the one-time addition of high salt concentrations. Considering the better performance of A or T ( Figure 1 ), it is expected that the introduction of A / T spacer sequences will reduce the difficulty of PS2M modification. When A6 or T6 spacer sequences are introduced, the aggregation state is significantly weakened and well dispersed. However, Cl- does not promote the complete dispersion of DNA / AuNP conjugates (wine red). The poor performance of Cl- is due to the difference between T and Cl -The similar affinity of the two makes the DNA / AuNP system “chaotic” and unfavorable for modification. Therefore, it is recommended to use the A spacer to achieve better modification. Similarly, for AuNPs modified with CEA aptamers, a good dispersion state can be maintained regardless of the introduction of A6 or T6 spacers. This may be because the CEA aptamer does not form a complex conformation to achieve protection of AuNPs. Therefore, when coupled with AuNPs, the introduction of A6 or T6 spacers into the DNA sequence improves the binding efficiency and stability, especially for those sequences with poor tolerance to high salt concentrations.

[0060] Figure 3 This indicates that halide ion concentration plays a key role in the loading capacity of DNA aptamers on AuNPs. - As the concentration increases from 50 to 300 mM, the PS2M / -A / -T modified AuNPs maintain good dispersion (wine red) ( Figure 3 A), DNA loading capacity increased accordingly based on gel electrophoresis results, indicating that higher concentrations of F - The DNA binding on AuNP was effectively promoted. - / Br - With the increase of concentration, CEA / -A / -T modified AuNPs also maintained a good dispersion state and obtained a higher loading capacity ( Figure 3 In B), there is almost no difference between each AuNP sample in the microcentrifuge tube, all of which are wine red. Compared with the CEA / -A sequence, the increase in halide ion concentration has a more obvious enhancement on the loading capacity of the CEA-T sequence, which is attributed to the low affinity of the flexible T sequence and Br - Filling performance.

[0061] In order to obtain stable DNA / AuNP conjugates, the present invention has studied more modification conditions in detail. When DNA is mixed with AuNP without any treatment, the solution maintains good dispersibility. However, when the mixture is subjected to electrophoresis, a tailing band appears, indicating that the binding of DNA to AuNP is uneven. After centrifugation, AuNPs are obviously aggregated and deposited and cannot migrate in the gel, indicating that the DNA binding efficiency is low and salt addition is essential for the formation of Au-S bonds between DNA and AuNPs. When Tween-20 is introduced, the tailing band still exists before centrifugation. After centrifugation, AuNPs are obviously aggregated and turned blue, and large blue bands appear in the gel, indicating that Tween-20 alone is not enough to stably modify AuNPs. When NaF is added alone, large bands appear without tailing. After centrifugation, AuNPs are slightly aggregated and a red-purple band appears, confirming the unique stability and promoted modification performance of NaF. When both Tween-20 and NaF were added, AuNPs could maintain the wine red color and obtain uniform bands, which indicated that Tween-20 and NaF had a stable combined function on DNA-modified AuNPs.

[0062] In addition to the effect of halide ions, the introduction of surfactants can further promote the stability of DNA / AuNP conjugates. The present invention studies the types of surfactants. Figure 6 As shown in the figure, PS2M-modified AuNPs showed good dispersibility with the addition of Tween-20 or SDS, while the addition of Triton X-100 could not provide a stable environment for the dispersion of AuNPs. Even with the introduction of A6 or T6 spacer sequences, the dispersion state was not sufficiently improved. This indicates that the G-quadruplex conformation formed by the G-rich sequence cannot stabilize AuNPs. In addition, compared with Tween-20 and SDS, Triton X-100 has the lowest molecular weight and contains a more hydrophobic phenyl group, so it is ineffective in stabilizing DNA / AuNP conjugates. As for CEA / -A / -T-modified AuNPs, they can maintain stable dispersion regardless of the surfactant used, thanks to the specific structure and affinity of A and T.

[0063] The present invention studies the influence of NaF / NaBr concentration and modification time. Figure 3 As shown in Figure C, the loading capacity of PS2M increases with the increase of NaF concentration and reaches equilibrium at a concentration of 200 mM, which is consistent with the electrophoresis results ( Figure 3 As for the modification of CEA aptamer, the loading capacity almost reached the maximum value at 200 mM NaBr ( Figure 3D), so DNA coupling does not require a higher concentration of salt. In addition, due to the change in sequence base composition, the loading capacity is higher than PS2M. Generally, even at a reaction time of 12 hours, the loading capacity in a short period of time (0.5 to 1 hour) is much lower than the loading capacity of high salt concentrations. When the concentration is 200 or 300mM, there is no obvious relationship between the loading capacity and time, so a higher concentration of salt is not required. In addition, the salt concentration used in the present invention is basically the same as that of the traditional salt aging method, or even lower.

[0064] Considering the potential effect of AuNP size on DNA loading capacity, the present invention analyzes the corresponding conditions in detail (at a preferred halide ion concentration of 200 mM). Figure 3 As shown in EJ, for the same DNA sequence, after adding Br - In the case of AuNP, it shows a higher - Better DNA loading capacity, which may be attributed to Br - Helps DNA to form an upright conformation on AuNPs and further generate more binding sites. In general, for AuNPs of different sizes, the loading capacity of CEA aptamers (18% to 60%) is greater than that of PS2M (19% to 45%), indicating the correlation between sequence composition and loading capacity. This may be due to the larger steric hindrance of G and the formation of G-quadruplexes.

[0065] For the PS2M sequence group, the introduction of the A6 / T6 spacer sequence improved tolerance ( Figure 2 ), but in most cases, the loading capacity was reduced due to the increased steric hindrance of the extended chain or the introduced A6 spacer binding to the AuNP surface. - The loading capacity of the induced PS2M-T was not reduced, indicating that F - The modification of T-rich sequences was advantageous, confirming the improvement of T over the modification of G-rich sequences with lower affinity. For the modification of CEA aptamers with better tolerance, NaCl was used and compared with the effects of two other halide salts. - In the case of - and Cl - Better DNA loading capacity, and the loading capacity increases with the increase of AuNP size. In addition, the overall loading capacity seems to decrease after the introduction of A6 / T6 spacers, indicating that the original A / T-rich sequences do not require further compositional changes.

[0066] Generally speaking, with F - In comparison, Br - The introduction of Br increases the DNA loading capacity even more, thanks to -Filling characteristics. - It helps DNA to stretch on the AuNP surface, thus greatly expanding the DNA binding area. In contrast, F- can only induce random coiled DNA to be arranged on the AuNP surface, so the DNA binding area is relatively reduced. In addition, the DNA loading capacity increases with the increase of AuNP size, indicating that a larger surface area can load more DNA chains. It is worth noting that based on the surface differences of AuNPs of different sizes, the use of initial DNA and the modification time should be changed in practice.

[0067] In summary, the above results indicate that the improvement of DNA-AuNP binding by introducing oligoA / T spacer sequences depends on the original sequence composition. G-rich sequences require the introduction of oligoA / T spacer sequences to improve DNA binding efficiency, while A / T-rich sequences have sufficient binding capacity to obtain stable DNA / AuNP conjugates without any spacer sequences.

[0068] Comparative Example 1

[0069] Salt aging method: First, the thiol DNA (PS2M, PS2M-A, PS2M-T, CEA, CEA-A and CEA-T shown in Table 1, respectively) solution was treated with TCEP (molar ratio of 1:100) at room temperature for 2 h to eliminate the disulfide bonds between DNA chains. The treated thiol DNA was fully mixed with gold nanoparticles (the molar ratio of thiol DNA to 13 nm AuNP was 150:1, the molar ratio to 20 nm AuNP was 250:1, and the molar ratio to 50 nm AuNP was 2000:1) and reacted for 16 h. Phosphate buffer (PB) with a final concentration of 9 mM and sodium dodecyl sulfate (SDS) with a final concentration of 0.1% were added. The mixture was shaken on a shaker at room temperature for 30 min. 2 M sodium chloride was added in 6 portions to make the final concentration of the reaction system 0.2 M, and aged at room temperature for 1 day.

[0070] Comparative Example 2

[0071] pH 3 method: First, the thiol DNA (PS2M, PS2M-A, PS2M-T, CEA, CEA-A and CEA-T shown in Table 1, respectively) solution was treated with TCEP (molar ratio of 1:100) at room temperature for 2 h to eliminate the disulfide bonds between DNA chains. The treated thiol DNA was mixed with gold nanoparticles (the molar ratio of thiol DNA to 13 nm AuNP was 150:1, the molar ratio to 20 nm AuNP was 250:1, and the molar ratio to 50 nm AuNP was 1000:1) for 30 min, and then 500 mM citric acid-HCl buffer (pH 3.0) was quickly added to make the final concentration of the buffer in the reaction system 25 mM, and incubated at room temperature (13 nm and 20 nm AuNP were incubated for 1 h, and 50 nm AuNP was incubated for 4 h).

[0072] In order to prove that the DNA / AuNP prepared by the method of Example 2 of the present invention has better dispersibility and stability, the present invention compares it with other methods (Comparative Examples 1-2). The results show that the DNA loading amount ( Figure 4 ) and colloidal stability ( Figure 5 ) are superior to other methods. The pH 3 method showed poor modification results in some cases ( Figure 4 ). The tailing bands in the gel electrophoresis indicated that the loading capacity was relatively low or uneven, indicating poor modification efficiency. In particular, the modification performance of the pH 3-based method decreased after the introduction of the oligo T spacer sequence. The salt aging method achieved comparable loading capacity and satisfactory quality as this method. However, repeated addition of NaCl was not practically beneficial and time-consuming, and the amount of DNA used was much greater than that of this method when modifying 50 nm AuNPs. In addition, Br - The loading capacity of CEA aptamer exceeded that of the other two methods. Therefore, this method provides a better method for DNA modification of AuNPs due to higher loading capacity, shorter time and easier operation.

[0073] According to the DLS analysis results ( Figure 5 ), the hydrated particle size of DNA / AuNP synthesized by the method of the present invention is comparable to that of the other two methods. For the 13 nm AuNP modified with DNA, a relatively large size can be observed, which indicates that the method of the present invention has a better modification ability for small-sized AuNPs. In addition, the method of the present invention obtains an excellent dispersion DNA / AuNP system based on its lower polydispersity index (PDI) value (<0.4), while the pH 3 method has a larger PDI for 50 nm AuNP with T6 spacer DNA. This result is consistent with the tailing band in the agarose gel electrophoresis analysis ( Figure 4 ), confirming the excellent modification performance using the method of the present invention.

[0074] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A method for rapidly preparing nucleic acid-modified nanogold probes mediated by halide ions, characterized in that: The following steps are involved: Using a thiol reducing agent to react with thiol nucleic acid to eliminate the disulfide bonds between nucleic acid chains to obtain treated thiol nucleic acid; After mixing the treated thiol nucleic acid, surfactant and gold nanoparticles, a halide salt is immediately added to carry out a mixing reaction, and then the nucleic acid-modified nanogold probe is separated; The halide salt is a metal bromide or a metal fluoride.

2. The method according to claim 1, characterized in that The nucleic acid is DNA or RNA.

3. The method according to claim 1, characterized in that The metal bromide is NaBr.

4. The method according to claim 1, characterized in that: The metal fluoride is NaF.

5. The method according to claim 1, characterized in that The thiol reducing agent is tris(2-carboxyethyl)phosphine.

6. The method according to claim 1, characterized in that The surfactant is Tween-20.

7. The method according to claim 1, characterized in that The particle size of the gold nanoparticles is 13-50nm; the gold nanoparticles are prepared by trisodium citrate reduction method.

8. The method according to claim 1, characterized in that The molar ratio of the treated thiol nucleic acid to the gold nanoparticles is 150-1000:

1.

9. The method according to claim 1, characterized in that: The mixing reaction time is 0.5-2h.

10. The method according to claim 1, characterized in that The molar ratio of the thiol reducing agent to the thiol nucleic acid is 20 to 100:1.

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