WO2016145925A1 - High salt-resistant metal nanoparticle assembly and manufacturing method thereof - Google Patents

Abstract

A manufacturing method of a high salt-resistant metal nanoparticle assembly comprises: obtaining a metal nanoparticle a and ssDNA, adding the same to a mixture a of a coating agent, a buffer solution and a salt, and separating and purifying the same after mixing and culturing, thus obtaining a product a; obtaining a metal nanoparticle b and sscDNA, adding the same to a mixture b of the coating agent, the buffer solution and the salt, and separating and purifying the same after mixing and culturing, thus obtaining a product b; and mixing the product a and the product b, dissolving the same in a mixed solution of the coating agent, the buffer solution and the salt, and centrifuging and washing the same after agitating and culturing the same, thus obtaining the high salt-resistant metal nanoparticle assembly. Disclosed is a high salt resistant metal nanoparticle assembly manufactured by the above method. The method is simple, highly efficient and has high controllability. The resulting product has uniform appearance, good dispersibility and high salt resistance.

Description

High salt tolerance metal nanoparticle assembly and preparation method thereof

Technical field

 The invention belongs to the technical field of nano materials, and particularly relates to a high salt tolerance metal nanoparticle assembly and a preparation method thereof.

Background technique

In recent years, metal nanoparticle assemblies have found wide applications in nanodevices, nanosensors, and nanomedicine. Metal nano-assemblies not only have excellent optical, electrical, catalytic and other properties of isolated nanoparticles, but also have some new properties, such as surface-enhanced Raman properties, due to assembly, which greatly broadens the field of nano-particles in nanotechnology. The scope of application within. With the increasing application of metal nanoparticle assemblies, especially in biomedicine, the salt tolerance is very high. Metal nanoparticles, including metal nanoparticle assemblies, are colloids that are particularly sensitive to electrolyte solutions such as salt solutions. The addition of a small amount of salt makes it easy to agglomerate the nanoparticles, and the concentration of the salt in the living body is high. Therefore, in order to apply the metal nanoparticle assembly in the biomedical field, it is necessary to prepare a highly salt-tolerant assembly.

At present, the invention patent for high salt-tolerant metal nanoparticle assemblies in China is still blank. In fact, the technical invention of isolated salt-tolerant metal nanoparticles is also very rare. Application number is

 201010286955.X Chinese invention patent "A method for preparing high-stability and functionalized gold nanoparticles" discloses a use A method of DNA-assisted synthesis of gold nanoparticles. The gold nanoparticles prepared by the method have regular shape, uniform particle size and salt tolerance up to 20 times, but the method is only for isolated gold nanoparticles.

Summary of the invention

In order to overcome the shortcomings and deficiencies of the prior art, the primary object of the present invention is to provide a method for preparing a metal nanoparticle assembly having high salt tolerance, which is simple and efficient, has uniform product morphology, and good dispersibility.

 Another object of the present invention is to provide a metal nanoparticle assembly obtained by the above production method, and to carry out an experiment for salt tolerance of the assembly.

 The object of the present invention is achieved by the following technical solutions.

 A method for preparing a high salt tolerance metal nanoparticle assembly, comprising the following steps:

 (1) taking metal nanoparticles a and ssDNA, adding a mixture of a coating agent, a buffer solution and a salt a In the mixed culture, separation and purification, to obtain the product a;

 (2) taking metal nanoparticles b and sscDNA, adding to a mixture of a coating agent, a buffer solution and a salt b Medium, mixed and purified, separated and purified to obtain product b;

 (3) The products a and products b of steps (1) and (2) After mixing, it is dissolved in a mixed solution of a coating agent, a buffer solution and a salt, stirred and cultured, and then centrifuged to obtain a highly salt-tolerant metal nanoparticle assembly.

 In the above method, the metal nanoparticle a according to the step (1) is Au, Ag or Cu. One or two of the metals; the size of the metal nanoparticles is 1 to 100 nm; and the molar ratio of the metal nanoparticles a to ssDNA is 1:40 to 400.

 In the above method, the coating agent in the step (1) is sodium dodecyl sulfate (SDS); the coating agent and the metal nanoparticles are added The mass ratio of a to ssDNA mixture a is 0.01%~1%; the buffer solution is phosphate buffered saline (PBS), pH=7.3 The buffer solution has a concentration of 10 to 100 mmol/L in a mixture a of metal nanoparticles a and ssDNA; the salt is added with metal nanoparticles a and The concentration in the mixture of ssDNA a gradually increased from 6 to 24 h to the final 50 to 500 mmol/L.

 In the above method, the mixing culture time described in the step (1) is 6 to 24 h. The separation and purification method is centrifugal separation or dialysis.

 In the above method, the metal nanoparticles b described in the step (2) and the metal nanoparticles in the step (1) a The metal nanoparticles b are one or two of Au, Ag or Cu; the metal nanoparticles b have a size of 1 to 100 nm; the metal nanoparticles b The molar ratio to sscDNA is 1:0.2~20.

 In the above method, the encapsulating agent in the step (2) is SDS, and the wrapping agent is added with metal nanoparticles b and The mass ratio of the mixture b of sscDNA is 0.01%~1%; the buffer solution is PBS, pH=7.3, and the buffer solution is added with metal nanoparticles b and The concentration of the mixture of sscDNA b is 10~100 mmol/L; the concentration of the salt in the mixture b of metal nanoparticles b and sscDNA is 50-500. Mmmol/L ;

 In the above method, the mixing culture time described in the step (2) is 6 to 24 h. The separation and purification method is centrifugal separation or dialysis.

 In the above method, the molar ratio of the product a to the product b in the step (3) is 1:10 to 400; SDS; the mass ratio of the package to the mixed solution of product a and product b is 0.01%~1%; the buffer solution is PBS, pH=7.3 The concentration of the buffer solution in the mixed solution in which the product a and the product b are mixed is 10 to 100 mmol/L; the concentration of the salt in the mixed solution in which the product a and the product b are mixed is 50~500 mmol/L; the mixed culture time is 6~24 h; the centrifugal speed is 5000~20,000 rpm; the centrifugation time is 5~30 min, centrifugation 1~3 times.

 Salt tolerance test of the above metal nanoparticle assembly, including stability in physiological saline and sodium chloride at different concentrations ( NaCl Stability in solution.

 The present invention has the following advantages and effects over the prior art:

The method is easy to operate, has strong controllability and high assembly efficiency, and the obtained product has uniform structure and good dispersibility, and the product has strong salt tolerance and can withstand a salt concentration up to three times the concentration of physiological saline. Therefore, the metal nanoassemblies obtained by the preparation method have potential advantages in biological applications.

DRAWINGS

 Figure 1 is a transmission electron micrograph (TEM) of the gold nanoparticle assembly obtained in Example 2;

 Figure 2 is a transmission electron micrograph of the gold nanoparticle assembly obtained in Example 2 after incubation for 12 h in physiological saline ( TEM );

 Figure 3 is a gold nanoparticle assembly obtained in Example 2 at different concentrations of NaCl The salt tolerance test result in the solution, that is, the ultraviolet absorption spectrum.

detailed description

 The present invention will be further described below by way of embodiments and the accompanying drawings, but the embodiments of the invention are not limited thereto.

 The metal nanoparticles used in the method of the present invention are commercially available or can be prepared by themselves using the prior art.

 The ssDNA and sscDNA used in the following examples were purchased from Shanghai Shenggong Bioengineering Co., Ltd.

 Its serial number is as follows:

ssDNA : 5' -HS-(CH ₂ ) ₆ ATC CTG ACA TCG GCA CGA GTA TTT CTA CCA TGT ATC-3'

sscDNA: 5'-HS-(CH ₂ ) ₆ GAT ACA TGG TAG AAA TAC TCGTGC CGA TGT CAG GAT-3'.

 Example 1

 ( 1 ) 18 nm gold nanoparticles and ssDNA according to 1:100 The molar ratio is added to the final concentration (ie, relative to the concentration of the mixture of gold nanoparticles and ssDNA added, the same below) is 0.2% (mass fraction) SDS, 20 mM PBS (pH= 7.3) In a mixture of 300 mM NaCl, mix for 20 h, centrifuge at 15000 rpm for 3 times for 15 min each time. Remove the lower layer of sediment;

 ( 2 ) Add 4 nm gold nanoparticles to sscDNA in a molar ratio of 1:10 to the final concentration of 0.2% (mass fraction) SDS, 20 mM PBS (pH= 7.3) and 100 mM NaCl mixture b, mixed culture for 6 h and then dialyzed against dialysis bags 20 h to purify;

 (3) The products of steps (1) and (2) are mixed at a molar ratio of 1:20 and added to a final concentration of 0.2. % (mass fraction) SDS, 20 mM PBS (pH = 7.3) and 150 mM NaCl in a mixed solution, slowly stirred for 20 h, then at 15,000 Centrifuge 3 times at rpm for 15 min each time to obtain a gold nanoparticle assembly.

 Example 2

 (1) Add 18 nm gold nanoparticles to ssDNA at a molar ratio of 1:100 to the final concentration of 0.2% (mass fraction) SDS, 20 mM PBS (pH = 7.3) and 300 mM NaCl mixture a, mixed culture for 20 h, 1, Centrifuge 3 times at 5000 rpm for 15 min each time. Remove the lower layer of sediment;

 ( 2 ) Add 4 nm gold nanoparticles to sscDNA in a molar ratio of 1:10 to the final concentration of In a mixture of 0.2% SDS, 20 mM PBS (pH= 7.3) and 100 mM NaCl, mixed culture for 6 h and then dialyzed for 20 h with dialysis bags. To purify;

 (3) The products of steps (1) and (2) are mixed at a molar ratio of 1:40 and added to a final concentration of 0.2. In a mixed solution of % SDS, 20 mM PBS (pH = 7.3) and 150 mM NaCl, slowly stir for 20 h, then centrifuge at 15,000 rpm. Three times, each time 15 min, a gold nanoparticle assembly was obtained.

 The obtained gold nanoparticle assembly was subjected to TEM characterization, and the results are shown in Fig. 1. Can be seen from Figure 1 4 nm The gold nanoparticles were successfully connected to the surface of 18 nm gold nanoparticles to form a 'nuclear-satellite' assembly. The average number of satellites was nine, and the assembly was well dispersed and uniform in size.

 Example 3

 (1) Add 18 nm gold nanoparticles to ssDNA at a molar ratio of 1:100 to the final concentration of 0.2% (mass fraction) SDS, 20 mM PBS (pH = 7.3) and 300 mM NaCl mixture a, mixed culture for 20 h, 1, Centrifuge 3 times at 5000 rpm for 15 min each time. Remove the lower layer of sediment;

 ( 2 ) Add 4 nm gold nanoparticles to sscDNA in a molar ratio of 1:10 to 0.2% SDS, 20 mM PBS (pH = 7.3) and 100 mM NaCl mixture b, mixed culture for 6 h, then dialyzed with dialysis bag for 20 h to purify;

 (3) The products of steps (1) and (2) are mixed at a molar ratio of 1:80 and added to a final concentration of 0.2. In a mixture of % SDS, 20 mM PBS (pH = 7.3) and 150 mM NaCl, slowly stir for 20 h, then centrifuge at 15,000 rpm. Three times, each time 15 min, a gold nanoparticle assembly was obtained.

 Example 4

 (1) Add 18 nm gold nanoparticles to ssDNA at a molar ratio of 1:100 to the final concentration of 0.2% (mass fraction) SDS, 20 mM PBS (pH = 7.3) and 300 mM NaCl mixture a, mixed culture for 20 h, 1, Centrifuge 3 times at 5000 rpm for 15 min each time. Remove the lower layer of sediment;

 ( 2 ) Add 4 nm gold nanoparticles to sscDNA in a molar ratio of 1:10 to 0.2% SDS, 20 mM PBS (pH = 7.3) and 100 mM NaCl mixture b, mixed culture for 6 h, then dialyzed with dialysis bag for 20 h to purify;

 (3) mixing the products of steps (1) and (2) in a molar ratio of 1:160, and adding to the final concentration In a mixed solution of 0.2% SDS, 20 mM PBS (pH = 7.3) and 150 mM NaCl, slowly stir for 20 h, then at 15,000 rpm. The gold nanoparticle assembly was obtained by centrifugation three times for 15 minutes each time.

 Salt tolerance test

 (1) The gold nanoparticle assembly of Example 2 was cultured in physiological saline for 12 hours, and then subjected to transmission electron microscopy (TEM). ) characterization. Its TEM image is shown in Figure 2.

 It can be seen from Fig. 2 that the gold nanoparticle assembly prepared by the method of the present invention is cultured in physiological saline for up to 12 h. It still retains its intact morphology, indicating its good stability at 0.9% salt concentration.

 (2) The gold nanoparticle assembly of Example 2 was taken at 0.9%, 1.8%, 3.0%, 3.3, respectively. Salt tolerance tests were performed in % and 4.5% (mass fraction) NaCl solutions, and the initial color change of the solution was observed and UV characterized (results shown in Figure 3).

The stability of the gold nanoparticle assembly is easily affected by the salt concentration. A normally uniformly dispersed aqueous solution of the assembly is generally ruby red, and agglomeration may occur when it is in a high concentration of salt solution. This is reflected in the appearance of the color change of the solution (usually purple), which is reflected in the ultraviolet spectrum as the red shift of the maximum absorption peak. Therefore, the stability of the solution can be verified by the color of the solution and the ultraviolet absorption spectrum. Color change display: in In the 0.9% NaCl solution (corresponding to the concentration of physiological saline), the gold nanoparticle assembly obtained in Example 2 did not agglomerate, and the color was the original ruby red color, which was tested with salt tolerance (1) The results are consistent with each other; when the salt concentration is increased by about two times (3.0%), there is still no agglomeration, and the solution is still ruby red, indicating that the assembly still has good stability at this time. From Figure 3 It can be seen that when the salt concentration is as high as 3.0% At the time, the maximum absorption peak of the gold nanoparticle assembly still has no significant red shift, which is consistent with the aforementioned color change results. This indicates that the product has high salt tolerance and is expected to be applied under physiological conditions.

The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and combinations thereof may be made without departing from the spirit and scope of the invention. Simplifications should all be equivalent replacements and are included in the scope of the present invention.

Claims (9)

1. A method for preparing a high salt tolerance metal nanoparticle assembly, comprising the steps of:

   (1) taking metal nanoparticles a and ssDNA, added to a mixture of a coating agent, a buffer solution and a salt, mixed and cultured, and then isolated and purified to obtain a product a;

   (2) taking metal nanoparticles b and sscDNA, added to the mixture b of the package, buffer solution and salt, mixed culture and separation and purification, to obtain product b;

   (3) mixing the product a and the product b of the steps (1) and (2), dissolving in a mixed solution of a coating agent, a buffer solution and a salt, stirring and culturing, and then centrifuging to obtain a high salt-tolerant metal nanoparticle assembly. body.
2. The method for preparing a high salt-tolerant metal nanoparticle assembly according to claim 1, wherein the metal nanoparticle a according to the step (1) is one or two of Au, Ag or Cu. The size of the metal nanoparticles is 1~100 Nm; the molar ratio of the metal nanoparticles a to ssDNA is 1:40-400.
3. The method for preparing a high salt tolerance metal nanoparticle assembly according to claim 1, wherein the coating agent in the step (1) is sodium dodecyl sulfate (SDS); the wrapping agent The mass ratio of the mixture a with the metal nanoparticles a and ssDNA is 0.01 %~1%; the buffer solution is phosphate buffered saline (PBS), pH=7.3, and the concentration of the buffer solution in the mixture a of metal nanoparticles a and ssDNA is 10~100. Mmmol / L; the concentration of the salt in the mixture a of the metal nanoparticles a and ssDNA added increased from 6 to 24 h to the final 50 to 500 mmol / L.
4. The method for preparing a high salt tolerance metal nanoparticle assembly according to claim 1, wherein the mixing culture time in the step (1) is 6 to 24 h; The separation and purification method is centrifugal separation or dialysis.
5. The method for preparing a high salt tolerance metal nanoparticle assembly according to claim 1, wherein the metal nanoparticles b in the step (2) are the same as the metal nanoparticles a in the step (1) Or different, the metal nanoparticles b are one or two of Au, Ag or Cu; the size of the metal nanoparticles b is 1~100 The molar ratio of the metal nanoparticle b to the sscDNA is 1:0.2-20.
6. The method for preparing a high salt-tolerant metal nanoparticle assembly according to claim 1, wherein the wrapping agent in the step (2) is SDS, and the wrapping agent is added with the metal nanoparticle b and The mass ratio of the mixture b of sscDNA is 0.01 %~1%; the buffer solution is PBS, pH=7.3, and the buffer solution has a concentration of 10~100 in the mixture b of the metal nanoparticles b and sscDNA. Mmmol/L; the salt has a concentration of 50 to 500 mmol/L in a mixture b of metal nanoparticles b and sscDNA.
7. The method for preparing a high salt tolerance metal nanoparticle assembly according to claim 1, wherein the mixing culture time in the step (2) is 6 to 24 h; The separation and purification method is centrifugal separation or dialysis.
8. The method for preparing a high salt-tolerant metal nanoparticle assembly according to claim 1, wherein the molar ratio of the product a to the product b in the step (3) is 1:10 to 400; The wrapping agent is SDS; the mass ratio of the wrapping agent to the mixed solution of product a and product b is 0.01%~1 %; the buffer solution is PBS, pH = 7.3; the concentration of the buffer solution in the mixed solution of product a and product b is 10 to 100 Mmmol / L; the salt in the mixed solution of product a and product b in a concentration of 50 ~ 500 mmol / L; the mixed culture time is 6 ~ 24 h; the centrifugal speed is 5000~20,000 rpm; the centrifugation time is 5~30 min, and the centrifugation is performed 1~3 times.
9. The high salt-tolerant metal nanoparticle assembly is prepared according to the preparation method according to any one of claims 1 to 8.