Sulfur atom doped gold-silver alloy nanocluster capable of emitting light in near-infrared second region and preparation method and application of sulfur atom doped gold-silver alloy nanocluster

Abstract

The invention relates to a sulfur atom-doped gold-silver alloy nanocluster capable of emitting light in a near-infrared second region as well as a preparation method and application of the sulfur atom-doped gold-silver alloy nanocluster. The nano-cluster is a water phase Au25 nano-cluster co-doped with Ag (silver) and S (sulfur), namely Au13-16Ag12-9S2. The near-infrared second-region photoluminescence property of the nanocluster at 1030 nm is remarkably improved, and the intensity is improved by 10 times or more compared with that of a classic nanocluster without doped water phase Au25; meanwhile, the nanocluster not only has higher imaging contrast in the aspect of photoluminescence imaging of near-infrared second regions of lymphatic vessels and inflammatory lymph nodes, but also shows lower tissue accumulation and higher in-vivo clearing efficiency after intravenous injection compared with non-doped similar nanoclusters. Therefore, the accurate sulfur atom doped gold-silver alloy nanocluster is expected to provide a new insight for near-infrared fluorescence performance regulation and control and a structure-performance relationship of the metal nanocluster, and has a wide application prospect in the field of biological imaging.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedical imaging, and particularly relates to a sulfur atom-doped gold-silver alloy nanocluster emitting in the second near-infrared region, a preparation method thereof, and an application thereof. Background Art

[0002] Photoluminescent materials are a class of materials that can emit light of a specific wavelength after being excited by light radiation. Such materials have a wide range of applications in the biomedical field, such as for biomedical imaging, disease diagnosis, drug delivery, etc., bringing many conveniences and breakthroughs to biomedical research and clinical practice. Among them, in the field of biomedical imaging, because photoluminescent materials can generate fluorescent or phosphorescent signals of specific wavelengths, they can form a sharp contrast with the background signals in the living body, thereby realizing highly sensitive imaging detection. Moreover, some near-infrared photoluminescent materials can penetrate deeper biological tissues, reducing the absorption and scattering of light by tissues, helping to obtain image information of deeper tissues, and at the same time being able to maintain a relatively high resolution to a certain extent, providing the possibility for studying the deep structure and lesions of organisms.

[0003] As a class of novel photoluminescent materials, ultrasmall metal nanoclusters (MNCs) have attracted much attention and made remarkable progress in the field of nanomaterials due to their unique quantum size effect, surface plasmon resonance characteristics, good fluorescence tunability, and relatively low toxicity. In particular, MNCs show great potential in fluorescence imaging in the second near-infrared region because they can deeply penetrate biological tissues while causing less damage to normal tissues, which is more conducive to in vivo imaging. However, the photoluminescence (PL) efficiency of MNCs, especially the near-infrared second-region photoluminescence of aqueous MNCs, usually only has a NIR-II PL quantum yield of less than 1%. Therefore, how to develop highly efficient aqueous near-infrared second-region emitting MNCs is one of the key points and difficulties in current nanomaterial research. Summary of the Invention

[0004] The purpose of the present invention is to provide a sulfur atom-doped gold-silver alloy nanocluster emitting in the second near-infrared region, a preparation method thereof, and an application thereof.

[0005] To achieve the above purpose, the present invention adopts the following technical solutions: A sulfur atom-doped gold-silver alloy nanocluster emitting in the second near-infrared region is obtained by co-doping Ag and S in an aqueous Au 25 nanocluster through in-situ silver doping and sulfide treatment to obtain a nanocluster Au 13-16 Ag 12-9 S2, and the sum of the atomic numbers of Au and Ag is 25. The preparation method thereof includes the following steps: (1) Add the newly prepared AgNO3 solution, 6-mercaptohexanoic acid (MHA) solution, and ultrapure water into a reaction flask, and form an Ag(I)-MHA complex solution under stirring at 50 °C; (2) Adjust the pH of the Ag(I)-MHA complex solution synthesized in step (1) to 12 with NaOH solution, and the solution changes from turbid to clear and transparent; (3) Add the newly prepared NaBH4 solution to the solution synthesized in step (2). After reacting for 30 min, successively perform centrifugation and ultrafiltration to obtain Ag 25 (MHA) 18 and store it at 4 °C; (4) Mix the HAuCl4 solution and 6-mercaptohexanoic acid (MHA) solution, and adjust the pH value to 11 with NaOH solution to prepare an Au(I)-MHA complex solution; (5) Under stirring at room temperature, mix the Au(I)-MHA complex solution synthesized in step (4) with NaOH solution, and add it to the purified Ag 25 (MHA) 18 obtained in step (3). Adjust the pH to 11 with NaOH solution and react for 1 h to obtain Au 13- 16 Ag 12-9 (MHA) 18 ; (6) Add the Na2S solution to the Au 13-16 Ag 12-9 (MHA) 18 nanoclusters synthesized in step (5). React under stirring at room temperature for 4 - 6 h, and perform centrifugation and ultrafiltration operations to obtain Au 13-16 Ag 12-9 S2 nanoclusters.

[0006] Furthermore, in step (3), the rotation speed of the centrifugation is 12000 rpm and the time is 20 min.

[0007] Furthermore, in step (3), the molecular weight cut-off of the ultrafiltration membrane used is 50000 and 10000.

[0008] Furthermore, in step (5), the molar ratio of Au(I)-MHA to Ag 25 (MHA) 18 is 25:1.

[0009] Furthermore, in step (6), the rotation speed of the centrifugation is 12000 rpm and the time is 5 min.

[0010] Furthermore, in step (6), the molecular weight cut-off of the ultrafiltration membrane used is 50000 and 3000.

[0011] Furthermore, the sulfur atom-doped gold-silver alloy nanoclusters described in step (6) have a near-infrared second-region photoluminescence at 1030 nm that is more than 10 times higher than that of classical undoped aqueous-phase Au 25 nanoclusters.

[0012] Furthermore, the sulfur atom-doped gold-silver alloy nanoclusters described in step (6) have a relative photoluminescence quantum yield of 1.28% in aqueous solution (using IR20 dye as a reference).

[0013] Application: The application of the sulfur atom-doped gold-silver alloy nanoclusters with near-infrared second-region luminescence in photoluminescence imaging. Furthermore, the Au 13-16 Ag 12-9 S2 nanoclusters of the present invention can be used as near-infrared imaging nanoprobes, enabling high-contrast photoluminescence (PL) imaging of mouse lymphatic vessels and inflamed lymph nodes, and showing lower tissue accumulation and higher in vivo clearance efficiency than undoped homologous nanoclusters (Au 25 ) after intravenous injection.

[0014] Advantages of the present invention: The present invention uses doping silver elements into gold nanoclusters to enable sulfur atoms to enter the structure of the alloy nanoclusters, preparing Au 13-16 Ag 12-9 S2 nanoclusters with precise sulfur atom numbers. Among them, the near-infrared second-region photoluminescence performance of the nanoclusters is significantly improved, and the intensity is more than 10 times higher than that of classical undoped aqueous-phase Au 25 nanoclusters. This sulfur atom-doped gold-silver alloy nanocluster opens up a new and effective way for improving the near-infrared second-region photoluminescence performance of metal nanoclusters, provides new insights into the structure-property relationship of metal nanoclusters, and provides reference and inspiration for subsequent research.

[0015] Meanwhile, the Au 13-16 Ag 12-9 S2 nanoclusters exhibit excellent performance in in vivo imaging applications, enabling high-resolution and high-contrast imaging of lymphatic vessels and inflamed lymph nodes, and having the advantages of low tissue accumulation, high clearance efficiency, and low toxicity, laying a solid foundation for its long-term application and clinical transformation in the biomedical field. Description of the Drawings

[0016] Figure 1 For the Au prepared in the examples and comparative examples 25 、Au 13-16 Ag 12-9 、Au 13-16 Ag 12-9TEM image of S2.

[0017] Figure 2 In a, it is a comparison chart of the photoluminescence intensities of the nanoclusters prepared in the examples and comparative examples after being treated with Na2S solutions of different concentrations; in b, it is the ultraviolet absorption spectrum chart of the nanoclusters prepared in the examples and comparative examples; in c, it is the photoluminescence spectrum chart of the nanoclusters prepared in the examples and comparative examples before and after being treated with 200 μM Na2S.

[0018] Figure 3 It is a chart showing the cytotoxicity of the nanoclusters prepared in the examples.

[0019] Figure 4 For injecting ICG, Au 25 and Au 13-16 Ag 12-9 It is a comparison chart of the lymphatic imaging of the nude mouse's leg and the photoluminescence intensity of the lymphatic vessels in the nude mouse's leg 30 min after injecting Au2Ag2S2. Detailed implementation manners

[0020] A sulfur atom-doped gold-silver alloy nanocluster emitting in the second near-infrared region, and the nanocluster is an aqueous-phase Au nanocluster co-doped with Ag and S, namely Au2Ag2S2, which is prepared by in-situ silver doping and sulfide treatment. 25 nanocluster, that is, Au 13-16 Ag 12-9 S2, and is prepared by in-situ silver doping and sulfide treatment.

[0021] The preparation method of the sulfur atom-doped gold-silver alloy nanocluster emitting in the second near-infrared region includes the following steps: (1) Add 0.125 mL of freshly prepared 20 mM AgNO3 solution, 1 mL of 5 mM 6-mercaptohexanoic acid (MHA) solution and 3.775 mL of ultrapure water into a reaction flask, and form an Ag(I)-MHA complex solution under stirring at 50°C; (2) Adjust the pH of the Ag(I)-MHA complex solution synthesized in step (1) to 12 with 1 M NaOH solution, and the solution changes from turbid to clear and transparent; (3) Add 0.1 mL of freshly prepared 112 mM NaBH4 solution to the solution synthesized in step (2), after reacting for 30 min, successively carry out centrifugation and ultrafiltration to obtain Ag(MHA), and store it at 4°C; 25 (MHA) 18 and store it at 4°C; (4) Mix 0.5 mL of 40 mM HAuCl4 solution and 4 mL of 10 mM 6-mercaptohexanoic acid (MHA) solution, and adjust the pH value to 11 with 1 M NaOH solution to prepare an Au(I)-MHA complex solution; (5) Under stirring at room temperature, the Au(I)-MHA complex solution synthesized in step (4) was mixed with 4 mL of 10 mM NaOH solution and added to the purified Ag synthesized in step (3). 25 (MHA) 18 The pH was adjusted to 11 using 1 M NaOH solution and reacted for 1 h to obtain Au 13-16 Ag 12-9 (MHA) 18 ; (6) 20 μL of 0 - 200 μM Na2S solution was added to 980 μL of the Au 13-16 Ag 12-9 (MHA) 18 nanoclusters synthesized in step (5). The reaction was carried out under stirring at room temperature for 4 - 6 h. After centrifugation and ultrafiltration operations, Au 13-16 Ag 12-9 S2 nanoclusters were obtained.

[0022] Furthermore, in step (3), the rotation speed of the centrifugation was 12000 rpm and the time was 20 min.

[0023] Furthermore, in step (3), the molecular weight cut-off of the ultrafiltration membrane used was 50000 and 10000.

[0024] Furthermore, the molar ratio of Au(I)-MHA to Ag 25 (MHA) 18 used in step (5) was 25:1.

[0025] Furthermore, the concentration of the Na2S solution in step (6) was preferably 200 μM.

[0026] Furthermore, in step (6), the rotation speed of the centrifugation was 12000 rpm and the time was 5 min.

[0027] Furthermore, in step (6), the molecular weight cut-off of the ultrafiltration membrane used was 50000 and 3000.

[0028] Furthermore, for the sulfur atom-doped gold-silver alloy nanoclusters described in step (6), their near-infrared second-region photoluminescence at 1030 nm was more than 10 times higher than that of the classical undoped aqueous-phase Au 25 nanoclusters.

[0029] Furthermore, for the Au 13-16 Ag 12-9 S2 nanoclusters described in step (6), their relative photoluminescence quantum yield in aqueous solution was 1.28% (using IR20 dye as a reference).

[0030] Further, the application of sulfur atom-doped gold-silver alloy nanoclusters emitting in the second near-infrared region in photoluminescence imaging.

[0031] To make the content of the present invention more understandable, the technical solutions of the present invention will be further described below in conjunction with specific embodiments, but the present invention is not limited thereto.

[0032] Unless otherwise specified, the raw materials and reagents described in the examples are all commercially available products. Examples

[0033] Preparation of sulfur atom-doped gold-silver alloy nanoclusters, the specific steps are as follows: (1)Ag 25 (MHA) 18 Synthesis of nanoclusters: Mix the freshly prepared AgNO3 solution (20 mM, 0.125 mL), MHA solution (5 mM, 1 mL) and 3.775 mL of ultrapure water in a reaction flask, and form an Ag(I)-MHA complex solution under stirring at 50 °C. Then, adjust the pH of the Ag(I)-MHA complex solution to 12 with 1 M NaOH solution, and the solution changes from turbid to clear and transparent. Subsequently, add the freshly prepared NaBH4 solution (112 mM, 0.1 mL), react for 30 min, and obtain Ag 25 (MHA) 18 nanoclusters after centrifugation (12000 rpm, 20 min) and ultrafiltration (molecular weight cut-off 50000, 10000) in sequence, and store them at 4 °C for further use.

[0034] (2)Au 13-16 Ag 12-9 S2 nanocluster synthesis: First, mix the HAuCl4 solution (40 mM, 0.5 mL) and the MHA solution (10 mM, 4 mL), and adjust the pH value of the mixed solution to 11 with 1 M NaOH solution to prepare an Au(I)-MHA complex solution. Then stir at room temperature, mix the Au(I)-MHA complex solution with the NaOH solution (10 mM, 4 mL) and add it to the prepared Ag 25 (MHA) 18 and then adjust the pH of the above solution to 11 with 1 M NaOH solution. The molar ratio of Au(I)-MHA / Ag 25 (MHA) 18 is 25:1, and Au 13-16 Ag 12-9 (MHA) 18 is obtained after reacting for 1 h, that is, Au 13-16Ag 12-9 Finally, 20 μL of a 0 - 200 μM Na2S solution (preferably 200 μM) was added to 980 μL of Au 13-16 Ag 12-9 (MHA) 18 nanoclusters. After centrifugation (12,000 rpm, 5 min) and ultrafiltration (molecular weight cut-off 50,000, 3,000), Au 13-16 Ag 12-9 S2 nanoclusters were obtained.

[0035] Note: Au 13-16 Ag 12-9 is a mixture of Au 13 Ag 12 、Au 14 Ag 11 、Au 15 Ag 10 、Au 16 Ag9 nanoclusters; Au 13- 16 Ag 12-9 S2 nanoclusters are a mixture of Au 13 Ag 12 S2、Au 14 Ag 11 S2、Au 15 Ag 10 S2、Au 16 Ag9S2 nanoclusters.

[0036] Comparative Example Synthesis of monometallic Au 25 (MHA) 18 : HAuCl4 solution (20 mM, 0.5 mL) and MHA solution (50 mM, 0.4 mL) were added to a reaction flask containing 9.1 mL of ultrapure water. After adjusting the pH to approximately 12 using 1 M NaOH solution, the mixed solution was purged with CO gas for 2 min and then stirred at room temperature for 24 h to obtain Au 25 (MHA) 18 nanoclusters, i.e., Au 25 .

[0037] Figure 1 Figure Figure 1 shows the TEM images of the nanoclusters prepared in the examples and comparative examples. As can be seen from the figure, the change in the size of the nanoclusters after sulfide treatment is negligible, and the size of the obtained Au 13-16 Ag 12-9 S2 is approximately 2 nm, and the particle size distribution is relatively uniform.

[0038] Application Example 1: Optical Property Test A certain amount of the nanoclusters prepared in the examples and comparative examples was subjected to optical property tests. The ultraviolet absorption spectra and photoluminescence spectra are shown in Figure 2 .

[0039] As can be seen from Figure 2 a in 13-16 Ag 12-9 when treating Au 13- 16 Ag 12-9 with aqueous sodium sulfide solutions at different concentrations, the near-infrared photoluminescence intensity gradually increases in the concentration range of 0 - 200 μM, and then significant photoluminescence quenching occurs at a sulfide concentration of 250 μM. The ultraviolet absorption spectrum shows a weakening of the characteristic absorption peak at 475 nm, indicating that the optical properties of Au Figure 2 Ag 25 have changed after sulfide treatment. In particular, after treatment with 200 μM Na2S, its photoluminescence intensity is enhanced by more than 10 times at most, and is accompanied by a blue shift of about 70 nm, located at 1030 nm (

[0040] Application Example 2: Cytotoxicity Evaluation The nanoclusters prepared in the examples and comparative examples were made into solutions with different concentrations and co-incubated with human normal liver cells (L02) for 24 h, and then the cell viability was detected using a CCK8 kit. The results are shown in Figure 3 .

[0041] As can be seen from Figure 3 with the increase in concentration, the Au 13-16 Ag 12-9 nanoclusters used have little effect on the survival rate of L02 cells at a concentration of 0 - 500 μM (based on gold). This result proves that the sulfur atom-doped gold-silver alloy nanocluster material has very low cytotoxicity and also shows its good biocompatibility.

[0042] Application Example 3: Imaging of Nude Mouse Lymph Nodes (LN) Approximately 6-week-old, 20 g BALB / c healthy nude mice were taken. They were starved but given water 12 h before imaging. The nude mice were respectively injected with sulfur atom-doped gold-silver alloy nanoclusters (Au 13-16 Ag 12-9 S2, 1 mM, 200 μL), indocyanine green (ICG, 1 mM, 200 μL), and single-metal Au 25 (1 mM, 200 μL) via the plantar surface. After 30 min of injection, 808 nm laser irradiation (200 MW·cm -2) under photoluminescence imaging, the results are shown in Figure 4 .

[0043] It can be seen from Figure 4 that the migration of Au 13-16 Ag 12-9 S2 nanoclusters to the popliteal fossa and sciatic lymph nodes was observed, and the photoluminescence signal in the lymph nodes was much stronger than that of the injected ICG and single-metal Au 25 ( Figure 4 a). In addition, Au 13-16 Ag 12-9 S2 can also clearly observe lymphatic drainage, showing a significant contrast enhancement compared with ICG and single-metal Au 25 ( Figure 4 b).

[0044] The above results indicate that the sulfur atom-doped gold-silver alloy nanoclusters emitting in the second near-infrared region prepared by the present invention can achieve high-contrast near-infrared second-region photoluminescence imaging of lymphatic vessels and lymph nodes, and the performance is superior to that of ICG used clinically.

[0045] The above are only the preferred embodiments of the present invention, and all equivalent changes and modifications made according to the scope of the patent application of the present invention shall fall within the scope covered by the present invention.

Claims

1. A sulfur atom-doped gold-silver alloy nanocluster emitting near-infrared light in the second region, characterized in that: The nanoclusters are Ag and S co-doped aqueous Au 25 Nanoclusters, chemical formula Au 13-16 Ag 12-9 S2, and the sum of the atomic numbers of Au and Ag is 25, which is prepared by in-situ silver doping and sulfide treatment.

2. The method for preparing sulfur-doped gold-silver alloy nanoclusters emitting near-infrared light in the second region according to claim 1, characterized in that: The steps include: (1) Add the freshly prepared AgNO3 solution, 6-mercaptohexanoic acid (MHA) solution and ultrapure water into a reaction flask and form a Ag(I)-MHA complex solution under stirring at 50°C; (2) adjusting the pH of the Ag(I)-MHA complex solution synthesized in step (1) to 12 with a NaOH solution, and the solution changes from turbid to clear and transparent; (3) Add the newly prepared NaBH4 solution to the solution synthesized in step (2), react for 30 min, and then centrifuge and ultrafilter to obtain Ag 25 (MHA) 18 And store it at 4°C; (4) Mixing HAuCl4 solution and 6-mercaptohexanoic acid MHA solution, adjusting the pH value to 11 using NaOH solution, and preparing Au(I)-MHA complex solution; (5) Under stirring at room temperature, the Au(I)-MHA complex solution synthesized in step (4) was mixed with the NaOH solution and added to the purified Ag synthesized in step (3). 25 (MHA) 18 The pH was adjusted to 11 using NaOH solution and reacted for 1 h to obtain Au 13-16 Ag 12-9 (MHA) 18 ; (6) Add Na2S solution to the Au synthesized in step (5) 13-16 Ag 12-9 (MHA) 18 The reaction was carried out at room temperature for 4-6 hours under stirring, and Au was obtained by centrifugation and ultrafiltration. 13-16 Ag 12-9 S2 nanoclusters.

3. The preparation method according to claim 2, characterized in that The centrifugal speed in step (3) is 12000 rpm and the time is 20 min.

4. The preparation method according to claim 2, characterized in that: The ultrafiltration membrane used in step (3) has a molecular weight cut-off of 50,000 and 10,000.

5. The preparation method according to claim 2, characterized in that: Au(I)-MHA and Ag used in step (5) 25 (MHA) 18 The molar ratio is 25:

1.

6. The preparation method according to claim 2, characterized in that The centrifugation speed in step (6) is 12000 rpm and the time is 5 min.

7. The preparation method according to claim 2, characterized in that: The molecular weight cut-off of the ultrafiltration membrane used in step (6) is 50,000 and 3,000.

8. The preparation method according to claim 2, characterized in that The sulfur atom-doped gold-silver alloy nanoclusters described in step (6) have a near-infrared second-region photoluminescence at 1030 nm that is brighter than that of the classical undoped aqueous Au nanoclusters. 25 Nanoclusters improve more than 10 times.

9. The preparation method according to claim 2, characterized in that: The Au in step (6) 13-16 Ag 12-9 The relative photoluminescence quantum yield of S2 nanoclusters in aqueous solution is 1.28%, with IR20 dye as a reference.

10. Use of the sulfur atom-doped gold-silver alloy nanoclusters emitting near-infrared second region according to claim 1 in photoluminescence imaging.

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