Raman-fluorescence dual signal atomic precision metal nanoclusters and preparation and application thereof

Abstract

The application discloses a kind of atom precision metal nanoclusters with Raman-fluorescence dual signal and preparation and application.Atom precision metal nanoclusters with Raman-fluorescence dual signal are composed of two parts: one is atom precision metal core, and two is ligand containing alkynyl functional group.The atom precision metal core of the nanocluster described in the application and alkynyl functional ligand can play a synergistic effect, effectively enhance the fluorescence signal and Raman signal of cluster;In addition, due to the structure of super-small size metal core, the nanocluster has high efficient and rapid kidney clearance characteristics.The atom precision metal nanocluster with Raman-fluorescence dual signal has two kinds of mutually interfering fluorescence and Raman optical signals, wherein the fluorescence signal can be used to obtain macroscopic imaging information of whole body in vivo, and the Raman signal can be used to obtain microscopic molecular interaction microscopic imaging information.

Description

Technical Field

[0001] This invention belongs to the field of functional nanomaterials, specifically relating to atomically precise metal nanoclusters with Raman-fluorescence dual signals and their preparation and application. Background Technology

[0002] Gold nanoclusters (AuNCs) are a class of ultrasmall nanomaterials with a size of less than 2 nm, composed of several to hundreds of gold atoms. Due to their ultrasmall size, gold nanoclusters exhibit a strong quantum confinement effect and a discontinuous, size-dependent electronic energy level structure, thus displaying unique photoluminescence and other physicochemical properties. Compared with larger (5-200 nm) plasmon resonance nanoparticles, gold nanoclusters have good biocompatibility and are easily cleared by the kidneys due to their ultrasmall size, avoiding the risk of long-term accumulation in the body. They are considered highly promising "renal-clearing" nanomaterials in fields such as bioimaging, sensing, and diagnosis.

[0003] Currently, the most common strategy for stabilizing the atomic-level structure of gold nanoclusters is to use thiol ligands for surface protection via Au-S bonds. However, reviews indicate that thiol-protected AuNCs face stability challenges in complex biological environments. Au-S bonds may break or ligate may detach under harsh conditions such as the presence of oxidants, pH fluctuations, strong light irradiation, or thermal stress, leading to cluster disintegration or changes in properties. Furthermore, competitive binding by molecules such as proteins in biological systems can also affect ligand stability. These factors collectively limit the reliable application of AuNCs in bioimaging and sensing. Compared to thiol ligands, the sp-hybridized carbon atom in the alkynyl group can provide a strong electron density to the metal atom through its σ orbital, forming a robust Au-C≡C bond. Moreover, the π bond of the alkynyl group can synergistically interact with the empty d orbital of the metal. This σ double bonding mode is generally stronger and more stable than a single Au-S bond, thus significantly improving the robustness of the nanoframework.

[0004] On the other hand, fluorescent probes are widely used in optical imaging and detection technologies, but their signals are easily interfered with by the autofluorescence, light scattering, and spectral overlap of biological systems. As this review states, in traditional visible light and near-infrared I (NIR-I, 700-900 nm) imaging, the autofluorescence, light absorption, and light scattering of biological tissues significantly reduce imaging resolution and signal-to-background ratio. In contrast, near-infrared II (NIR-II, 1000-1700 nm) imaging has become a current research hotspot due to its significantly reduced autofluorescence and increased tissue penetration depth (J. Sun et al. Coordination Chemistry Reviews 533(2025) 216544). However, the emission wavelengths of most AuNCs are still concentrated in the visible light and NIR-I regions, limiting their application potential in deep tissue imaging. Complementary Raman spectroscopy, whose signal characteristics are insensitive to the excitation wavelength and almost unaffected by photobleaching and fluorescence quenching, can provide information that does not interfere with fluorescence, offering a new approach to overcoming the aforementioned optical interference problems.

[0005] In summary, existing technologies present the following prominent contradictions: on the one hand, the structural stability of metal nanoclusters with good biocompatibility faces challenges; on the other hand, the demand for highly stable, multimodal optical signal probes is increasingly urgent. Therefore, there is an urgent need to develop a novel metal nanocluster system that can both utilize more stable chemical bonding methods to achieve robust protection of atomically precise structures and integrate multimodal optical signals to meet the needs of high-precision, high-reliability diagnostic and therapeutic applications in complex biological environments. Summary of the Invention

[0006] To overcome the limitations of existing technologies, this invention provides an atomically precise metal nanocluster with Raman-fluorescence dual signals, its preparation, and its application. This atomically precise metal nanocluster with Raman-fluorescence dual signals has the following characteristics: (a) Fully alkyne-based protection: Utilizing the strong metal -C≡C bonds to ensure the stability of the framework under harsh environments; (b) Kidney clearance capacity: Excellent biocompatibility is achieved through its ultra-small size, avoiding long-term accumulation in the body; (c) Raman-fluorescence dual signal: provides complementary and interference-resistant multimodal optical information, suitable for high signal-to-noise ratio imaging and detection.

[0007] The objective of this invention is achieved through the following technical solution: This invention provides an atomically precise metal nanocluster with Raman-fluorescence dual signals, which consists of an atomically precise metal core and ligands containing alkynyl functional groups.

[0008] Furthermore, the atomically precise metal nucleus consists of M metal atoms, where M is 13, 25, 38, or 55, and the metal nucleus particle size is 0.5 nm to 5 nm; the metal atoms are at least one of gold (Au), silver (Ag), and copper (Cu).

[0009] Furthermore, the metal core consists of 25 gold atoms (Au25).

[0010] Furthermore, the ligand containing the alkynyl functional group is an alkynyl molecule RC≡CR containing a functional group R; wherein the functional group R is selected from at least one of the following: hydrogen group (-H), hydrocarbon group with different numbers of carbon atoms (such as -CH3, -C2H5, etc.), phenyl group (-C6H5), imidazole ring, sulfonic acid group (-SO3H), hydroxyl group (-OH), carboxyl group (-COOH), amino group (-NH2), mercapto group (-SH), fluorine group (-F), nitro group (-NO2), and methoxy group (-OCH3).

[0011] Furthermore, the ligand containing the alkynyl functional group is 4-alkynylbenzoic acid (pEBA).

[0012] This invention also provides a method for preparing the above-mentioned atomically precise metal nanoclusters with Raman-fluorescence dual signals, comprising the following steps: (1) Dissolve the ligand containing the alkynyl functional group in a mixed solvent of alcohol and amine to form a ligand solution; (2) Mix the soluble metal salt solution with the ligand solution to carry out a redox reaction; (3) After the redox reaction is completed, a reducing agent is added to carry out the reduction reaction; (4) After the reduction reaction is completed, the reaction solution is purified to obtain atomically precise metal nanoclusters with Raman-fluorescence dual signals.

[0013] Furthermore, in step (1), the alcohol is selected from C1. At least one of 6-alkyl alcohols and polyols; the amine is selected from C1. 8. At least one of alkylamines, diamines, triamines and alkanolamines.

[0014] Furthermore, the alcohol is methanol, and the amine is tripropylamine.

[0015] Furthermore, in step (2), the soluble metal salt is selected from at least one of chloroauric acid, silver nitrate and copper chloride; the molar ratio of the soluble metal salt to the ligand containing the alkynyl functional group is 1:0.5~1:4.

[0016] Furthermore, the soluble metal salt is chloroauric acid, and the molar ratio of chloroauric acid to the alkynyl ligand is 1:2.

[0017] Furthermore, in step (3), the reducing agent is selected from at least one of carbon monoxide, sodium borohydride, sodium citrate and ascorbic acid; the reduction reaction is carried out at a temperature of 15-35°C for 10-20 hours.

[0018] Furthermore, the reducing agent is carbon monoxide.

[0019] Furthermore, the reduction reaction is carried out at room temperature for 12-16 hours.

[0020] Furthermore, in step (4), purification is performed using an ultrafiltration tube with a molecular weight cutoff of 3-10 kDa, with repeated dilution-ultrafiltration 15 times to ensure complete removal of unreacted byproducts.

[0021] Furthermore, in step (4), purification is performed using an ultrafiltration tube with a molecular weight cutoff of 10 kDa.

[0022] This invention also provides the application of the above-mentioned atomically precise metal nanoclusters with Raman-fluorescence dual signals in optical imaging, including near-infrared II (NIR-II) fluorescence imaging and Raman imaging.

[0023] This invention also provides the application of the above-mentioned atomically precise metal nanoclusters with Raman-fluorescence dual signals in the preparation of diagnostic and therapeutic agents for rapid in vivo clearance, wherein the diagnostic and therapeutic agents are integrated formulations for the diagnosis and treatment of diseases.

[0024] Compared with the prior art, the present invention has the following advantages and effects: (1) High structural stability: This invention uses ligands containing alkynyl functional groups to protect the surface of metal nanoclusters through strong RC≡CR bonds. Compared with clusters protected by traditional thiol ligands, this σ-π double bonding mode has higher bond energy and chemical inertness, which can effectively resist the effects of oxidative environment, light or thermal stress, and ensure that the metal nanoclusters maintain atomically precise structure and stable optical properties in complex or harsh biological environments; (2) Good biosafety: Compared with traditional large-sized nanoprobes, the atomically precise metal nanoclusters with Raman-fluorescence dual signals provided by this invention can be rapidly excreted from the body through the kidneys, which greatly reduces the toxicity risk and immune burden that may be caused by long-term retention in the body, and provides a basis for achieving safe clinical translation; (3) Strong optical performance: This invention creatively integrates fluorescence and Raman scattering dual signal modes on the same precise atomic cluster. Compared with traditional single fluorescent probes, dual-modal signals have inherent complementarity and mutual verification: Raman signals are highly specific and unaffected by photobleaching; fluorescence signals have high sensitivity and are suitable for quantitative dynamic imaging. The combination of the two can effectively overcome biological background interference such as autofluorescence and light scattering of biological tissues, significantly improve the signal-to-noise ratio, accuracy and information richness of imaging, and provide a more reliable tool for high-requirement integrated diagnosis and treatment; (4) Simple preparation method: The synthetic route provided by this invention uses a mild reducing agent as the reducing agent, the steps are simple, the reaction conditions are mild, and the reproducibility is good. By precisely controlling parameters such as the precursor ratio and reaction time, the target product with a precise number of atoms and a certain number of ligands can be prepared efficiently and reproducibly, providing a reliable and easily scaled-up process route for obtaining cluster materials with uniform structure and stable performance. Attached Figure Description

[0025] Figure 1 Au in Example 1 25 (pEBA) 18 A schematic diagram of the synthesis of gold nanoclusters.

[0026] Figure 2 Au prepared in Example 1 25 (pEBA) 18 Fourier spectrum of gold nanoclusters.

[0027] Figure 3 Au prepared in Example 1 25 (pEBA) 18 Mass spectrum of gold nanoclusters.

[0028] Figure 4 Au prepared in Example 1 25 (pEBA) 18 TEM image of gold nanoclusters.

[0029] Figure 5 Au prepared in Example 1 25 (pEBA) 18 Fluorescence spectrum of gold nanoclusters.

[0030] Figure 6 Au prepared in Example 1 25 (pEBA) 18 UV-Vis spectrum of gold nanoclusters.

[0031] Figure 7 Au prepared in Example 1 25 (pEBA) 18 Raman spectra of gold nanoclusters.

[0032] Figure 8 Au prepared in Example 1 25 (pEBA) 18 In vivo fluorescence imaging of gold nanoclusters in the near-infrared II region.

[0033] Figure 9 Au prepared in Example 1 25 (pEBA) 18 UV-Vis absorption spectra of gold nanoclusters after being placed at room temperature for different times.

[0034] Figure 10 Au prepared in Example 1 25 (pEBA) 18 The trend of absorbance of gold nanoclusters at 700 nm over time. Detailed Implementation

[0035] The present invention will be further described in detail below with reference to the embodiments and accompanying drawings, but the embodiments of the present invention are not limited thereto.

[0036] In the following specific embodiments, the tetrachloroauric acid trihydrate (HAuCl) involved 4· 3H2O was purchased from Shanghai Mairui Chemical Technology Co., Ltd.; methanol, tripropylamine, and pEBA were purchased from Shanghai Bid Pharmaceutical Technology Co., Ltd.; none of the above chemical reagents required further purification before use. Ultrapure water was used in all experiments. All glassware used in the experiments was pre-soaked in aqua regia and then rinsed with deionized water at least three times.

[0037] The instruments used in this invention to investigate the properties of gold nanoclusters mainly include: Mettler Toledo Technologies ME104E / 02 electronic analytical balance; Hunan Xiangyi Laboratory Instrument Development Co., Ltd. L530R benchtop low-speed refrigerated centrifuge; PerkinElmer LS-55 fluorescence / phosphorescence / luminescence spectrophotometer; Shimadzu UV-2600 UV-Vis absorption spectrometer; Changchun New Industries Optoelectronic Technology Co., Ltd. FC-W-1064-10W laser; Thermo Fisher Scientific inductively coupled plasma mass spectrometry; ZephIR 1.7; and Photon NIR-II imaging system equipped with a deeply cooled InGaAs camera for fluorescence imaging. Example 1 The following is an embodiment of the atomically precise metal nanoclusters with Raman-fluorescence dual signals prepared by the present invention: (1) Synthesis of Au 25 (pEBA) 18 Gold nanoclusters Figure 1 Au in Example 1 25 (pEBA) 18 A schematic diagram of the synthesis of gold nanoclusters.

[0038] 0.210 mmol of 4-ethynylbenzoic acid (pEBA) was dissolved in a mixture of 20 mL of methanol and 1.2 mL of tripropylamine. After stirring to dissolve, 0.105 mmol of aqueous chloroauric acid was added, and the mixture was stirred at room temperature for 1 hour to carry out a redox reaction. After the redox reaction was completed, carbon monoxide was bubbled into the mixture for 4 minutes until saturation, and the mixture was sealed and stirred at 500 rpm at room temperature for 14 hours to carry out a reduction reaction. After the reduction reaction was completed, the reaction solution was mixed with 4 times the volume of deionized water, and the solution was purified by dilution-ultrafiltration 15 times using a 10 kDa ultrafiltration tube to finally obtain Au. 25 (pEBA) 18 Gold nanoclusters in aqueous solution, Au 25 (pEBA) 18 Gold nanoclusters are atomically precise metal nanoclusters with both Raman and fluorescence signals.

[0039] (2) Au 25 (pEBA) 18 Structural characterization of gold nanoclusters Au prepared in Example 1 25 (pEBA) 18 The gold nanoclusters were characterized by Fourier transform infrared spectroscopy, ESI-MS, and TEM. The test results are as follows: Figure 2 , Figure 3 , Figure 4 As shown.

[0040] Figure 2 Au prepared in Example 1 25 (pEBA) 18 Fourier transform infrared spectrum of gold nanoclusters, located in the 2100-2000 cm⁻¹ region. 1 The characteristic absorption peaks appearing within this range are attributed to the stretching vibrations of the alkynyl group, located in the 3000-3500 cm⁻¹ range. 1 The characteristic absorption peaks appearing within this range are attributed to the stretching vibration of ≡CH in the terminal alkynyl group (-C≡CH); located at 3000-3100 cm⁻¹. -1 The absorption peaks within the range are attributed to the =CH stretching vibration on the aromatic ring of the pEBA ligand; while those located at 3400-3500 cm⁻¹ are attributable to the =CH stretching vibration. -1The nearby broad peaks may be attributed to the OH stretching vibration of the carboxyl group (-COOH) in the ligand or the OH vibration of adsorbed water on the sample surface. Compared to the free pEBA ligand, this peak shows a significant red shift and intensity change, indicating that the sp-hybridized carbon atom of the alkynyl group provides electron density to the gold atom through a σ bond, while its π orbital interacts with the empty d orbital of gold, forming a stable Au-C≡C coordinate bond. Furthermore, in the spectrum at approximately 1400 cm⁻¹... 1 With 1550cm 1 The absorption peaks appearing nearby correspond to the symmetric and asymmetric stretching vibrations of the carboxyl group, indicating that the ligand binds to the cluster surface in a deprotonated form through the carboxyl group. FTIR results confirm that the pEBA ligand is successfully anchored to the surface of the gold nanoclusters through a dual coordination mode of alkyne and carboxyl groups, achieving all-alkyne chemical protection, which is completely consistent with the structural design proposed in this invention.

[0041] Figure 3 Au prepared in Example 1 25 (pEBA) 18 The mass spectrum of gold nanoclusters obtained in negative ion mode shows that the mass-to-charge ratio (m / z) of the main signal peak cluster is distributed in the range of 3766 to 3770. The experimentally measured isotopic distribution is in excellent agreement with the calculated values. These data conclusively confirm that the synthesized product is Au with accurate atomic number and stoichiometry. 25 (pEBA) 18 Gold nanoclusters.

[0042] Figure 4 Au prepared in Example 1 25 (pEBA) 18 TEM images of the gold nanoclusters show their morphology and size. The images reveal that the gold nanoclusters are approximately spherical and highly dispersed, with no particles adhering to each other. Referring to the 10nm scale bar in the image, statistical analysis indicates that the average core diameter of the gold nanoclusters is approximately 1.0 ± 0.3 nm, and the size distribution is very narrow, which is completely consistent with the structural design proposed in this invention.

[0043] (3) Au 25 (pEBA) 18 Determining the optical properties of gold nanoclusters Examine the Au prepared in Example 1 25 (pEBA) 18 The fluorescence spectrum, UV-Vis spectrum, and Raman spectrum of gold nanoclusters are shown in the following figures. Figure 5 , Figure 6 and Figure 7 As shown.

[0044] Figure 5 Au prepared in Example 1 25 (pEBA) 18 Fluorescence spectrum of gold nanoclusters under 808 nm near-infrared excitation, Au 25 (pEBA) 18 The gold nanoclusters exhibit a sharp and symmetrical emission peak near 1100 nm. After concentration normalization, the fluorescence intensity at 1100 nm exceeds 600,000 (au), indicating that the material has extremely high fluorescence brightness.

[0045] Figure 6 Au prepared in Example 1 25 (pEBA) 18 The UV-Vis spectrum of gold nanoclusters shows characteristic absorption peaks at approximately 415 nm and 640 nm. The spectral morphology is consistent with that of atomically precise Au nanoclusters reported in the literature. 25 The characteristic absorptions of the clusters (typically located around 670 nm and 450 nm) are qualitatively consistent. Among them, the main absorption peaks are consistent with those of traditional thiols protecting Au. 25 The clusters exhibit a blue shift, which is consistent with the cluster regulation effect of stronger electronic interactions of the alkynyl ligand (pEBA).

[0046] Figure 7 Au prepared in Example 1 25 (pEBA) 18 Raman spectra of gold nanoclusters, comparing the Raman spectral signals of four samples. The free pEBA ligands are observed in the 1950-2250 cm⁻¹ range. -1 No obvious Raman characteristic peaks were observed within the range. In contrast, when pEBA and Au... 25 Metal nuclei combine to form Au 25 (pEBA) 18 After the complex was formed, at approximately 2020 cm -1 A sharp and high-intensity characteristic peak appeared at this location. This characteristic peak is located in the Raman silent region of the biological sample. Within this region, complex biological fluids such as blood and urine typically do not exhibit endogenous Raman signal interference. Calculations and comparisons show that Au... 25 (pEBA) 18 The Raman signal intensity of the gold nanoclusters at this location was increased by approximately 22 times compared to the background baseline. This indicates that Au 25 (pEBA) 18 The core exhibits a significant surface-enhanced Raman scattering (SERS) effect on the surface ligands. Experimental results show that the Au of this invention... 25 (pEBA) 18Gold nanoclusters possess excellent SERS enhancement properties, and their characteristic signals are located in the biological quiescent region, effectively avoiding interference from biological background fluorescence and Raman signals. They are very suitable as biomedical imaging probes or detectors with high sensitivity and high signal-to-noise ratio.

[0047] (4) Au 25 (pEBA) 18 Assessment of renal clearance of gold nanoclusters Female BALB / c mice aged 4-6 weeks underwent full-body hair removal. They were then fixed to a transparent plastic plate using medical pressure-sensitive tape. After wiping the mouse skin clean with a damp paper towel, they were placed in an NIR-II imaging system to acquire their own background fluorescence signal. Au 25 (pEBA) 18 Gold nanoclusters were injected into mice via the tail vein. Fluorescence signals were collected from the mice 5 minutes after injection, and NIR-II in vivo fluorescence imaging images were acquired under excitation by an 808nm light-emitting diode laser.

[0048] Examine the Au prepared in Example 1 25 (pEBA) 18 In vivo fluorescence imaging of gold nanoclusters in the near-infrared II region yielded the following results: Figure 8 As shown.

[0049] Figure 8 Au prepared in Example 1 25 (pEBA) 18 In vivo fluorescence imaging of gold nanoclusters shows that after 5 minutes of injection into mice, the gold nanoclusters were not detected in the liver but had a strong fluorescence signal in the bladder, indicating that the gold nanoclusters of the present invention have good renal clearance.

[0050] (5) Au 25 (pEBA) 18 Stability analysis of gold nanoclusters Regarding the Au synthesized in Example 1 25 (pEBA) 18 The stability of gold nanoclusters was analyzed, and the results are as follows: Figure 9 , Figure 10 As shown.

[0051] Figure 9 Au prepared in Example 1 25 (pEBA) 18 The UV-Vis absorption spectra of gold nanoclusters placed at room temperature for different times showed a high degree of consistency and overlap across different time points (0, 1, 2, 3, 4, 8, 12, and 16 days) within a 16-day testing period. This indicates that Au...25 (pEBA) 18 Gold nanoclusters exhibit excellent chemical stability in solution environments, maintaining their structural integrity over long periods without significant oxidation, dissociation, or aggregation.

[0052] Figure 10 Au prepared in Example 1 25 (pEBA) 18 The absorbance of gold nanoclusters at 700 nm over time is shown in the graph. The data indicates that within the 16-day testing period (days 0, 1, 2, 3, 4, 8, 12, and 16), the absorbance at this wavelength remained consistently around 0.14, exhibiting a flat linear trend without significant decrease or fluctuation. Based on the combined spectral and quantitative data analysis, it can be concluded that Au... 25 (pEBA) 18 Gold nanoclusters exhibit excellent antioxidant and anti-aggregation capabilities in solution environments.

[0053] Example 2 The difference between this embodiment and Example 1 is that the molar ratio of chloroauric acid to pEBA is 1:0.5, the reduction reaction temperature is 15°C, and the reduction reaction time is 10 hours. All other reaction conditions and operating procedures are the same as in Example 1.

[0054] Characterization and performance testing showed that the product obtained in Example 2 was basically consistent with that in Example 1 in terms of structural characteristics, morphological distribution, and key performance indicators. Although the reaction temperature was slightly reduced, the amount of ligand was decreased, and the reaction time was appropriately extended, the prepared material still exhibited similar catalytic activity, stability, and retention of alkyne functional groups, indicating that the expected structure construction and functional expression could be achieved under these conditions, achieving technical effects comparable to those in Example 1.

[0055] Example 3 The difference between this embodiment and Example 1 is that the molar ratio of chloroauric acid to pEBA is 1:4, the reduction reaction temperature is 35°C, and the reduction reaction time is 20 hours. All other reaction conditions and operating procedures are the same as in Example 1.

[0056] Characterization and performance testing showed that the product obtained in Example 3 was basically consistent with that in Example 1 in terms of structural characteristics, morphological distribution, and key performance indicators. Even with increased ligand dosage, higher reaction temperature, and appropriately extended reaction time, the prepared material still exhibited similar catalytic activity, stability, and retention of alkynyl functional groups, indicating that the expected structural construction and functional expression could be achieved under these conditions, resulting in technical effects comparable to those of Example 1.

[0057] The above is a further detailed description of the present invention and should not be considered as a limitation on the specific implementation of the present invention. For those skilled in the art, simple deductions or substitutions without departing from the concept of the present invention are all within the protection scope of the present invention.

Claims

1. An atomically precise metal nanocluster with Raman-fluorescence dual signals, characterized in that, The atomically precise metal nanoclusters with Raman-fluorescence dual signals consist of an atomically precise metal core and ligands containing alkynyl functional groups.

2. The atomically precise metal nanoclusters with Raman-fluorescence dual signals according to claim 1, characterized in that, The atom-precise metal core consists of M metal atoms, where M is 13, 25, 38, or 55, and the metal core particle size is 0.5 nm to 5 nm. The metal atom is at least one of gold (Au), silver (Ag), and copper (Cu).

3. The atomically precise metal nanoclusters with Raman-fluorescence dual signals according to claim 1, characterized in that, The ligand containing the alkynyl functional group is an alkynyl molecule RC≡CR containing a functional group R; wherein the functional group R is selected from at least one of hydrogen, hydrocarbon groups with different numbers of carbon atoms, phenyl, imidazole ring, sulfonic acid group, hydroxyl, carboxyl, amino, mercapto, fluorine, nitro and methoxy.

4. A method for preparing atomically precise metal nanoclusters with Raman-fluorescence dual signals as described in any one of claims 1-3, characterized in that, Includes the following steps: (1) Dissolve the ligand containing the alkynyl functional group in a mixed solvent of alcohol and amine to form a ligand solution; (2) Mix the soluble metal salt solution with the ligand solution to carry out a redox reaction; (3) After the redox reaction is completed, a reducing agent is added to carry out the reduction reaction; (4) After the reduction reaction is completed, the reaction solution is purified to obtain the atomically precise metal nanoclusters with Raman-fluorescence dual signals.

5. The preparation method according to claim 4, characterized in that, In step (1), the alcohol is selected from C1. At least one of alkyl alcohols and polyols of C6; the amine is selected from C1.

8. At least one of alkylamines, diamines, triamines and alkanolamines.

6. The preparation method according to claim 4, characterized in that, In step (2), the soluble metal salt is selected from at least one of chloroauric acid, silver nitrate and copper chloride; the molar ratio of the soluble metal salt to the ligand containing the alkynyl functional group is 1:0.5~1:

4.

7. The preparation method according to claim 4, characterized in that, In step (3), the reducing agent is selected from at least one of carbon monoxide, sodium borohydride, sodium citrate and ascorbic acid; the reduction reaction is carried out at a temperature of 15-35°C for 10-20 hours.

8. The preparation method according to claim 4, characterized in that, In step (4), the purification is performed by dilution-ultrafiltration using 3-10 kDa.

9. The application of atomically precise metal nanoclusters with Raman-fluorescence dual signals as described in any one of claims 1-3 in optical imaging, characterized in that, The optical imaging includes near-infrared II fluorescence imaging and Raman imaging.

10. The application of the atomically precise metal nanoclusters with Raman-fluorescence dual signals as described in any one of claims 1-3 in the preparation of rapidly in vivo clearance diagnostic and therapeutic agents, characterized in that, The therapeutic preparation is an integrated diagnostic and therapeutic preparation for the disease.

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