A long-lived, high-luminescence near-infrared II region fluorescent copper nanoclusters and their preparation and application

Abstract

This invention provides a method for preparing long-lifetime, high-emission, near-infrared II region fluorescent copper nanoclusters, comprising: adding an aqueous solution of ligand template molecules to an aqueous solution of copper nitrate to obtain a mixed solution; wherein the ligand template molecules are selected from any one or a mixture of two or more small molecule compounds, peptides, or proteins; and the ligand template molecules contain functional groups with thiol or disulfide bond structures; and removing the Cu in the mixed solution with an aqueous solution of sodium borohydride. 2+ The copper atoms are reduced to monovalent copper ions, and the reaction continues, causing copper atoms to grow and aggregate within the ligand template molecule to form copper nanoclusters with 20-45 copper atoms. The copper nanoclusters prepared by the method of this invention can emit near-infrared II fluorescence, exhibiting high fluorescence quantum yield, long fluorescence lifetime, and good biocompatibility, demonstrating potential applications in fluorescence detection, bioanalysis, imaging, and other fields.

Description

Technical Field

[0001] This invention relates to the field of near-infrared II region fluorescent emitting materials technology, and in particular to a long-lifetime near-infrared II region fluorescent copper nanoclusters and their preparation and application. Background Technology

[0002] In recent years, inorganic metal nanoclusters have attracted widespread attention due to their unique spectral and chemical properties, making them promising for applications in sensing, biolabeling, and bioimaging. In particular, metal nanoclusters, composed of only a few dozen metal atoms, are under strong quantum confinement, with their conduction and valence bands decomposed into discrete energy levels, allowing for fluorescence emission through interaction with electromagnetic radiation via electronic transitions. Gold and silver nanoclusters have become research hotspots due to their high quantum yield and tunable photoluminescence properties, but copper nanoclusters (CuNCs) possess significant application potential in bioimaging, sensing, and diagnostics due to their unique optical properties and biocompatibility. However, research on the synthesis and application of copper nanoclusters lags behind, mainly due to their poor stability in aqueous media, sensitivity to oxidation, and the difficulty in preparing small-sized copper-based particles.

[0003] Despite the immense potential of copper nanoclusters, the currently known types are limited, such as Cu13, Cu14, and Cu18, which restricts their comprehensive research and application exploration. To advance the development of copper nanoclusters, it is crucial to expand the existing range of copper nanoclusters, explore new synthetic methods and structure-property relationships, and evaluate their potential applications in sensing, bioimaging, and optical devices. In particular, extending the emission wavelength of copper nanoclusters from the first near-infrared window to the second near-infrared window will facilitate deeper tissue imaging and reduce photon scattering in bioimaging, which has significant application value in the biomedical field.

[0004] Although some copper clusters have been reported to emit in the near-infrared region, there are currently no reports of copper nanoclusters dispersed in aqueous phase emitting in the near-infrared-II window. With increasing demands for tissue imaging in scientific research and clinical medicine, there is an urgent need to develop new technologies to prepare copper nanoclusters that can stably emit light in the near-infrared II region (NIR-II) with high fluorescence quantum yield efficiency and long fluorescence lifetime, in order to meet the requirements of high resolution and low background interference for deep tissue imaging. Summary of the Invention

[0005] To address the aforementioned technical problems, the present invention aims to provide a long-lifetime, high-luminescence near-infrared II region fluorescent copper nanoclusters, as well as their preparation and application. The copper nanoclusters of the present invention can emit near-infrared II region fluorescence, are stable in water dispersion, and have good biocompatibility.

[0006] The first aspect of this invention provides a method for preparing long-lifetime, high-luminescence near-infrared II region fluorescent copper nanoclusters, comprising the following steps:

[0007] (1) Dissolve copper nitrate in water, and then add an aqueous solution of ligand template molecules to a light blue transparent aqueous solution of copper nitrate to obtain a mixed solution; wherein, the ligand template molecules are selected from any one or a mixture of two or more small molecule compounds, polypeptides or proteins; and the ligand template molecules contain functional groups with thiol or disulfide bond structures;

[0008] (2) Use sodium borohydride aqueous solution to remove Cu from the mixed solution described in (1). 2+ The copper atoms are reduced to monovalent copper ions, and then the reaction continues to allow copper atoms to grow and aggregate within the ligand template molecule to form copper nanoclusters, wherein the number of copper atoms in the copper nanoclusters is 20-45.

[0009] Furthermore, the small molecule compound mentioned in step (1) can be selected from LA-sulfobetaine, LA-PEG, etc. n -OCH3 or LA-PEG n Any one or more of -NH2.

[0010] The chemical structure of the LA-sulfobetaine is as follows:

[0011]

[0012] Furthermore, LA-PEG n -OCH3 and LA-PEG n The molecular weight of -NH2 can be 1000, 2000, 3000 or 5000.

[0013] Further, the polypeptide mentioned in step (1) may be EKEKEKEKC or EKEKEKEK(LA).

[0014] Further, in step (1), the protein may be selected from any one or a mixture of two or more of β-lactoglobulin, bovine ribonuclease-A, or bovine serum albumin (BSA).

[0015] Preferably, in step (1), the concentration of the copper nitrate aqueous solution is 5-10 mM; and the concentration of the ligand template molecule aqueous solution is 10 mg / mL-25 mg / mL.

[0016] In step (1), after the ligand template molecule is added, the Cu in the mixed solution 2+Ions are anchored in the ligand template molecules, providing space for the subsequent aggregation and reduction of copper atoms to form copper nanoclusters. As the concentration of ligand template molecules in the mixed solution increases, the dispersion of copper clusters in water increases, but the emission spectrum also exhibits a blue shift of 50-200 nm. Therefore, in a preferred embodiment of this invention, the ratio of copper nitrate to ligand template molecules in step (1) needs to be controlled according to the type of ligand template molecules. Specifically:

[0017] In one specific embodiment of the present invention, when the ligand template molecule in step (1) is a small molecule compound, the molar ratio of copper nitrate to the ligand template molecule in the mixed solution in step (1) is controlled to be 3-10:1. More preferably, when the small molecule compound is LA-sulfobetaine, the molar ratio of copper nitrate to LA-sulfobetaine is controlled to be 3-5:1; most preferably, the molar ratio of copper nitrate to LA-sulfobetaine is controlled to be 5:1.

[0018] In another specific embodiment of the present invention, when the ligand template molecule in step (1) is a polypeptide, the molar ratio of copper nitrate and ligand template molecule in the mixed solution in step (1) is controlled to be 10-25:1.

[0019] In another specific embodiment of the present invention, when the ligand template molecule in step (1) is a protein, the molar ratio of copper nitrate to ligand template molecule in the mixed solution in step (1) is controlled to be 25:18.

[0020] In step (2), sodium borohydride acts as a reducing agent. The amount of reducing agent added should be controlled within a suitable range. If the amount is too large, the reduction reaction will be too violent, resulting in a black precipitate in the mixed solution or an immediate blackening of the solution. This indicates that a large number of copper atoms aggregate to form elemental copper nanoparticles rather than clusters, making it difficult to form copper nanoclusters with near-infrared II fluorescence. Therefore, in the preferred embodiment of the present invention, the molar ratio of sodium borohydride in step (2) to copper nitrate in step (1) is preferably 1-1.8:1.

[0021] Further, in step (2), the Cu in the mixed solution of (1) is removed by using an aqueous solution of sodium borohydride. 2+ The specific steps for reducing Cu to monovalent copper ions are as follows: Sodium borohydride aqueous solution is added dropwise to the mixed solution described in (1) while stirring, so that Cu... 2+ The ions are reduced to Cu + ;

[0022] Furthermore, in step (2), the specific method for the continued reaction to allow copper atoms to grow and aggregate within the ligand template molecule to form copper nanoclusters can be: rapid aggregation to form copper clusters under microwave rapid heating conditions, with copper ions on the surface combining with the ligands to form a stable structure; or reacting at 25°C for 12-24 hours, or reacting at 4°C for 24-72 hours, can also slowly grow into copper clusters. Under these temperature conditions and reaction times, copper nanoclusters can grow relatively ideally.

[0023] Furthermore, the rapid aggregation of copper clusters under microwave rapid heating environment is preferably carried out at 100W, 50-100℃ (more preferably 80℃) for 90-900s (more preferably 900s), so that copper atoms grow and aggregate within the ligand template molecule to form copper nanoclusters, and the final reaction solution is a yellow transparent clear solution.

[0024] Furthermore, in the preferred step (2), after forming the copper nanoclusters, the process further includes using a dialysis bag with a molecular weight cutoff of 8000-14000 Da to remove unreacted ions (such as Cu). 2+ Na + NO3 - The steps are as follows.

[0025] The second aspect of the present invention claims a long-lifetime, high-luminescence near-infrared II region fluorescent copper nanoclusters prepared by the above preparation method, comprising copper nanoclusters and ligand template molecules attached to the surface of the copper nanoclusters, wherein the number of copper atoms in the copper nanoclusters is 20-45, and the ligand template molecules are any one or a combination of two of small molecule compounds, peptides or proteins; and the ligand template molecules contain thiol groups or disulfide bonds.

[0026] Preferably, the ligand template molecule is derived from a polypeptide or protein; and the polypeptide or protein contains amino acid units with thiol groups or disulfide bonds.

[0027] More preferably, the ligand template molecule is derived from the polypeptide EKEKEKEKC or EKEKEKEK(LA).

[0028] More preferably, the ligand template molecule is derived from any one or more of the following proteins: β-lactoglobulin, bovine ribonuclease-A, or bovine serum albumin (BSA).

[0029] A third aspect of the present invention provides the application of the copper nanoclusters described in the second aspect of the present invention (i.e., water-dispersible and stable copper nanoclusters modified with different thiol or disulfide compounds) in the preparation of fluorescence imaging formulations.

[0030] Furthermore, in the application described in this invention, the fluorescence imaging formulation is a formulation for in vivo non-invasive real-time near-infrared II region fluorescence imaging. The copper nanoclusters used to prepare the fluorescence imaging formulation are ultra-small copper cluster formulations. With their long lifespan and strong luminescence properties, they can achieve precise in vivo non-invasive fluorescence imaging in the near-infrared II region, and are particularly suitable for the early diagnosis of kidney and bladder cancer.

[0031] Furthermore, the formulation is an intravenous injection. The near-infrared II region copper nanoclusters of the present invention have good biocompatibility and safety, and can be administered intravenously.

[0032] In the application described in this invention, the targeted modification of the ligand template molecules in the copper nanoclusters enhances the accurate differentiation of normal tissues and tumor cells in vivo by the fluorescent imaging agent, improves the safety of copper-based nanobioproducts, and accelerates their application in the field of clinical imaging.

[0033] The long-lifetime, high-luminescence near-infrared II region fluorescent copper nanoclusters of the present invention have a fluorescence emission peak in the near-infrared II region, an emission wavelength corresponding to the emission peak of 900-1200 nm, a fluorescence lifetime of 10-30 microseconds, and a fluorescence quantum yield of up to 2.25%.

[0034] By means of the above solution, the present invention has the following advantages:

[0035] This invention employs an aqueous-phase synthesis method. Copper nitrate is dissolved in an aqueous solution, and a reducing agent is used to reduce divalent copper ions to copper atoms. Specific ligand templates are selected, including water-soluble small molecule compounds, peptides, and proteins containing thiol groups or disulfide bonds. Then, by changing conditions such as temperature and reaction time, copper atoms are induced to grow and aggregate within the corresponding ligand templates to form copper nanoclusters. The copper nanoclusters synthesized by this method are a novel ultra-small copper cluster formulation with long lifetime and strong luminescence properties, especially excellent fluorescence performance in the near-infrared II region, making it highly valuable for in vivo non-invasive fluorescence imaging. The ultra-small size of this formulation allows for rapid metabolism by the kidneys after intravenous administration and eventual accumulation in the bladder, providing a highly sensitive imaging method for the early diagnosis of renal or bladder cancer, and offering a safe and effective new approach for cancer detection in clinical practice. While oil-phase methods can also prepare luminescent materials with similar functions, subsequent processing is complex, the reaction requires high temperatures, and biocompatibility is poor, preventing direct in vivo use and necessitating subsequent ligand replacement or biocompatible molecular encapsulation. Compared to oil-phase preparation methods, this invention employs a one-pot aqueous-phase method, which can complete the reaction in 8 hours at 4-25°C. Furthermore, the use of a microwave-assisted synthesizer can shorten the reaction time to less than 1 minute. This synthesis method is simple, convenient, and safe to operate.

[0036] The above description is merely an overview of the technical solution of the present invention. In order to better understand the technical means of the present invention and to implement it in accordance with the contents of the specification, the following describes the preferred embodiments of the present invention in conjunction with detailed drawings. Attached Figure Description

[0037] Figure 1 This is a high-resolution transmission electron microscope image of the copper nanoclusters prepared in Example 1 of this invention.

[0038] Figure 2 This demonstrates the dynamic light-scattering hydration particle size distribution of the copper nanoclusters prepared in Example 1 of this invention.

[0039] Figure 3 The ultraviolet-visible absorption spectrum and fluorescence emission spectrum of the copper nanoclusters prepared in Example 1 of this invention are shown.

[0040] Figure 4 The time-resolved fluorescence spectrum of the copper nanoclusters prepared in Example 1 of this invention is shown.

[0041] Figure 5 This demonstrates the quantum yield of the copper nanoclusters prepared in Example 1 of this invention, calculated in comparison with IR-26 molecules.

[0042] Figure 6 The X-ray photoelectron spectroscopy spectrum of the copper nanoclusters prepared in Example 1 of this invention is shown.

[0043] Figure 7 The X-ray excited Auger electron spectrum of the copper nanoclusters prepared in Example 1 of this invention is shown.

[0044] Figure 8 This is a high-resolution transmission electron microscope image of the copper nanoclusters prepared in Example 5 of this invention.

[0045] Figure 9 The results of the test on the CCK8 toxicity of the copper nanoclusters prepared in Example 1 of this invention to HUVEC cells are presented.

[0046] Figure 10 The results demonstrate the real-time in vivo imaging monitoring of the copper nanoclusters prepared in Example 1 via intravenous injection in mice.

[0047] Figure 11 The results of in vitro imaging monitoring of major organs in mice after intravenous injection of copper nanoclusters prepared in Example 1 are presented.

[0048] Figure 12 The fluorescence emission spectra of copper nanoclusters synthesized by feeding copper nitrate and the small molecule ligand LA-sulfobetaine at different molar ratios are shown. Detailed Implementation

[0049] The specific embodiments of the present invention will be described in further detail below with reference to the examples. These examples are for illustrative purposes only and are not intended to limit the scope of the invention.

[0050] The materials used in the following embodiments of the present invention are as follows:

[0051] Deionized water (resistivity 18.2 mΩcm), copper nitrate (Cu(NO3)2, Maclean), sodium borohydride (NaBH4, Sinopharm), bovine serum albumin (BSA, Sinopharm).

[0052] Example 1

[0053] (1) The synthesized ligand template molecule LA-sulfobetaine was dissolved in water to prepare a ligand template solution with a concentration of 100 mM. In addition, copper nitrate was dissolved in deionized water to obtain a copper nitrate solution with a concentration of 10 mM.

[0054] (2) Take 500 μL of the copper nitrate solution prepared in step (1) and 10 μL of the template ligand solution prepared in step (1) and mix them evenly. Stir at room temperature to obtain a mixed solution with a molar ratio of copper nitrate and LA-sulfobetaine of 5:1.

[0055] (3) After mixing thoroughly for 5 minutes, add 60 μL of NaBH4 aqueous solution (200 mM concentration) to the above mixed solution to obtain a clear, brownish-red mixed solution. Place the mixed solution in a refrigerator at 4°C overnight until the solution turns into a clear, yellow solution, indicating that the reaction is complete and the solution contains a large number of copper nanoclusters LA-sulfobetaine@CuNCs.

[0056] The product obtained in step (3) was dissolved in water and dialyzed with a dialysis bag with a molecular weight cutoff of 8000-14000 Da to remove unreacted ions. It was then stored in a refrigerator at 4°C for later use.

[0057] Alternatively, the same copper nanoclusters can be prepared by using microwave-assisted synthesis. The mixed solution containing the reducing agent in step (3) is added to a microwave synthesizer with a reaction energy of 100W and a reaction temperature of 80℃ for 900s.

[0058] The copper nanoclusters prepared in this embodiment were subjected to performance testing and characterization, and the results are as follows: Figures 1-6 As shown, the specific description is as follows:

[0059] Figure 1This is a high-resolution transmission electron microscope (TEM) image of the copper nanoclusters prepared in Example 1. The image shows that the copper nanoclusters are spherical with an average particle size of approximately 2.95 nm. Furthermore, dynamic light scattering particle size distribution testing shows that the hydrated particle size of the copper nanoclusters is 4.18 ± 0.99 nm. Figure 2 ).

[0060] Figure 3 The absorption and emission spectra of the copper nanoclusters prepared in Example 1 are shown. When excited by an 808 nm laser, the emission peak is at 1000 nm, which is in the near-infrared II region.

[0061] Figure 4 The time-resolved fluorescence spectra of the copper nanoclusters prepared in Example 1, measured by time-correlated single-photon counting, are shown. Figure 4 The results show that the fluorescence lifetimes (τ) are 11 μs and 29 μs, respectively.

[0062] Figure 5 The figure shows the quantum yield of the copper nanoclusters prepared in Example 1 compared to IR-26 molecules. The "copper nanoclusters" in the figure represent the copper nanoclusters prepared in Example 1. Figure 5 As can be seen, with IR-26 as the standard control, the quantum yield of the copper nanoclusters LA-sulfobetaine@CuNCs in Example 1 is 2.25%.

[0063] Figure 6 The X-ray photoelectron spectra of the copper nanoclusters prepared in Example 1 are shown, exhibiting Cu2p at 952.2 and 932.4 eV, respectively. 1 / 2 and 2p 3 / 2 The energy difference between the spin-orbit splitting peaks in the Cu2p fine spectrum is 19.8 eV, indicating the presence of Cu(I).

[0064] Figure 7 The Cu-LMMX X-ray excited Auger electron spectrum of the copper nanoclusters prepared in Example 1 shows a significant peak at 916.4 eV, confirming that all copper atoms in the clusters are Cu(I).

[0065] Example 2

[0066] (1) The synthesized ligand template molecule LA-sulfobetaine was dissolved in water to prepare a ligand template solution with a concentration of 100 mM. In addition, copper nitrate was dissolved in deionized water to obtain a copper nitrate solution with a concentration of 10 mM.

[0067] (2) Take 500 μL of the copper nitrate solution prepared in step (1) and 15 μL of the template ligand solution prepared in step (1) and mix them evenly. Stir at room temperature to obtain a mixed solution with a molar ratio of copper nitrate and LA-sulfobetaine of 10:3.

[0068] (3) After mixing thoroughly for 5 minutes, add 60 μL of NaBH4 aqueous solution (200 mM concentration) to the above mixed solution to obtain a clear, brownish-red mixed solution. Place the mixed solution in a refrigerator at 4°C overnight until the solution turns into a clear, yellow solution, indicating that the reaction is complete and the solution contains a large number of copper nanoclusters LA-sulfobetaine@CuNCs.

[0069] The product obtained in step (3) was dissolved in water and dialyzed with a dialysis bag with a molecular weight cutoff of 8000-14000 Da to remove unreacted ions. It was then stored in a refrigerator at 4°C for later use.

[0070] Alternatively, the same copper nanoclusters can be prepared by using microwave-assisted synthesis. The mixed solution with reducing agent added in step (3) is added to a microwave synthesizer, the reaction energy is 100W, the reaction is carried out at 80℃ for 900 seconds.

[0071] Example 3

[0072] (1) The synthesized ligand template molecule LA-sulfobetaine was dissolved in water to prepare a ligand template solution with a concentration of 100 mM. In addition, copper nitrate was dissolved in deionized water to obtain a copper nitrate solution with a concentration of 10 mM.

[0073] (2) Take 500 μL of the copper nitrate solution prepared in step (1) and 5 μL of the template ligand solution prepared in step (1) and mix them evenly. Stir at room temperature to obtain a mixed solution with a molar ratio of copper nitrate and LA-sulfobetaine of 10:1.

[0074] (3) After mixing thoroughly for 5 minutes, add 60 μL of NaBH4 aqueous solution (200 mM concentration) to the above mixed solution to obtain a clear, brownish-red mixed solution. Place the mixed solution in a refrigerator at 4°C overnight until the solution turns into a clear, yellow solution, indicating that the reaction is complete and the solution contains a large number of copper nanoclusters LA-sulfobetaine@CuNCs.

[0075] The product obtained in step (3) was dissolved in water and dialyzed with a dialysis bag with a molecular weight cutoff of 8000-14000 Da to remove unreacted ions. It was then stored in a refrigerator at 4°C for later use.

[0076] Alternatively, the same copper nanoclusters can be prepared by using microwave-assisted synthesis. The mixed solution with reducing agent added in step (3) is added to a microwave synthesizer, the reaction energy is 100W, the reaction is carried out at 80℃ for 900 seconds.

[0077] Examples 4-9

[0078] Copper nanoclusters were prepared according to the method of Example 1, except that LA-sulfobetaine was replaced with equimolar amounts of β-lactoglobulin (Example 4), bovine serum albumin (Example 5), ribonuclease (Example 6), and polyethylene glycol thioctic acid (LA-PEG). n -OCH3 or LA-PEG n -NH2)(Examples 7-8) and bioengineered custom peptides (with the amino acid sequence structure EKEKEKEK(LA)) (Example 9).

[0079] The morphology of the copper nanoclusters prepared in Example 5 with BSA as a ligand is shown in [example 5]. Figure 8 .

[0080] Comparative Example 1 - Comparative Example 2

[0081] Copper nanoclusters were prepared according to the method in Example 1, except that LA-sulfobetaine was replaced with equimolar amounts of glutathione and dihydrolipoic acid to obtain copper nanoclusters.

[0082] Experimental Example 1. Fluorescence Performance Test

[0083] The near-infrared II region fluorescence properties of several copper nanoclusters prepared in the above examples were tested, and the results are shown in Table 1.

[0084] Table 1. Near-infrared II fluorescence properties of copper nanoclusters prepared with different ligand template molecules.

[0085]

[0086] As shown in Table 1, all copper nanoclusters prepared with the nine ligand molecules can emit infrared fluorescence, and the ligands used include β-lactoglobulin, bovine serum albumin, ribonuclease, and polyethylene glycol thioctic acid (LA-PEG). n -OCH3 or LA-PEG n The fluorescence emission peak of copper nanoclusters prepared with -NH2 and the bioengineered custom peptide EKEKEKEK(LA) is in the near-infrared II region (NIR-II), while the fluorescence peak of copper nanoclusters prepared with dihydrolipoic acid and glutathione as ligands in Comparative Examples 1 and 2 is slightly blue-shifted and is in the near-infrared I region.

[0087] Experimental Example 2. Biosafety Test

[0088] The biosafety of the copper nanoclusters prepared in Example 1 was tested. The toxicity of high concentrations of copper nanoclusters to human umbilical vein endothelial cells (HUVECs) was assessed using a CCK8 assay. The results showed that the copper nanoclusters prepared in Example 1 did not exhibit significant toxicity even at a high concentration of 400 μg / mL. Figure 9 ).

[0089] Experimental Example 3. Mouse In vivo imaging experiment

[0090] The copper nanoclusters prepared in Example 1 were administered to normal mice via tail vein. After hair removal from the abdomen of the mice, each mouse was injected with a dose of 4 mg / kg. Figure 10 In vivo real-time imaging results showed that within 15 minutes of injection, copper nanoclusters rapidly accumulated in the kidneys and were efficiently cleared into the bladder via the kidneys. This rapid renal clearance not only reduces the toxicity of long-term accumulation of imaging agents but also increases the fluorescence contrast between lesions and normal tissues. Figure 11 The in vitro imaging results of major organs also showed the in vivo distribution of the copper nanoclusters prepared in Example 1 within 15 minutes of injection.

[0091] Experimental Example 4. Determination of the yield of copper nanoclusters under different feed ratios

[0092] Following the method in Example 1, copper nanoclusters were prepared by feeding ligand template molecule LA-sulfobetaine and copper nitrate at molar ratios of 3:1, 5:1, 7:1, 9:1, 11:1, and 15:1, respectively. The product solutions before dialysis were subjected to photofluorescence spectroscopy using an Edinburgh-Steady-State / Transient Fluorescence Spectrometer FLS1000. The excitation light was an 808 nm laser, and the emission spectrum range was 850-1300 nm. The results are shown in [Figure 1]. Figure 12 .from Figure 12 As can be seen, when the molar ratio of copper nitrate to ligand LA-sulfobetaine is 3:1, 5:1, and 11:1, the fluorescence intensity of the product solution is above 10,000, indicating that the product solution contains a large amount of the target product and the corresponding synthesis process has a high yield of the target product. In particular, the synthesis efficiency of the target product copper nanoclusters is the highest at the feed ratio of 5:1, and the fluorescence intensity of the product solution is as high as 30,000. However, at feed ratios of 7:1, 9:1, and 15:1, the synthesis efficiency of copper nanoclusters is lower, and the fluorescence intensity of the product solution is basically below 10,000.

[0093] It is evident that, under the condition that the ligand template molecule is the same, the effect of the feed ratio of the synthesized copper nanoclusters in this invention on the yield of the target cluster has a certain degree of unpredictability.

[0094] In the copper nanocluster structure of this invention, modification with various ligands enables the copper nanoclusters to specifically target renal cell carcinoma or bladder cancer, improving the accuracy of early cancer detection. Furthermore, the copper nanoclusters of this invention possess long-lifetime, strong near-infrared II fluorescence emission capabilities, which can enhance the depth and resolution of non-invasive optical imaging, enabling the detection of deep or small-sized renal cell carcinoma or bladder cancer lesions.

[0095] The above are merely preferred embodiments of the present invention and are not intended to limit the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the technical principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for preparing long-lifetime, high-luminescence near-infrared II region fluorescent copper nanoclusters, comprising the following steps: (1) Dissolve copper nitrate in water to obtain an aqueous solution of copper nitrate, then add an aqueous solution of a small molecule compound to the aqueous solution of copper nitrate to obtain a mixed solution; control the molar ratio of copper nitrate to the small molecule compound in the mixed solution to be 3-10:1; or, Copper nitrate is dissolved in water to obtain a copper nitrate aqueous solution. Then, an aqueous solution of the polypeptide is added to the copper nitrate aqueous solution to obtain a mixed solution. The molar ratio of copper nitrate to polypeptide in the mixed solution is controlled to be 10-25:

1. Alternatively, Copper nitrate is dissolved in water to obtain an aqueous solution of copper nitrate. Then, an aqueous solution of protein is added to the aqueous solution of copper nitrate to obtain a mixed solution. The molar ratio of copper nitrate to protein in the mixed solution is controlled to be 25:

18. The small molecule compound, polypeptide, or protein contains a functional group having a thiol or disulfide bond structure; the protein is any one or a mixture of two or more of β-lactoglobulin, bovine ribonuclease, or bovine serum albumin; the polypeptide is EKEKEKEKC or EKEKEKEK(LA); the small molecule compound is selected from LA-sulfobetaine and LA-PEG with structures as shown in formula (I). n -OCH3 or LA-PEG n Any one or more of -NH2; （I）； (2) Use sodium borohydride aqueous solution to remove Cu from the mixed solution in (1). 2+ The sodium borohydride is reduced to monovalent copper ions, and the molar ratio of sodium borohydride to copper nitrate is 1-1.8:

1. Then, the reaction continues to allow copper atoms to grow and aggregate within the ligand template molecule to form copper nanoclusters, wherein the number of copper atoms in the copper nanoclusters is 20-45.

2. The preparation method according to claim 1, characterized in that: The concentration of the copper nitrate aqueous solution in step (1) is 5-10 mM; the concentration of the aqueous solution of the ligand template molecule is 10 mg / mL-25 mg / mL.

3. The preparation method according to claim 1, characterized in that: The small molecule compound mentioned in step (1) is LA-sulfobetaine, and the molar ratio of copper nitrate and LA-sulfobetaine in the mixed solution mentioned in step (1) is controlled to be 3-5:

1.

4. The preparation method according to claim 3, characterized in that: The molar ratio of copper nitrate and LA-sulfobetaine in the mixed solution described in the control step is 5:

1.

5. The preparation method according to claim 1, characterized in that: Step (2) involves using an aqueous solution of sodium borohydride to remove Cu from the mixed solution described in (1). 2+ The specific steps for reducing Cu to monovalent copper ions are as follows: Sodium borohydride aqueous solution is added dropwise to the mixed solution described in (1) while stirring, so that Cu... 2+ The ions are reduced to Cu + .

6. The preparation method according to claim 1, characterized in that: The specific method for the continued reaction described in step (2) to grow and aggregate copper atoms within the ligand template molecule to form copper nanoclusters is as follows: copper clusters are rapidly aggregated in a microwave rapid heating environment, and the copper ions on the surface combine with the ligand to form a stable structure; or the reaction is carried out at 25°C for 12-24 h; or the reaction is carried out at 4°C for 24-72 h.

7. The preparation method according to claim 6, characterized in that: The rapid aggregation of copper clusters under microwave rapid heating environment is achieved by reacting for 90-900 seconds under microwave intensity of 100W and temperature of 50-100℃.

8. The preparation method according to claim 6, characterized in that: The rapid aggregation of copper clusters under microwave rapid heating environment is achieved by reacting at 100W microwave intensity and 80℃ for 900s.

9. A long-lifetime, high-luminescence near-infrared II region fluorescent copper nanoclusters prepared by the method according to any one of claims 1-8, comprising copper nanoclusters and ligand template molecules attached to the surface of the copper nanoclusters, wherein the number of copper atoms in the copper nanoclusters is 20-45, and the ligand template molecules are any one or a combination of two of small molecule compounds, peptides or proteins; and the ligand template molecules contain thiol groups or disulfide bonds; the emission wavelength corresponding to the emission peak of the copper nanoclusters is 1000-1200 nm, and the fluorescence lifetime is 10-30 microseconds.

10. The copper nanoclusters as described in claim 9, characterized in that: The ligand template molecule is any one or a combination of two or more of EKEKEKEKC, EKEKEKEK(LA), β-lactoglobulin, bovine ribonuclease, or bovine serum albumin.

11. The use of the copper nanoclusters according to any one of claims 9-10 in the preparation of formulations for fluorescence imaging.

12. The application as described in claim 11, characterized in that: The fluorescence imaging described is in vivo, non-invasive, real-time near-infrared II region fluorescence imaging.

13. The application as described in claim 11, characterized in that: The aforementioned fluorescence imaging formulation is used for the early diagnosis of kidney and bladder cancer.

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