Long-service-life strong-luminescence near-infrared II-region fluorescent copper nanocluster and preparation and application thereof

Abstract

The invention provides a preparation method of a long-life strong-luminescence near-infrared II-region fluorescent copper nanocluster, which comprises the following steps: adding an aqueous solution of ligand template molecules into an aqueous solution of copper nitrate to obtain a mixed solution; wherein the ligand template molecule is selected from any one or a mixture of more than two of a small molecule compound, polypeptide or protein; the ligand template molecule contains a functional group with a sulfydryl or disulfide bond structure; cu < 2 + > in the mixed solution is reduced into cuprous ions by using a sodium borohydride aqueous solution, and then the reaction is continued, so that copper atoms grow and gather in ligand template molecules to form the copper nanocluster with the copper atom number of 20-45. The copper nanocluster prepared by the method disclosed by the invention can emit near-infrared II-region fluorescence, has higher fluorescence quantum yield, long fluorescence lifetime and good biological safety, and has various application potentials in the fields of fluorescence detection, biological analysis, imaging and the like.

Description

Technical Field

[0001] The invention relates to the technical field of near-infrared II region fluorescent emitting materials, and in particular to a long-lifetime near-infrared II region fluorescent copper nanocluster and preparation and application thereof. Background Art

[0002] In recent years, inorganic metal nanoclusters have attracted extensive attention due to their unique spectral and chemical properties, which make them have a wide range of application potentials in sensing, biolabeling and bioimaging. In particular, metal nanoclusters are composed of only tens of metal atoms, which are under strong quantum confinement, and the conduction band and valence band are decomposed into discrete energy levels, allowing the generation of fluorescence emission through the interaction with electromagnetic radiation through electronic transitions. Gold and silver nanoclusters have become a hot topic of research due to their high quantum yield and tunable photoluminescence properties, but copper nanoclusters (CuNCs) have significant application potentials in bioimaging, sensing and diagnosis due to their unique optical properties and biocompatibility. However, the synthesis and application research of copper nanoclusters is relatively backward, mainly due to their poor stability in aqueous media, sensitivity to oxidation and the difficulty in preparing small-sized copper-based particles.

[0003] Although copper nanoclusters show great potential, there are only a limited number of known copper nanoclusters, such as Cu13, Cu14, Cu18, etc., which restricts the exploration of their comprehensive research and application. In order to promote the development of copper nanoclusters, it is particularly important to expand the existing types of copper nanoclusters, explore new synthesis 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 be beneficial for achieving deeper tissue imaging and reducing photon scattering in bioimaging, which has important application value in the biomedical field.

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

[0005] In order to solve the above technical problems, the purpose of the present invention is to provide a long-lifetime, strong-luminescence near-infrared II region fluorescent copper nanocluster and its preparation and application. The copper nanocluster of the present invention can emit near-infrared II region fluorescence, is stably dispersed in water and has good biological safety.

[0006] The first aspect of the present invention provides a method for preparing a long-lifetime, strong-luminescent near-infrared II region fluorescent copper nanocluster, comprising the following steps:

[0007] (1) dissolving copper nitrate in water, and then adding an aqueous solution of a ligand template molecule to the light blue transparent copper nitrate aqueous solution to obtain a mixed solution; wherein the ligand template molecule is selected from any one or a mixture of two or more of a small molecule compound, a polypeptide or a protein; and the ligand template molecule contains a functional group having a thiol or disulfide bond structure;

[0008] (2) using sodium borohydride aqueous solution to remove the Cu in the mixed solution of (1) 2+ The copper ions are reduced to monovalent copper ions, and then the reaction is continued so that the copper atoms grow and aggregate in the ligand template molecules to form copper nanoclusters. The number of copper atoms in the copper nanoclusters is 20-45.

[0009] Furthermore, in step (1), the small molecule compound can be selected from LA-sulfobetaine, LA-PEG n -OCH3 or LA-PEG n -NH2 or a mixture of two or more thereof.

[0010] The chemical structure of LA-sulfobetaine is

[0011]

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

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

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

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

[0016] In step (1), after adding the ligand template molecule, the Cu 2+The ions are anchored in the ligand template molecules, providing space for subsequent copper atoms to aggregate and reduce to form copper nanoclusters. As the concentration of the ligand template molecules in the mixed solution increases, the dispersion of the copper clusters in water will increase, but the emission spectrum will also have a blue shift of 50-200nm. Therefore, in the preferred embodiment of the present invention, it is necessary to control the ratio of copper nitrate and ligand template molecules in step (1) according to the type of ligand template molecules. Specifically as follows:

[0017] In a 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. In a more preferred embodiment, 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 to ligand template 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 the 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 an appropriate range. If the amount is too much, the reduction reaction will be too violent, which will cause a black precipitate to appear in the mixed solution or turn black immediately, indicating that a large number of copper atoms aggregate to form single copper nanoparticles rather than clusters, making it difficult to form copper nanoclusters with near-infrared II region fluorescence effect. 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] Furthermore, in step (2), the sodium borohydride aqueous solution is used to remove the Cu 2+ The specific steps of reducing to monovalent copper ions are: adding sodium borohydride aqueous solution dropwise to the mixed solution described in (1) while stirring, so that Cu 2+ ions are reduced to Cu + ;

[0022] Furthermore, in step (2), the specific method of continuing the reaction so that the copper atoms grow and aggregate in the ligand template molecules to form copper nanoclusters can be: rapidly aggregate to form copper clusters in a microwave rapid heating environment, and the copper ions on the surface combine with the ligands to form a stable structure; or react at 25°C for 12-24h, or react at 4°C for 24-72h, and slowly grow into copper clusters. Under these temperature conditions and reaction times, copper nanoclusters can grow relatively ideally.

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

[0024] Furthermore, in the preferred step (2), after the copper nanoclusters are formed, a dialysis bag with a molecular weight cutoff of 8000-14000Da is used to dialyze and remove unreacted ions (such as Cu 2+ ,Na + ,NO3 - ) steps.

[0025] The second aspect of the present invention claims protection for a long-lifetime, strong-luminescence, near-infrared II region fluorescent copper nanocluster prepared by the above-mentioned preparation method, which includes a copper nanocluster and a ligand template molecule attached to the surface of the copper nanocluster, the number of copper atoms in the copper nanocluster is 20-45, the ligand template molecule is any one or a combination of two of a small molecule compound, a polypeptide or a protein; and the ligand template molecule contains a thiol group or a disulfide bond.

[0026] Preferably, the ligand template molecule is derived from a polypeptide or a protein; and the polypeptide or the protein comprises an amino acid unit having a thiol group or a disulfide bond.

[0027] Further 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 (RNase-A) or bovine serum albumin (BSA).

[0029] The third aspect of the present invention provides the use of the copper nanoclusters described in the second aspect of the present invention (ie, copper nanoclusters stably dispersed in water modified with different thiol or disulfide bond compounds) in the preparation of fluorescent imaging agents.

[0030] Furthermore, in the application described in the present invention, the fluorescent imaging preparation is a preparation for in vivo non-invasive real-time near-infrared II zone fluorescence imaging, and the copper nanoclusters used to prepare the fluorescent imaging preparation are ultra-small copper cluster preparations, which can achieve accurate in vivo non-invasive fluorescence imaging in the near-infrared II zone due to their long life and strong luminescence characteristics, and are particularly suitable for early diagnosis of kidney and bladder cancer.

[0031] Furthermore, the preparation is an intravenous injection. The near-infrared II zone copper nanoclusters of the present invention have good biocompatibility and safety, and can enter the body through intravenous administration.

[0032] In the application of the present invention, the targeted modification of the ligand template molecules in the copper nanoclusters enhances the accurate distinction of normal tissues and tumor cells by the fluorescent imaging preparation in vivo, improves the safety of copper-based nanobiological products, and accelerates their application in the field of clinical imaging.

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

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

[0035] The present invention adopts an aqueous phase synthesis method, dissolves copper nitrate in an aqueous solution, and uses a reducing agent to reduce divalent copper ions to copper atoms, selects a specific ligand template, which includes a water-soluble small molecule compound containing a thiol group or a disulfide bond, a polypeptide and a protein, and then changes the temperature and reaction time and other conditions to make the copper atoms grow and aggregate in the corresponding ligand template to form copper nanoclusters. The copper nanoclusters synthesized by the method of the present invention are a new type of ultra-small copper cluster preparation, which has long life and strong luminescence characteristics, especially excellent fluorescence performance in the near-infrared second region, so that it has significant application value in the field of in vivo non-invasive fluorescence imaging. The ultra-small size characteristics of the preparation allow it to be rapidly metabolized by the kidneys and eventually aggregated in the bladder after intravenous administration, providing a highly sensitive imaging method for early diagnosis of renal cancer or bladder cancer, and also providing a safe and effective new way for cancer detection in the clinic. Although the oil phase method can also prepare luminescent materials with similar functions, the subsequent processing is complicated, the reaction requires high temperature, and the biological safety is poor. It cannot be directly used in the body and needs to be subsequently replaced by ligands or encapsulated by biocompatible molecules. Compared with the oil phase preparation method, the present invention adopts the water phase one-pot method, which can complete the reaction in 8 hours at 4-25°C, and the microwave-assisted synthesizer can shorten the reaction time to less than 1 minute. The synthesis method is simple, convenient and safe.

[0036] The above description is only an overview of the technical solution of the present invention. In order to more clearly understand the technical means of the present invention and implement it according to the contents of the specification, the following is a description of the preferred embodiments of the present invention in conjunction with detailed drawings. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0038] Figure 2 It reflects the dynamic light scattering hydrated particle size distribution of the copper nanoclusters prepared in Example 1 of the present invention.

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

[0040] Figure 4 It reflects the time-resolved fluorescence spectrum of the copper nanoclusters prepared in Example 1 of the present invention.

[0041] Figure 5 It reflects the quantum yield of the copper nanoclusters prepared in Example 1 of the present invention after calculation in comparison with the IR-26 molecules.

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

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

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

[0045] Fig. 9 The results of the CCK8 toxicity test of the copper nanoclusters prepared in Example 1 of the present invention on HUVEC cells are shown.

[0046] Fig.10 The results of real-time imaging monitoring of mice injected intravenously with the copper nanoclusters prepared in Example 1 are shown.

[0047] Fig.11 The results show the in vitro imaging monitoring results of the main organs of mice injected intravenously with the copper nanoclusters prepared in Example 1.

[0048] Fig.12 It reflects the fluorescence emission spectra of copper nanoclusters synthesized by adding copper nitrate and small molecule ligand LA-sulfobetaine in different molar ratios. DETAILED DESCRIPTION

[0049] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention.

[0050] In the following examples of the present invention, the materials used are as follows:

[0051] Deionized water (resistivity 18.2 mΩcm), copper nitrate (Cu(NO3)2, McLean), 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) 500 μL of the copper nitrate solution prepared in step (1) and 10 μL of the template ligand solution prepared in step (1) were mixed evenly, and stirred at room temperature to obtain a mixed solution with a molar ratio of copper nitrate to LA-sulfobetaine of 5:1.

[0055] (3) After being thoroughly mixed for 5 minutes, 60 μL of reducing agent NaBH4 aqueous solution (concentration 200 mM) was added dropwise to the mixed solution to obtain a brown-red transparent clear mixed solution. The mixed solution was placed in a 4°C refrigerator overnight until the solution turned into a yellow transparent clear solution, indicating that the reaction was complete and the solution contained a large amount of copper nanoclusters LA-sulfobetaine@CuNCs.

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

[0057] The same copper nanoclusters can also be prepared by using a microwave-assisted synthesis method, where the mixed solution containing the reducing agent in step (3) is added into a microwave synthesizer, the reaction energy is 100 W, and the reaction time is 900 s at 80° C.

[0058] The copper nanoclusters prepared in this example were tested and characterized for their performance. Figure 1-Figure 6 The specific description is as follows:

[0059] Figure 1This is a high-resolution transmission electron microscopy image of the copper nanoclusters prepared in Example 1. It can be seen from the figure that the copper nanoclusters are spherical and have an average particle size of about 2.95 nm. In addition, the dynamic light scattering particle size distribution test 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 808 nm laser, the emission peak is at 1000 nm, which is within the near-infrared II region.

[0061] Figure 4 The time-resolved fluorescence spectrum of the copper nanoclusters prepared in Example 1 measured by time-correlated single photon counting method is shown. Figure 4 It can be seen 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 after comparison with the IR-26 molecules. The "copper nanoclusters" in the figure represent the copper nanoclusters prepared in Example 1. Figure 5 It can be seen that, taking IR-26 as the standard control sample, the quantum yield of the copper nanoclusters LA-sulfobetaine@CuNCs in Example 1 is 2.25%.

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

[0064] Figure 7 The Cu-LMMX ray-excited Auger electron spectrum of the copper nanoclusters prepared in Example 1 shows a significant peak at 916.4 eV, confirming that all the 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) 500 μL of the copper nitrate solution prepared in step (1) and 15 μL of the template ligand solution prepared in step (1) were mixed evenly, and stirred at room temperature to obtain a mixed solution with a molar ratio of copper nitrate to LA-sulfobetaine of 10:3.

[0068] (3) After being thoroughly mixed for 5 minutes, 60 μL of reducing agent NaBH4 aqueous solution (concentration 200 mM) was added dropwise to the mixed solution to obtain a brown-red transparent clear mixed solution. The mixed solution was placed in a 4°C refrigerator overnight until the solution turned into a yellow transparent clear solution, indicating that the reaction was complete and the solution contained a large amount of copper nanoclusters LA-sulfobetaine@CuNCs.

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

[0070] The same copper nanoclusters can also be prepared by using a microwave-assisted synthesis method, where the mixed solution to which the reducing agent is added in step (3) is added into a microwave synthesizer, the reaction energy is 100 W, and the reaction is carried out at 80° C. 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), mix them evenly, and stir at room temperature to obtain a mixed solution with a molar ratio of copper nitrate to LA-sulfobetaine of 10:1.

[0074] (3) After being thoroughly mixed for 5 minutes, 60 μL of reducing agent NaBH4 aqueous solution (concentration 200 mM) was added dropwise to the mixed solution to obtain a brown-red transparent clear mixed solution. The mixed solution was placed in a 4°C refrigerator overnight until the solution turned into a yellow transparent clear solution, indicating that the reaction was complete and the solution contained a large amount of copper nanoclusters LA-sulfobetaine@CuNCs.

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

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

[0077] Example 4-Example 9

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

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

[0080] Comparative Example 1-Comparative Example 2

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

[0082] Experimental Example 1. Fluorescence performance test

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

[0084] Table 1 Test results of near-infrared II fluorescence performance of copper nanoclusters prepared by different ligand template molecules

[0085]

[0086] As can be seen from Table 1, the copper nanoclusters prepared by the nine ligand molecules can emit infrared fluorescence, and the copper nanoclusters prepared by using β-lactoglobulin, bovine serum albumin, ribonuclease, lipoic acid polyethylene glycol (LA-PEG n -OCH3 or LA-PEG n -NH2) and bioengineered customized polypeptide EKEKEKEKEK (LA) have a fluorescence emission peak in the near-infrared II region (NIR-II), while the fluorescence peak in the near-infrared II region of the 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 copper nanoclusters prepared in Example 1 were subjected to a biosafety test, and the toxicity of high-concentration copper nanoclusters to human umbilical vein endothelial cells (HUVEC) was tested using CCK8. The results showed that the copper nanoclusters prepared in Example 1 did not show obvious toxicity even at a high concentration of 400 μg / mL ( Fig. 9 ).

[0089] Experimental Example 3. In vivo imaging experiment in mice

[0090] Normal mice were treated with the copper nanoclusters prepared in Example 1 through tail vein administration. After the abdominal hair of the mice was removed, the injection dose for each mouse was 4 mg / kg. Fig.10 The results of in vivo real-time imaging showed that within 15 minutes of injection, copper nanoclusters were rapidly accumulated in the kidneys and efficiently cleared into the bladder through 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. Fig.11 The in vitro imaging results of major organs also reflect 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 at different feed ratios

[0092] Referring to the method of Example 1, the ligand template molecule LA-sulfobetaine and copper nitrate were respectively fed at molar ratios of 3:1, 5:1, 7:1, 9:1, 11:1 and 15:1 to prepare copper nanoclusters. The photoluminescence spectrum of the obtained product solution before dialysis was scanned using an Edinburgh-steady-state / transient fluorescence spectrometer FLS1000. The excitation light was 808 nm laser and the emission spectrum range was 850-1300 nm. The results are shown in FIG. Fig.12 .from Fig.12 It can be seen that when the molar ratio of copper nitrate and ligand LA-sulfobetaine is 3:1, 5:1 and 11:1, the fluorescence intensity of the product solution is above 10000, indicating that the product solution contains a large amount of target product, and the yield of the target product in the corresponding synthesis process is high, especially at a feed ratio of 5:1, the efficiency of synthesizing the target product copper nanoclusters is the highest, and the fluorescence intensity of the product solution is as high as 30000; while at a feed ratio of 7:1, 9:1 and 15:1, the efficiency of synthesizing copper nanoclusters is low, and the fluorescence intensity of the product solution is basically below 10000.

[0093] It can be seen that, under the condition that the ligand template molecules are the same, the effect of the feed ratio of the present invention on the yield of the target clusters has a certain degree of unpredictability.

[0094] In the copper nanocluster structure of the present invention, the modification of multiple ligands can make the copper nanocluster specifically target kidney cancer or bladder cancer, thereby improving the accuracy of early cancer detection. In addition, the copper nanocluster of the present invention has the ability of long-life strong near-infrared second-zone fluorescence, which can enhance the depth and resolution of non-invasive optical imaging and can be used to detect deep or small kidney cancer or bladder cancer lesions.

[0095] The above are only preferred embodiments of the present invention and are not intended to limit the present invention. It should be pointed out that a person skilled in the art can make several improvements and modifications without departing from the technical principles of the present invention, and these improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A method for preparing a long-lifetime, strong-luminescence near-infrared II region fluorescent copper nanoclusters, comprising the following steps: (1) dissolving copper nitrate in water to obtain a copper nitrate aqueous solution, and then adding an aqueous solution of a ligand template molecule to the copper nitrate aqueous solution to obtain a mixed solution; wherein the ligand template molecule is selected from any one or a mixture of two or more of a small molecule compound, a polypeptide or a protein; and the ligand template molecule contains a functional group having a thiol or disulfide bond structure; the small molecule compound is preferably LA-sulfobetaine, LA-PEG n -OCH3 or LA-PEG n -NH2, any one or a mixture of two or more thereof; the polypeptide is preferably EKEKEKEKC or EKEKEKEK(LA); the protein is preferably any one or a mixture of two or more thereof selected from β-lactoglobulin, bovine ribonuclease (RNase-A) or bovine serum albumin (BSA); (2) using sodium borohydride aqueous solution to remove the Cu in the mixed solution of (1) 2+ Reduction to monovalent copper ions, preferably the molar ratio of sodium borohydride to copper nitrate is 1-1.8:1; then the reaction is continued so that the copper atoms grow and aggregate in the ligand template molecules to form copper nanoclusters, and 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 ligand template molecule in step (1) is a small molecule compound, and the molar ratio of copper nitrate to ligand template molecule in the mixed solution of step (1) is controlled to be 3-10:1; preferably, the small molecule compound is LA-sulfobetaine, and the molar ratio of copper nitrate to LA-sulfobetaine in the mixed solution of step (1) is controlled to be 3-5:1; most preferably, the molar ratio of copper nitrate to LA-sulfobetaine in the mixed solution of step (1) is controlled to be 5:

1.

4. The preparation method according to claim 1, characterized in that: The ligand template molecule in step (1) is a polypeptide, and the molar ratio of copper nitrate to the ligand template in the mixed solution in step (1) is controlled to be 10-25:

1.

5. The preparation method according to claim 1, characterized in that: The ligand template molecule in step (1) is a protein, and the molar ratio of copper nitrate to the ligand template molecule in the mixed solution in step (1) is controlled to be 25:

18.

6. The preparation method according to claim 1, characterized in that: In step (2), the Cu in the mixed solution of (1) is treated with sodium borohydride aqueous solution. 2+ The specific steps of reducing to monovalent copper ions are: adding sodium borohydride aqueous solution dropwise to the mixed solution described in (1) while stirring, so that Cu 2+ ions are reduced to Cu + .

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

8. The preparation method according to claim 7, characterized in that: The rapid aggregation to form copper clusters under a microwave rapid heating environment is carried out under a microwave intensity of 100W and a temperature of 50-100°C (preferably 80°C) for 90-900s (preferably 900s).

9. A long-lifetime, strong-luminescence, near-infrared II region fluorescent copper nanocluster, comprising a copper nanocluster and a ligand template molecule attached to the surface of the copper nanocluster, wherein the number of copper atoms in the copper nanocluster is 20-45, and the ligand template molecule is any one or a combination of two of a small molecule compound, a polypeptide or a protein; and the ligand template molecule contains a thiol group or a disulfide bond; the ligand template molecule is preferably any one or a combination of two or more of EKEKEKEKEKC, EKEKEKEK (LA), β-lactoglobulin (β-lactoglobulin), bovine ribonuclease (RNase-A) or bovine serum albumin (BSA).

10. Use of the copper nanoclusters according to claim 9 in the preparation of a preparation for fluorescence imaging; the fluorescence imaging is preferably in vivo non-invasive real-time near-infrared II region fluorescence imaging; and is further preferably used for early diagnosis of kidney and bladder cancer.

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