Near-infrared II-region fluorescent silver nanocluster as well as preparation and application thereof

Abstract

The invention provides a preparation method of a near-infrared II-zone silver nanocluster, which comprises the following steps: preparing silver nitrate powder into an aqueous solution, then adding ligand template molecules to obtain a mixed solution, and adding an alkaline aqueous solution into the mixed solution for reaction to obtain an alkaline mixed solution with the pH value of 10-11; the ligand template molecule is any one or a composition of more than two of an organic small molecule compound, amino acid, a polypeptide fragment, a high-molecular compound or protein; the ligand template molecule contains sulfydryl or simultaneously contains sulfydryl and a conjugated structure; ag < 1 + > in the alkaline mixed solution is reduced into silver atoms Ag0 by using a sodium borohydride aqueous solution, and then the reaction is continued, so that the silver atoms grow and gather in ligand template molecules to form silver nanoclusters with the silver atom number of 18-25, and the ligands are coated with the silver nanoclusters. The silver nanocluster disclosed by the invention can emit near-infrared II-region fluorescence and can be used for biological living body imaging.

Description

Technical Field

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

[0002] Near-infrared II fluorescence imaging technology refers to the use of infrared lasers (>808nm) to excite contrast agent materials to emit near-infrared II fluorescence (1000-1700nm) for real-time, dynamic detection at the level of living organisms. This technology originated from an important scientific hypothesis in Carroll et al.'s 2006 fluorescent quantum dot bioimaging study: the near-infrared II region (900nm-1700nm) fluorescence bioimaging effect is better than the visible light region and near-infrared I region (400-900nm). Once proposed, this hypothesis has been recognized by many scholars. Compared with traditional optical imaging technology, near-infrared II region imaging has high sensitivity (detection limit: 10 -15 mol / L), high time (<50ms) and spatial resolution (<25μm), deep tissue penetration (>2cm), low cost, no radiation, and low interference from biological tissue self-luminescence, etc., and have become a hot area in biological imaging research in recent years. Since 2018, top engineering materials journals such as Nature, Nature Nanotechnology, and Nature Materials have successively reported on the synthesis and preparation of new near-infrared II materials and their imaging applications in vivo (such as brain, kidney, liver, etc.). Near-infrared II nanoimaging has become a hot research field in biological imaging technology that is quite exploratory and challenging.

[0003] At present, research has found that inorganic nanomaterials with near-infrared II fluorescence imaging performance mainly include the following two categories: rare earth doped materials and heavy metal quantum dots. These two nanomaterials have different luminescence mechanisms.

[0004] Rare earth element doped nanomaterials are usually composed of a matrix and an activator. The matrix is ​​a compound that serves as the main body of the material (e.g. Y 2 O 3、 La 2 O 3 or Gd 2 O 3 ), the activator is a small amount of doped ions (especially trivalent rare earth ions, such as Sm 3+ 、Eu 3+ , Tb 3+ 、Dy 3+ 、Nd 3+ 、Ho 3+ , Er 3+ 、Tm3+ or Yb 3+ ), for near-infrared II rare earth-doped nanoparticles, the emission wavelength depends on the element type of the doped activator. For example, Ho, Pr, Tm and Er can emit fluorescence at 1185nm, 1310nm, 1475nm and 1525nm, respectively. This material has excellent optical properties such as long fluorescence lifetime, narrow spectral line, wide coverage area of ​​fluorescence emission wavelength, etc., but the quantum yield is low (0.1%-3%). Moreover, rare earth-doped nanofluorescent materials have poor biological stability. Rare earth elements are easily combined with phosphoric acid and converted into rare earth phosphate compounds, resulting in fluorescence quenching, triggering cell NLRP3 inflammasome activation and releasing inflammatory factors. Therefore, the biosafety of near-infrared II rare earth materials is the main obstacle to clinical application.

[0005] Heavy metal quantum dots are mainly composed of IV, II-VI, IV-VI or III-V group elements, containing a limited number of atoms to form a lattice, and the three dimensions are all in the nanometer order of magnitude, and the excitons are bound in three spatial directions. The luminescence mechanism of heavy metal quantum dots lies in the action of excitons. The luminescence process is that the negatively charged electrons in the valence band are excited to jump to the conduction band, leaving a positively charged hole to form an electron-hole combination. When the exciton is deactivated, it will show fluorescence characteristics. At the same time, as the size of the quantum dot lattice changes, the band gap between the valence band and the conduction band will change, thereby affecting the change of the luminescence wavelength from visible light to infrared light. As a novel semiconductor nanomaterial, quantum dots have many unique optical properties, especially in the range of near-infrared II fluorescence (1000-1700nm). Compared with other fluorescent materials, quantum dots have the characteristics of large absorption coefficient and high quantum yield (QY>15%), and have extremely high research value in near-infrared II fluorescence imaging. However, near-infrared II fluorescent quantum dots are mostly composed of active heavy metals, such as PbS, Ag 2 Se, CdTe / HgTe, these heavy metal elements are easily released in biological media and cause cytotoxicity. At present, some scholars have proved that the heavy metal ions released by intravenous injection of quantum dots containing heavy metal ions have significant toxic side effects on nerve cells.

[0006] The significant cytotoxic side effects of the above two types of near-infrared II materials are precisely the shortcomings and deficiencies of the existing technology. Therefore, how to synthesize and prepare near-infrared II region nano-imaging materials with good biocompatibility is an important scientific issue in the current field of nano-bioimaging research.

[0007] Silver nanomaterials have microwave absorption performance, high surface activity, optical properties, conductive properties and photoelectrochemical properties, and also have optical conversion characteristics (absorption, emission, scattering and plasma resonance). Although silver nanomaterials have potential application value in a variety of biomedical imaging due to their unique physical and chemical properties, the existing silver cluster nanomaterials cannot effectively achieve fluorescence emission in the near-infrared II region, but are limited to applications in the visible light emission region, such as the silver nanoclusters disclosed in patent document CN114790216A. Due to the very limited penetration depth of visible light for tissues, the above-mentioned existing silver nanoclusters are only suitable for in vitro biological indicators, and cannot be used for real-time tracking imaging of living bodies.

[0008] Therefore, in order to meet the needs of real-time tracking imaging of living bodies in biomedical imaging, it is urgent to develop silver cluster nanocontrast agents that can achieve fluorescence emission in the near-infrared II region. Summary of the invention

[0009] The purpose of the present invention is to provide a near-infrared II region silver nanocluster and its preparation and application. The silver nanocluster of the present invention can emit near-infrared II region fluorescence and has good biological safety and fluorescence stability.

[0010] The first aspect of the present invention provides a method for preparing near-infrared II region silver nanoclusters, comprising the following steps:

[0011] (1) Silver nitrate powder (AgNO 3 ) is prepared into an aqueous solution until the silver nitrate aqueous solution becomes colorless and transparent, then the ligand template molecule aqueous solution is added to the colorless and transparent silver nitrate aqueous solution to obtain a mixed solution, and an alkaline aqueous solution is added to the mixed solution for reaction to obtain an alkaline mixed solution with a pH value of 10-11; wherein the ligand template molecule can be any one of an organic small molecule compound, an amino acid, a polypeptide peptide, a polymer compound or a protein, or a combination of two or more thereof; and the ligand template molecule contains a functional group having a thiol structure; preferably, the ligand template molecule also contains a conjugated structure;

[0012] (2) using sodium borohydride aqueous solution to remove Ag in the alkaline mixed solution of (1) 1+ Reduced to silver atoms Ag 0 , and then continue the reaction so that the silver atoms grow and aggregate in the ligand template molecules to form silver nanoclusters, wherein the number of silver atoms in the silver nanoclusters is 18-25.

[0013] In the preparation method of the present invention, the ligand template molecular structure at least contains a functional group having a thiol structure, and in a more preferred ligand template molecular structure, in addition to the functional group containing a thiol structure, it also contains a functional group having a conjugated structure. This means that in the scheme of the present invention, a single compound molecule containing a thiol structure can be used as the ligand template molecule, a single compound molecule containing both a thiol group and a conjugated structure can be used as the ligand template molecule, and a mixture of one or more thiol-containing compound molecules and one or more conjugated compound molecules can be used as the ligand template molecule.

[0014] Furthermore, the organic small molecule compound in step (1) is selected from any one or more of mercaptoethanol, thioglycolic acid, mercaptoethylamine, cysteine, histidine or tyrosine.

[0015] Furthermore, the polypeptide peptide segment in step (1) is selected from glutathione; the protein preferably contains a conjugated functional group, such as a protein containing a histidine or tyrosine structure, and can be specifically selected from one or more of bovine ribonuclease (RNase-A), bovine serum albumin (BSA) or β-lactoglobulin. Most preferably, the protein is RNase-A.

[0016] Furthermore, the alkaline aqueous solution described in step (1) is preferably a sodium hydroxide (NaOH) aqueous solution. More preferably, the concentration of the sodium hydroxide aqueous solution is 1 M. In step (1), the role of the alkaline condition is to deprotonate the functional groups of the sulfhydryl structure of the ligand template molecule, which is more conducive to the ligand molecule and the silver ions on the surface of the silver cluster to form a stable structure. If the mixed solution of the ligand template molecule and silver nitrate flocculates, add more sodium hydroxide to make the pH of the mixed solution greater than or less than the isoelectric point of the protein.

[0017] Preferably, in step (1), the concentration of the silver nitrate aqueous solution is 10 mM; the concentration of the aqueous solution of the ligand template molecule is 10 mg / mL-25 mg / mL.

[0018] In step (1), after adding the ligand template molecule, the Ag in the mixed solution 1+ The ions are freely distributed in the ligand template molecules, providing space for the subsequent aggregation of silver atoms to form silver nanoclusters. As the concentration of the ligand template molecules in the mixed solution increases, the fluorescence intensity of the silver clusters will increase, but the emission spectrum will have a blue shift of 50-100nm. Therefore, in the preferred embodiment of the present invention, it is necessary to control the ratio of silver nitrate and ligand template molecules in step (1) according to the type of ligand template molecules. Specifically as follows:

[0019] In a specific embodiment of the present invention, when the ligand template molecule in step (1) is a small molecule compound or an amino acid, the molar ratio of silver nitrate to the ligand template molecule in the mixed solution in step (1) is controlled to be 25:18;

[0020] In another specific embodiment of the present invention, when the ligand template molecule in step (1) is a polypeptide peptide or protein, the molar ratio of silver nitrate to the ligand template molecule in the mixed solution in step (1) is controlled to be 20-25:1.

[0021] In step (1), the silver nitrate aqueous solution needs to be added with the ligand template molecule first, mixed evenly, and then the sodium hydroxide solution is added to avoid the premature formation of white silver hydroxide precipitate.

[0022] In step (2), sodium borohydride acts as a reducing agent and opens the dithiol group. 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 gray precipitate to appear in the mixed solution or turn black immediately, indicating that a large number of silver atoms aggregate to form silver nanoparticles rather than clusters, making it difficult to form silver 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 silver nitrate in step (1) is preferably 0.02-0.1:1.

[0023] Furthermore, in step (2), the Ag in the alkaline mixed solution of (1) is removed by using a sodium borohydride aqueous solution. 1+ Reduced to silver atoms Ag 0 The specific steps are preferably: adding sodium borohydride aqueous solution dropwise to the mixed solution so that Ag 1+ The ions are reduced to Ag 0 .

[0024] Due to Ag 0 The high surface energy will quickly aggregate to form clusters in the microwave heating environment, and the silver ions on the surface will combine with the ligands to form a stable structure. Therefore, in the preferred scheme, the method of continuing the reaction in step (2) to make the silver atoms grow and aggregate in the ligand template molecules to form silver nanoclusters can be specifically: quickly aggregate to form silver clusters in the microwave rapid heating environment, or react at room temperature 25°C for 24-72h to make Ag 0 Slowly grow into silver clusters.

[0025] Furthermore, the rapid aggregation to form silver clusters under a microwave rapid heating environment is preferably carried out under a microwave of 100W, 50-100°C (more preferably 70°C) for 30-90s (more preferably 60s), so that the silver atoms grow and aggregate in the ligand template molecules to form silver nanoclusters, and the reaction solution finally becomes a brown transparent clear solution.

[0026] Furthermore, in the preferred step (2), after the silver nanoclusters are formed, the method further comprises using a dialysis bag with a molecular weight cutoff of 20,000 Da to dialyze and remove unreacted ions (such as Ag 1+ 、Na + and Cl - ) steps.

[0027] The second aspect of the present invention provides a near-infrared II region fluorescent silver nanocluster, which is prepared by the method described in the first aspect of the present invention, and comprises a silver nanocluster and a ligand template molecule attached to the surface of the silver nanocluster, wherein the number of silver atoms in the silver nanocluster is 18-25, and the ligand template molecule is selected from any one or a combination of two or more of small molecule compounds, amino acids, polypeptide peptides, polymer compounds or proteins; and the ligand template molecular structure contains a thiol group; preferably, the ligand template molecule also contains a conjugated structure.

[0028] In the silver nanoclusters of the present invention, the ligand template molecular structure contains at least a functional group having a thiol structure, and in a more preferred ligand template molecular structure, in addition to the functional group containing the thiol structure, it also contains a functional group having a conjugated structure. This means that in the scheme of the present invention, a single compound molecule containing a thiol structure can be used as a ligand template molecule, a single compound molecule containing both a thiol group and a conjugated structure can be used as a ligand template molecule, and a mixture of one or more thiol-containing compound molecules and one or more conjugated structure-containing compound molecules can also be used as a ligand template molecule.

[0029] Preferably, the organic small molecule compound may be any one or more of mercaptoethanol, thioglycolic acid, mercaptoethylamine, cysteine, histidine or tyrosine.

[0030] Preferably, the polypeptide peptide segment may be glutathione;

[0031] Preferably, the protein is a protein containing a conjugated functional group, such as a protein containing a histidine or tyrosine structure, and can be specifically selected from one or more of bovine ribonuclease (RNase-A), bovine serum albumin (BSA) or β-lactoglobulin; the most preferred protein is RNase-A.

[0032] The silver nanoclusters described in the present invention are preferably spherical nanoclusters with a diameter of 1.8 to 2.2 nm; more preferably, the diameter of the spherical nanoclusters is 2.0 nm.

[0033] The silver nanoclusters of the present invention have the following optical properties: the fluorescence emission wavelength is between 900 and 1400 nm, and the luminescence quantum yield is greater than 1.5%.

[0034] The third aspect of the present invention provides the use of the near-infrared II region fluorescent silver nanoclusters described in the second aspect of the present invention in the preparation of near-infrared II region fluorescent imaging preparations.

[0035] Furthermore, the preparation is an intravenous injection. The near-infrared II region silver nanoclusters of the present invention have good biocompatibility and safety, and can enter the body through intravenous administration.

[0036] Further, imaging agents are used to detect liver diseases such as hepatotoxicity and liver cancer.

[0037] The near-infrared II zone silver nanoclusters of the present invention can be used to effectively study the biosafety structure-activity relationship of silver nanoclusters, provide guidance for the safety design of silver-based nanobiological products, and accelerate the promotion of silver nanoproducts to clinical imaging applications.

[0038] The near-infrared II region silver nanoclusters of the present invention can emit near-infrared II region fluorescence, and the luminescence mechanism is caused by the combined action of two theories, namely, the quantum size effect and the ligand-to-metal charge transfer theory. The electron-donating properties of the ligand affect the silver ions (Ag 1+ ) to the internal silver atoms (Ag 0 ) The electron excitation transition energy level, the cluster to the ligand energy level orbit affects the relaxation phenomenon of the electron returning to the ground state, thereby changing the emission wavelength. Specifically, the principle is as follows:

[0039] When the number of silver atoms contained in the silver nanoclusters N is less than 30, the luminescence principle basically conforms to the quantum size effect, and the emission peak position is positively correlated with the number of silver atoms. The emission wavelength E ev =E Fermi / N 1 / 3 ; where E Fermi is the Fermi energy of silver; N is the number of core atoms in the cluster. This conclusion shows that the electronic structure of the cluster depends on the free electron density of the metal and the size of the cluster. However, the quantum size theory alone cannot fully explain the near-infrared II fluorescent silver nanoclusters, and the properties of the ligands must also be considered.

[0040] At the same time, according to the charge transfer transition theory from metal to ligand, the generation of fluorescence is mainly through the charge transfer of atoms in the ligand to the central silver atom. The essence of this process is the redox process of the metal. Its transition process needs to absorb specific energy to show specific emission. Charge transition will only occur when the orbital energy level of the ligand matches the empty orbital energy level of the metal. Therefore, the ligand template molecule of the present invention includes a thiol-containing molecule, which greatly affects the electronic structure on the silver cluster when combined with the Ag ions on the surface of the silver cluster, resulting in the generation of complex molecular orbitals. The electron-donating properties of the ligand are changed without changing the core ligand to adjust the luminous intensity and wavelength.

[0041] In summary, the silver nanoclusters of the present invention are silver clusters with multiple atoms as the core, which are constructed based on atomic magic number sequences. Based on the quantum size effect and wave function theory, the multi-atom sequence clusters can compress the HOMO-LUMO orbit to red-shift the emitted light. At the same time, the present invention innovatively adopts ligands containing thiol groups, or "cocktail" mixed ligands containing both thiol groups and conjugated structures. The ligands containing thiol groups are used to enhance the stability of the overall physical structure of the clusters, and the aromatic ligands containing conjugated structures are used to further enhance the delocalization range of excited electrons, thereby further compressing the HOMO-LUMO orbit, thereby achieving near-infrared II region emission of the silver clusters.

[0042] By means of the above scheme, compared with the prior art, the present invention has the following advantages:

[0043] Compared with the existing oil phase synthesis method, the present invention adopts the water phase synthesis method to synthesize silver nitrate AgNO 3 The ligand template molecules including thiol-containing and conjugated structure functional groups are dissolved in an alkaline aqueous solution, and a reducing agent is used to convert the positive monovalent silver ion Ag into 1+ Reduced to silver atoms Ag 0 , this process can change the temperature and reaction time and other conditions through a microwave-assisted synthesis device, so that the silver atoms grow and aggregate in the corresponding ligand template to form silver nanoclusters. Although the oil phase method can also prepare luminescent materials with similar functions, the subsequent treatment is complicated, the reaction temperature needs to be greater than 200°C, and the biological safety is poor, and it cannot be directly used in the body. Compared with the oil phase preparation method, the present invention adopts an aqueous phase one-pot method, which can complete the reaction in 24-72 hours at room temperature, and the aqueous phase synthesis system can be treated with microwave assistance to shorten the time to 1 minute. Therefore, the synthesis method of the present invention is simple to operate, convenient and safe.

[0044] Compared with existing heavy metal quantum dot nanomaterials, the present invention adopts biologically inert element Ag to synthesize near-infrared II nanomaterials, which can effectively improve the biocompatibility of nanoclusters and provide a safe and bio-friendly new nanomaterial for the near-infrared II imaging field.

[0045] Compared with the existing silver nanoclusters that can only achieve emission in the visible light region, the present invention selects ligand template molecules with specific structures and prolongs the reduction reaction time, regulates the magic number sequence of silver atoms and the charge transfer from ligand to metal, so that the silver atoms grow and aggregate in the ligand template molecules to form silver nanoclusters containing a large number of silver atoms (18-25), and encapsulate mixed ligands. Based on multiple principles, the HOMO-LUMO orbit is significantly compressed, the emission light is red-shifted, and the silver nanoclusters can emit near-infrared II region fluorescence with a wavelength range of 900-1400nm, making it possible for their industrial application in real-time tracking and imaging of living organisms.

[0046] 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

[0047] Figure 1 The absorption and emission spectra of the silver nanoclusters prepared in Example 1 are shown.

[0048] Figure 2 The high-resolution transmission electron microscopy and particle size distribution test results of the silver nanoclusters prepared in Example 1 are reflected.

[0049] Figure 3 The atomic force microscopy characterization results of the silver nanoclusters prepared in Example 1 are shown.

[0050] Figure 4 It reflects the quantum yield of silver clusters calculated in comparison with IR-26 molecules.

[0051] Figure 5 It reflects the live and dead staining results of different cells by different silver clusters in the near-infrared II region.

[0052] Figure 6 The results of real-time imaging monitoring of tumors in mice using tail vein administration of silver nanoclusters prepared in Example 1 are shown. DETAILED DESCRIPTION

[0053] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention.

[0054] In the following examples of the present invention, the materials used are as follows:

[0055] Deionized water (resistivity 18.2 mΩcm), silver nitrate (AgNO 3 , ≥99.9% Ag basis), sodium hydroxide (NaOH), sodium borohydride (NaBH 4 , ≥98.0%), BSA (MW: 66.4kDa; purity 98%).

[0056] Example 1

[0057] (1) Dissolve the ligand template molecule BSA in water to prepare a ligand template solution with a concentration of 25 mg / mL. Also, dissolve silver nitrate in water to obtain a silver nitrate solution with a concentration of 10 mM. Prepare 500 μL of both solutions.

[0058] (2) 500 μL of the BSA ligand template solution prepared in (1) was fully mixed with 500 μL of the silver nitrate solution prepared in (1), and the solution was observed to turn white. Then 25 μL of a 1 M sodium hydroxide aqueous solution was added dropwise to the mixed solution, and the white solution was observed to fade to a colorless and clear alkaline mixed solution. The pH value of the alkaline mixed solution was about 11.

[0059] (3) After thorough mixing for 10 minutes, 20 μL of reducing agent NaBH 4 The aqueous solution (concentration 20 mM) was added dropwise to the mixed solution. The mixed solution was placed in a refrigerator at 4°C overnight to obtain a brown transparent mixed solution, indicating that the reaction was complete and the solution contained a large amount of silver nanoclusters BSA@AgNCs.

[0060] The product obtained in step (3) was dissolved in water, dialyzed using a dialysis bag with a molecular weight cutoff of 20,000 Da to remove unreacted ions and salts, and then stored in a refrigerator at 4°C.

[0061] Alternatively, a microwave-assisted synthesis method may be used, 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 70° C. for 60 seconds. Silver nanoclusters may also be prepared. In this way, the finished product can be prepared in 1 minute without waiting overnight.

[0062] The performance of the silver nanoclusters prepared in this example was tested and characterized. Figure 1-Figure 4 The specific description is as follows:

[0063] Figure 1 This is the emission spectrum of the silver nanoclusters prepared in Example 1. The emission light peak is at 1078 nm, with a half-peak width of 223 nm, which is within the near-infrared II region.

[0064] Figure 2 This is the high-resolution transmission electron microscopy and particle size distribution test results of the silver nanoclusters prepared in Example 1. It can be seen from the figure that the silver nanoclusters are spherical and have an average particle size of 2.0±0.2 nm.

[0065] In addition, atomic force microscopy showed that the particle size of the silver nanoclusters prepared in Example 1 was 2.0±0.3nm ( Figure 3 ).

[0066] from Figure 4 It can be seen that the quantum yield of silver nanoclusters BSA@AgNCs is 1.8%. Figure 4 IR26 is the standard reference sample.

[0067] Example 2-Example 8

[0068] Examples 2 to 8 all prepared silver nanoclusters by referring to the method of Example 1, except that BSA was replaced by equimolar amounts of β-lactoglobulin, bovine ribonuclease (RNase-A), mercaptoethanol, thioglycolic acid, mercaptoethylamine, cysteine ​​and glutathione, respectively.

[0069] Example 9-Example 12

[0070] Examples 9 to 12 were all prepared by referring to the method of Example 1, except that BSA was replaced by the following mixtures in equal molar amounts:

[0071] a mixture of cysteine ​​and histidine;

[0072] a mixture of cysteine ​​and tyrosine;

[0073] a mixture of thioglycolic acid and histidine;

[0074] A mixture of thioglycolic acid and tyrosine.

[0075] The fluorescence properties of several silver nanoclusters prepared in the above examples in the near-infrared II region were tested, and the results are shown in Table 1.

[0076] Table 1 Test results of near-infrared II fluorescence properties of silver nanoclusters prepared by different ligand template molecules

[0077]

[0078]

[0079] As can be seen from Table 1, all 12 ligand molecules can emit near-infrared II fluorescence, and the fluorescence emission peaks of the silver nanoclusters prepared using β-lactoglobulin, bovine ribonuclease (RNase-A), mercaptoethanol, thioglycolic acid, mercaptoethylamine, cysteine, and glutathione (containing thiol) as ligand template molecules are all in the near-infrared II region (NIR-II), but the near-infrared II region fluorescence peaks of the silver nanoclusters prepared using mercaptoethanol, thioglycolic acid, mercaptoethylamine, and cysteine ​​as ligand template molecules are slightly blue-shifted, because although the thiol groups in bovine serum albumin (BSA), β-lactoglobulin, and bovine ribonuclease (RNase-A) will preferentially bind to Ag, other amino acids (including histidine or tyrosine, etc.) with lone pairs of electrons or conjugated systems can also coordinate with Ag to create a more complex electronic transition environment, so that the fluorescence emitted by the overall cluster is red-shifted. When the ligand template molecule contains both thiol and conjugated structure, its fluorescence emission peak can reach the range of 1000-1075nm, which has stronger penetration into biological tissues and is more suitable for real-time tracking and imaging of living organisms.

[0080] Experimental Example 1. Safety Test

[0081] The toxicity of the silver nanoclusters prepared in Example 1 to B16 mouse melanoma cells, MCF-7 human breast cancer cells, and 4T1 mouse breast cancer cells was tested. The results showed that the silver nanoclusters prepared in Example 1 had no toxicity at a dose of 500 ug / mL ( Figure 5 ).

[0082] Experimental Example 2. In vivo imaging experiment in mice

[0083] The silver nanoclusters prepared in Example 1 were intravenously administered to mice bearing breast cancer subcutaneous tumors. The mice's abdomens needed to be depilated, and no anesthesia was required. The injection dose for each mouse was 2 mg / kg. The mice were monitored by in vivo real-time imaging. The results were as follows: Figure 6 As shown in the figure, it can be seen that the silver nanoclusters are still significantly enriched in the tumor site and emit fluorescence 6 hours after injection, indicating that the silver nanoclusters of the present invention not only have stable near-infrared difluorescence luminescence ability, but also can be used for passive targeted tumor experiments in living mice, and have the potential for in vivo diagnosis of tumors.

[0084] 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 silver nanoclusters in the near-infrared II region, comprising the following steps: (1) preparing silver nitrate powder into an aqueous solution until the silver nitrate aqueous solution becomes colorless and transparent, then adding an aqueous solution of a ligand template molecule to the colorless and transparent silver nitrate aqueous solution to obtain a mixed solution, adding an alkaline aqueous solution to the mixed solution for reaction, and obtaining an alkaline mixed solution with a pH value of 10-11; wherein the ligand template molecule is any one of an organic small molecule compound, an amino acid, a polypeptide peptide, a polymer compound or a protein, or a combination of two or more thereof; and the ligand template molecule contains a thiol group, or contains a thiol group and a conjugated structure at the same time; the organic small molecule compound is preferably any one or more of mercaptoethanol, thioglycolic acid, mercaptoethylamine, cysteine, histidine or tyrosine; the polypeptide peptide is preferably glutathione; the protein is preferably one or more of bovine ribonuclease (RNase-A), bovine serum albumin (BSA) or β-lactoglobulin (β-lactoglobulin), more preferably RNase-A; the alkaline aqueous solution is preferably a sodium hydroxide (NaOH) aqueous solution, and more preferably a 1M sodium hydroxide aqueous solution; (2) using sodium borohydride aqueous solution to remove Ag in the alkaline mixed solution of (1) 1+ Reduced to silver atoms Ag 0 Then, under the action of sodium borohydride reducing agent, the reaction is continued at room temperature 25° C. for 24-72 hours, or under microwave treatment at 100 W, 50-100° C. (more preferably 70° C.) for 30-90 seconds (more preferably 60 seconds), so that the silver atoms grow and aggregate in the ligand template molecules and are coated by the ligand template molecules to obtain a reaction solution containing silver nanoclusters, wherein the number of silver atoms in the silver nanoclusters is 18-25.

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

3. The method according to claim 1, characterized in that: The ligand template molecule in step (1) is a small molecule compound or an amino acid, and the molar ratio of silver nitrate to the ligand template molecule in the mixed solution in step (1) is controlled to be 25:

18.

4. The method according to claim 1, characterized in that: The ligand template molecule in step (1) is a polypeptide peptide or protein, and the molar ratio of silver nitrate to the ligand template molecule in the mixed solution in step (1) is controlled to be 20-25:

1.

5. The method according to claim 1, characterized in that: The molar ratio of the sodium borohydride in step (2) to the silver nitrate in step (1) is 0.02-0.1:

1.

6. The method according to claim 1, characterized in that: The reaction solution containing silver nanoclusters is a brown transparent clear solution.

7. A near-infrared II region fluorescent silver nanocluster, prepared by the method described in any one of claims 1 to 6, comprising a silver nanocluster and a ligand template molecule attached to the surface of the silver nanocluster, wherein the number of silver atoms in the silver nanocluster is 18-25, and the ligand template molecule is selected from any one or a combination of two or more of a small molecule compound, an amino acid, a polypeptide peptide, a polymer compound or a protein; and the ligand template molecular structure contains a sulfhydryl group, or contains a sulfhydryl group and a conjugated functional group at the same time; the organic small molecule compound is preferably any one or more of mercaptoethanol, thioglycolic acid, mercaptoethylamine, cysteine, histidine or tyrosine; the polypeptide peptide is preferably glutathione; the protein is a protein containing a conjugated functional group, preferably a protein containing a histidine or tyrosine structure, more preferably one or more of bovine ribonuclease (RNase-A), bovine serum albumin (BSA) or β-lactoglobulin (β-lactoglobulin), and the most preferred protein is RNase-A.

8. Use of the near-infrared II region fluorescent silver nanoclusters according to claim 7 in the preparation of near-infrared II region fluorescent imaging preparations.

9. The use according to claim 8, characterized in that: The preparation is an intravenous injection.

10. The use according to claim 8, characterized in that: The imaging agent is used for detecting liver diseases, preferably hepatotoxicity or liver cancer.

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