A mannose-modified nano-silver cluster, a preparation method and application thereof as a probe

Abstract

The application belongs to the technical field of medicines, and discloses a mannose-modified nano silver cluster, a preparation method and application of the mannose-modified nano silver cluster as a probe, wherein the mannose-modified nano silver cluster is obtained by coupling bovine serum albumin-nano silver clusters and modified mannose through acid-amine condensation reaction, 14 modified mannose molecules are coupled on each bovine serum albumin molecule, the mannose-modified nano silver cluster can be applied as a probe in biological recognition and target labeling, a new visual tool is provided for receptor-ligand interaction research and drug delivery monitoring, and the application development of the sugar probe in biomedical detection and diagnosis and treatment is promoted.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, and in particular to a mannose-modified silver nanocluster, its preparation method, and its application as a probe. The silver nanocluster is a mannose-modified silver nanocluster. The mannose-functionalized silver nanocluster fluorescent probe of this invention possesses high affinity, stable fluorescence, and good targeting properties, providing a novel and efficient tool for studying glycoprotein interactions, and has significant application value in the fields of biolabeling and molecular recognition. Background Technology

[0002] The interaction between carbohydrates and proteins is a crucial molecular recognition process in life activities, playing a vital role in biological research such as cell communication, immune responses, and pathogen infection. Today, many advanced techniques, such as metabolic oligosaccharide engineering, nuclear magnetic resonance spectroscopy, carbohydrate microarray technology, and fluorescent probe methods, are used to elucidate the mechanisms of carbohydrate-protein interactions. Among these techniques, fluorescent probe methods have become a widely used research method in this field due to their advantages of high visualization and sensitivity.

[0003] Fluorescent probes achieve specific recognition and quantitative analysis of target molecules through the generation and changes of fluorescence signals. Their unique photophysical properties make fluorescent probes irreplaceable in chemical and biological research. In the design and application of fluorescent probes, the selection of fluorophores and fluorescent backbones is particularly important.

[0004] As a new class of fluorescent groups, nanofluorescent materials possess excellent optical properties and have found increasingly widespread applications in the biomedical field. Metallic fluorescent nanomaterials (such as gold and silver nanoclusters) exhibit good photostability and chemical inertness, and their fluorescence signals do not easily attenuate under continuous laser irradiation, meeting the requirements for long-term continuous bioimaging. Multiple chemical modification sites on the surface of metallic fluorescent nanomaterials provide an ideal platform for constructing multifunctional intelligent sensing systems, enabling precise coupling of targeted molecules, integration of signal transduction units, and modulation of fluorescence signal output simultaneously. This demonstrates unique advantages in biomedical research fields such as disease biomarker detection, live cell dynamic tracking, and drug delivery monitoring.

[0005] As the molecular carrier of fluorescent probes, the fluorescent backbone not only determines the overall stability, fluorescence properties, and biocompatibility of the probe, but its structural design and material selection also directly affect the probe's physicochemical properties and biological functions. The nanoscale cavities in bovine serum albumin (BSA) molecules can effectively encapsulate fluorescent molecules through non-covalent forces such as hydrophobic interactions and hydrogen bonds, reducing solvent-induced fluorescence quenching. The amino and carboxyl groups distributed on the surface of BSA molecules provide a high density of reaction sites for the directional coupling of fluorescent groups. As a natural protein, BSA possesses excellent biocompatibility and low immunogenicity, making it an ideal protein backbone for constructing high-performance fluorescent probes.

[0006] Glycosyl fluorescent probes construct a dual-function molecular recognition system by covalently coupling a glycoligand to a backbone carrying a fluorescent chromophore. When the glycoligand specifically binds to the target receptor, photophysical effects such as electron transfer or fluorescence resonance energy transfer alter the fluorescence properties, thereby achieving highly sensitive detection.

[0007] This invention focuses on silver nanoclusters. A mannose-silver nanocluster fluorescent probe (MBAg) was successfully constructed by covalently coupling mannose to the surface of bovine serum albumin (BSA)-modified silver nanoclusters using disuccinimidyl adipate as a linker. The probe was then characterized. The BSA framework ensures the biosafety of the probe system; the metallic silver nanomaterials provide a stable fluorescence signal; and the mannose units endow the probe with specific recognition capabilities. Through this multi-component synergistic design, a novel class of glycosylated fluorescent probes with simple preparation, strong specificity, and high binding efficiency was successfully developed, providing a powerful tool for studying glycoprotein interactions. Based on this, the biorecognition and targeting labeling functions of this probe were investigated.

[0008] Through searching, we found published documents related to this invention patent application (including but not limited to the following): 1. Conference Paper: Chen Zhen, et al. Synthesis of Bovine Serum Albumin-Encapsulated Silver Nanoclusters and Their Application as a Novel Fluorescent Probe for the Determination of Biothiols, 2013, 8th Conference on Organic Chemistry of the Chinese Chemical Society and the 1st International Symposium on Organic Chemistry in Chongqing. This paper presents the convenient synthesis of water-soluble, red-fluorescent silver nanoclusters (BSA-AgNCs) using bovine serum albumin as the fluorescent backbone and NaBH4 as the reducing agent in the presence of NaOH. The authors subsequently applied this novel fluorescent probe to the determination of biothiols. This literature does not report on the coupling of these bovine serum albumin-encapsulated silver nanoclusters with glycosyl donors or their related applications.

[0009] 2. Journal Article: Bovine Serum Albulmin Protein-Templated Silver Nanocluster (BSA-Ag13): An Effective Singlet Oxygen Generator for Photodynamic Cancer Therapy [J]. Advanced Healthcare Materials, 2016, (5), 2528-2535. Yong Yu et al. used bovine serum albumin as the backbone and silver nitrate as the silver source, and obtained bovine serum albumin silver nanoclusters with 13 silver atoms attached by reduction with sodium borohydride under alkaline conditions. The silver nanoclusters have good biocompatibility and strong ability to generate singlet oxygen, which can be used for photodynamic therapy to kill cancer cells. However, this literature also did not report on the coupling of the bovine serum albumin silver nanoclusters with glycosyl donors and their related applications.

[0010] 3. Dissertation: Wei Xiaxia. Synthesis and Labeling Study of Mannose-Gold Nanocluster Fluorescent Probe, 2020, Tianjin University of Science and Technology. Our research group previously designed and synthesized a mannose-gold nanocluster fluorescent probe with bovine serum albumin as the backbone. A series of fluorescence experiments verified that this fluorescent probe can specifically recognize and fluorescently label concanavalin A, E. coli K12, and human breast cancer cells (MCF-7). Related experimental results indicate that this probe can be used to study the interaction between sugar and receptor, and for detecting E. coli K12 and imaging cancer cells. However, the probe reported in that paper is a gold nanocluster. Although the related fluorescence experiments are similar, it is fundamentally different from the silver nanoclusters that this invention focuses on. Furthermore, the method used in that paper to couple mannose to the surface of bovine serum albumin is also different from that in this patent.

[0011] 4. Chinese Patent: Liu Xin, et al. A mannose-functionalized protein-gold nanocluster and its preparation method and application, Publication No. CN111057537B, Publication Date May 19, 2023. This patent discloses a mannose-functionalized bovine serum albumin-gold nanocluster and its preparation method. The authors then used the bovine mannose serum albumin-gold nanocluster to detect the lower limit of concanavalin A. The results showed that the nanocluster was non-toxic, biocompatible, well-dispersible, highly fluorescent, and had high detection sensitivity. However, the probe reported in this patent is a gold nanocluster, which is fundamentally different from the silver nanocluster described in this invention. In addition, this patent only detected concanavalin A and human breast cancer cells (MCF-7), and did not conduct experiments on specific recognition and fluorescent labeling of E. coli K12, which is significantly different from this invention.

[0012] By comparison, this invention differs fundamentally from the aforementioned published literature. This invention utilizes disuccinimide adipate as a linker to multivalently couple mannose to the surface of bovine serum albumin-modified silver nanoclusters, successfully constructing a mannose-silver nanocluster fluorescent probe. Verification using fluorescence spectroscopy and confocal microscopy revealed that the obtained mannose-silver nanocluster fluorescent probe possesses specific recognition and labeling capabilities for concanavalin A, E. coli K12, and MCF-7 cells, demonstrating its application potential in biomedical fields such as protein recognition and cell labeling. Therefore, the preparation of a novel mannose-silver nanocluster fluorescent probe is the motivation behind this invention. Summary of the Invention

[0013] The purpose of this invention is to address the technical deficiencies in the prior art by providing a mannose-modified silver nanocluster, its preparation method, and its application as a probe.

[0014] The technical solution adopted to achieve the purpose of this invention is: A mannose-modified silver nanocluster is obtained by coupling bovine serum albumin-silver nanoclusters and modified mannose via an acid-amine condensation reaction, wherein the structural formula of the modified mannose is: .

[0015] In the above technical solution, 14 modified mannose molecules are coupled to each bovine serum albumin molecule.

[0016] Another aspect of the present invention includes a method for preparing the mannose-modified silver nanoclusters, comprising the following steps: Step 1, full acetylation: Mannose 1 undergoes a full acetylation reaction with acetic anhydride to obtain fully acetylated mannose 2; Step 2, selective removal of the 1-acetyl group: the 1-acetyl group of the fully acetylated mannose 2 is removed to give product 3, which is deacetylated at the C-1-position; Step 3, trichloroacetylimine esterification: The product 3, which has been deacetylated at the C-1 position, reacts with trichloroacetonitrile to undergo trichloroacetylimine esterification, yielding the C-1-trichloroacetylimine esterified product 4; Step 4, Glycosylation: The C-1-trichloroacetylimine esterification product 4 reacts with 6-azido-1-hexanol under the action of a glycosylation promoter to obtain glycosylated product 5; Step 5, Deacetylation: Glycosylated product 5 undergoes deacetylation of all acetyl groups under the action of a catalyst to obtain deacetylated product 6; Step 6, reduction reaction: Deacetylated product 6 is reduced under the action of palladium on carbon and hydrogen, and the azide group is reduced to amino group to obtain amino product 7; Step 7, Connecting the connecting arm: Disuccinimid adipate reacts with amino product 7 to obtain mannose 8 with the connecting arm attached; Step 8: Prepare bovine serum albumin-silver nanoclusters (BSA-AgNCs); Step 9, Preparation of mannose-modified silver nanoclusters: Bovine serum albumin-silver nanoclusters BSA-AgNCs and mannose 8 with attached linker arms were stirred and reacted in PBS, dialyzed in a dialysis bag and freeze-dried under vacuum to obtain mannose-modified silver nanoclusters. The reaction pathway diagram is as follows:

[0017] In the above technical solution, in step 1, mannose is dissolved in pyridine, 6-10 equivalents of acetic anhydride are added and reacted for 8-12 hours. The reaction system is diluted with dichloromethane and washed with dilute acid to remove pyridine. The organic phase is concentrated to obtain fully acetylated mannose 2. In step 2, fully acetylated mannose 2 is dissolved in tetrahydrofuran, and 1.0-1.5 equivalents of N,N-dimethylethylenediamine are added and reacted for 8-12 hours. The reaction system is diluted with an appropriate amount of dichloromethane and washed with dilute acid to remove organic amines. After the organic phase is concentrated, product 3 with deacetylated group at C-1-position is obtained. In step 3, the product from the previous step is reacted with 2-5 equivalents of trichloroacetonitrile in the presence of a catalytic amount of base for 4-8 hours. The reaction system is then concentrated to dryness and separated by rapid column chromatography to obtain C-1-position trichloroacetylimine esterified product 3. In step 4, the C-1-trichloroacetylimine esterification product 3 and 1.0-1.5 equivalents of 6-azido-1-hexanol are dissolved in anhydrous dichloromethane. A catalytic amount of glycosylation promoter is added at -40~-20℃, and the reaction is continued for 20-40 min. The reaction system is then washed with sodium bicarbonate solution, saturated brine, and dried with anhydrous sodium sulfate. After concentration, it is separated by rapid column chromatography to obtain glycosylated product 5. In step 5, glycosylated product 5 is dissolved in an appropriate amount of anhydrous methanol, and a catalytic amount of sodium methoxide solution is added. The mixture is stirred at room temperature for 2-6 hours. After the reaction is completed, the pH of the system is adjusted to neutral using a cation exchange resin. The cation exchange resin is removed by filtration. After the filtrate is concentrated, it is separated by rapid column chromatography to obtain deacetylated product 6. In step 6, the deacetylated product 6 is dissolved in methanol, a catalytic amount of palladium on carbon is added, and the mixture is reacted at room temperature under hydrogen atmosphere. After the reaction is complete, the palladium on carbon is removed, and the filtrate is dried by rotary evaporation to obtain the amino product 7. In step 7, disuccinimide adipate is dissolved in dimethyl sulfoxide, and then an organic base is added. Subsequently, the dimethyl sulfoxide solution of amino product 7 is slowly added dropwise, and the reaction is carried out at room temperature for 2-6 hours. The reaction solution is placed in an ice-water bath, PBS is added and stirred, then an appropriate amount of chloroform is added and stirred vigorously for 5-20 minutes. The reaction solution is then centrifuged, and the supernatant is mannose 8 with the connecting arm attached. In step 8, BSA is dissolved in deionized water, silver nitrate solution is added, and the mixture is stirred at room temperature. The pH of the solution is adjusted to 8.5-9.5 with 0.1-1.0M sodium hydroxide solution. Then, the color of the solution is adjusted from colorless to reddish-brown with 5-10mM sodium borohydride solution and red fluorescence is observed under ultraviolet light. After stirring for 0.5-1.5 hours, the reaction solution is placed in a dialysis bag with a molecular weight cutoff of 8000-12000 Da. Dialysis is performed with ultrapure water for 8-20 hours. The liquid in the dialysis bag is then freeze-dried to obtain bovine serum albumin-silver nanoclusters BSA-AgNCs. In step 9, mannose 8 with the connecting arm is stirred with BSA-AgNCs in PBS at room temperature for 20-40 hours. The reaction solution is then dialyzed in a dialysis bag for 8-20 hours. The dialysate is then freeze-dried under vacuum to finally obtain the mannose-modified silver nanocluster probe.

[0018] In the above technical solution, in steps 1 and 2, the dilute acid is dilute sulfuric acid, dilute hydrochloric acid, dilute nitric acid, or dilute phosphoric acid, preferably dilute phosphoric acid.

[0019] In the above technical solution, in step 3, the catalytic base is potassium carbonate, cesium carbonate, sodium carbonate, triethylamine, N,N-diisopropylethylamine, or DBU, preferably DBU.

[0020] In the above technical solution, in step 4, the glycosylation promoter is trifluoromethanesulfonic acid, trimethylsilyl trifluoromethanesulfonic acid, or boron trifluoride ethyl ether, preferably boron trifluoride ethyl ether.

[0021] In the above technical solution, in step 7, the organic base is N-methylmorpholine, triethylamine, N,N-diisopropylethylamine, DBU, etc., preferably triethylamine.

[0022] Another aspect of the present invention includes the application of the mannose-modified silver nanoclusters as probes in biorecognition and targeted labeling.

[0023] Another aspect of the present invention includes the application of the mannose-modified silver nanoclusters as probes in the biorecognition or targeted labeling of concanavalin A, Escherichia coli, or human breast cancer cell line MCF-7.

[0024] Another aspect of the present invention includes the application of the mannose-modified silver nanoclusters as probes in drug delivery monitoring and biomedical detection.

[0025] Compared with the prior art, the beneficial effects of the present invention are: 1. The mannose-modified silver nanoclusters of the present invention have good water solubility and biocompatibility, and high safety.

[0026] 2. The mannose-modified silver nanoclusters of the present invention have good optical stability.

[0027] 3. The mannose-modified silver nanoclusters of the present invention, as probes, have good biological recognition and targeted binding specificity.

[0028] 4. The mannose-modified silver nanoclusters of the present invention, as probes, enable precise identification of specific cell surface receptors, providing a novel visualization tool for receptor-ligand interaction research and drug delivery monitoring, and promoting the application of sugar probes in biomedical detection and diagnosis. Attached Figure Description

[0029] Figure 1 This is a flowchart illustrating the synthesis of the mannose-modified silver nanoclusters MBAG according to the present invention.

[0030] Figure 2 The 1H NMR spectrum of 1,2,3,4,6-pentaacetyl-D-pyranomannose; Figure 3 The carbon NMR spectrum of 1,2,3,4,6-pentaacetyl-D-pyranomannose; Figure 4 The 1H NMR spectrum of 2,3,4,6-tetraacetyl-D-mannopyranose; Figure 5 The carbon NMR spectrum of 2,3,4,6-tetraacetyl-D-mannopyranose; Figure 6 The 1H NMR spectrum of 2,3,4,6-tetraacetyl-1-O-(trichloroacetylimino)-D-mannopyranose; Figure 7 The carbon NMR spectrum of 2,3,4,6-tetraacetyl-1-O-(trichloroacetylimino)-D-mannopyranose; Figure 8 The 1H NMR spectrum of 2,3,4,6-tetraacetyl-1-O-(6-azidohexyl)-D-mannopyranose; Figure 9 The carbon NMR spectrum of 2,3,4,6-tetraacetyl-1-O-(6-azidohexyl)-D-mannopyranose; Figure 10The 1H NMR spectrum of 1-O-(6-azidohexyl)-D-mannopyranose; Figure 11 The carbon NMR spectrum of 1-O-(6-azidohexyl)-D-mannopyranose; Figure 12 The 1H NMR spectrum of 1-O-(6-aminohexyl)-D-mannopyranose; Figure 13 The carbon NMR spectrum of 1-O-(6-aminohexyl)-D-mannopyranose; Figure 14 MALDI-TOF-MS image of bovine serum albumin (BSA); Figure 15 MALDI-TOF-MS image of bovine serum albumin-silver nanoclusters (BSA-AgNCs); Figure 16 MALDI-TOF-MS image of mannose-modified silver nanoclusters (MBAg); Figure 17 The fluorescence emission spectra of three samples, BSA, BSA-AgNCs, and MBAg, are shown at an excitation wavelength of 340 nm. Figure 18 The fluorescence emission spectra of MBAG and concanavalin A at different concentrations are shown. Figure 19 This is a real-world image of the fluorescence of concanavalin A after it binds to and aggregates with MBAG under ultraviolet light excitation. Figure 20 The fluorescence emission spectra of MBAg and Escherichia coli K12 are shown. Figure 21 Images are taken from a fluorescence confocal microscope; (a) is a bright-field image of MBAg and Escherichia coli; (b) is a fluorescence image of MBAg and Escherichia coli; (c) is a superimposed image of (a) and (b). Figure 22 Images are confocal fluorescence microscopy images of MBAg, MCF-7, and lysosomal green fluorescent probes; (a) bright field image; (b) fluorescence image at 520 nm; (c) fluorescence image at 680 nm; (d) is a superimposed image of (a), (b), and (c). Detailed Implementation

[0031] The present invention will be further described in detail below with reference to specific embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

[0032] Example 1 Unless otherwise specified, the biological materials, pharmaceuticals, and raw materials used in the specific embodiments are all from domestic reagent companies.

[0033] Specifically, the detailed experimental steps for the preparation of mannose-silver nanoclusters and subsequent receptor labeling studies are as follows: Experimental Example 1 like Figure 1 As shown, a mannose-modified silver nanocluster (MBAg) is prepared through the following steps: Step 1: Synthesis of 1,2,3,4,6-pentaacetyl-D-pyranomannose (compound 2): Mannose (5 g, 27.75 mmol) was dissolved in 30 mL of pyridine. Acetic anhydride (44.32 mL) was slowly added dropwise under an ice-water bath. After the addition was complete, the reaction mixture was stirred at room temperature for 12 h. The reaction mixture was diluted with 200 mL of dichloromethane and slowly poured into excess dilute phosphoric acid diluted with ice water. The aqueous phase was extracted with dichloromethane 2 to 3 times. The organic phases were combined and washed successively with saturated sodium bicarbonate solution and saturated sodium chloride solution. After drying with anhydrous sodium sulfate, dichloromethane was removed by rotary evaporation to obtain a pale yellow oily liquid compound 2 (10.48 g, 96.1%). 1 H NMR (400 MHz, CDCl3) δ 5.95 (d, J = 2.0 Hz, 1H), 5.32 - 5.04 (m, 3H), 4.15 (dd, J = 12.4, 4.8 Hz, 1H), 4.04 - 3.86 (m, 2H), 2.20 - 1.72 (m, 15H); 13 CNMR (101 MHz, CDCl3) δ 170.34, 169.72, 169.48, 169.31, 167.85, 90.38, 70.39, 68.56, 68.13, 65.30, 61.87, 20.62, 20.54, 20.48, 20.43, 20.40. The 1H NMR spectrum of 1,2,3,4,6-pentaacetyl-D-mannopyranose is shown below. Figure 2 The carbon NMR spectrum is shown below. Figure 3 .

[0034] Step 2, Synthesis of 2,3,4,6-Tetraacetyl-D-mannopyranose (Compound 3): Compound 2 (10.48 g, 26.85 mmol) was dissolved in 50 mL of tetrahydrofuran, and N,N-dimethylethylenediamine (2.84 g, 32.22 mmol) was slowly added. The mixture was stirred at room temperature for 12 h. Thin-layer chromatography (petroleum ether:ethyl acetate = 1:1) confirmed that the reactants had reacted completely. The tetrahydrofuran was concentrated under reduced pressure to remove the reactants. The residue was dissolved in 200 mL of dichloromethane, and the pH was adjusted to approximately 4 with dilute phosphoric acid. The aqueous phase was extracted with dichloromethane 2 to 3 times. The organic phases were combined and washed with saturated sodium chloride solution. After drying with anhydrous sodium sulfate, the solution was evaporated to dryness to obtain a yellow oily liquid, Compound 3 (8.84 g, 94.5%). 1 H NMR (400 MHz, CDCl3) δ 5.26 (dd, J = 10.0, 3.1Hz, 1H), 5.19 - 5.01 (m, 3H), 4.86 (d, J = 4.3 Hz, 1H), 4.16 - 4.06 (m, 2H), 4.02 - 3.94 (m, 1H), 2.10 - 1.82 (m, 12H); 13 C10 NMR (100 MHz, CDCl3) δ 170.34, 169.72, 169.48, 169.31, 167.85, 90.38, 70.39, 68.56, 68.13, 65.30, 61.87, 20.62, 20.54, 20.48, 20.43, 20.40. The 1H NMR spectrum of 2,3,4,6-tetraacetyl-D-mannopyranose is shown below. Figure 4 The carbon NMR spectrum is shown below. Figure 5 .

[0035] Step 3: Synthesis of 2,3,4,6-tetraacetyl-1-O-(trichloroacetylimino)-D-mannopyranose (compound 4): Compound 3 (5.3 g, 15.22 mmol) was dissolved in 50 mL of anhydrous dichloromethane. Under ice-water bath conditions, 0.3 mL of DBU was added, followed by slow addition of trichloroacetonitrile (4.4 g, 30.43 mmol). The mixture was stirred overnight at room temperature. Thin-layer chromatography (petroleum ether:ethyl acetate = 2:1, V / V) was used to monitor the reaction until complete. After dichloromethane was removed under reduced pressure, the mixture was purified by rapid column chromatography to obtain a pale yellow liquid (6.2 g, 82.7%), which was compound 4. 1 H NMR (400 MHz, CDCl3) δ 8.78 (s, 1H), 6.23 (d, J= 1.9 Hz, 1H), 5.45 - 5.39 (m, 1H), 5.35 (dd, J = 5.4, 2.0 Hz, 2H), 4.23 (dd, J = 11.9, 4.7 Hz, 1H), 4.12 (m, J = 11.8, 10.6, 3.7 Hz, 2H), 2.15 (s,3H), 2.02 (d, J = 5.5 Hz, 6H), 1.96 (s, 3H); 13 C10 NMR (100 MHz, CDCl3) δ 170.55, 169.79, 169.71, 169.61, 159.67, 94.48, 90.49, 71.19, 68.79, 67.83, 65.35, 62.02, 20.78, 20.69, 20.61. The 1H NMR spectrum of 2,3,4,6-tetraacetyl-1-O-(trichloroacetylimino)-D-mannopyranose is shown below. Figure 6 The carbon NMR spectrum is shown below. Figure 7 .

[0036] Step 4: Synthesis of 2,3,4,6-tetraacetyl-1-O-(6-azidohexyl)-D-mannopyranose (compound 5): Compound 4 (3.77 g, 7.65 mmol) and 6-azido-1-hexanol (1.31 g, 9.18 mmol) were dissolved in 50 mL of anhydrous dichloromethane. The reaction flask was placed in a cryogenic pump at -20 °C, and 0.5 mL of boron trifluoride diethyl ether was slowly added. The reaction system gradually turned orange-pink. The mixture was stirred for 20 min, and the reaction was monitored by thin-layer chromatography (petroleum ether:ethyl acetate = 2:1, V / V) until the reactants were completely reacted. The reaction solution was washed successively with saturated sodium bicarbonate solution and saturated brine. After concentration, the solution was purified by rapid column chromatography to obtain a white solid (2.8 g, 77.28%), which was compound 5. 1H NMR (400 MHz, CDCl3) δ 5.33 (dd, J =10.0, 3.4 Hz, 1H), 5.29 (d, J = 2.2 Hz, 1H), 5.25 - 5.20 (m, 1H), 4.79 (d, J= 1.7 Hz, 1H), 4.27 (dd, J = 12.2, 5.3 Hz, 1H), 4.09 (dd, J = 12.2, 2.5 Hz, 1H), 3.96 (m, J = 9.8, 5.3, 2.5 Hz, 1H), 3.68 (m, J = 9.6, 6.6 Hz, 1H), 3.44(m, J = 9.7, 6.4 Hz, 1H), 3.27 (t, J = 6.9 Hz, 2H), 2.07 (dd, J = 44.1, 20.9Hz, 12H), 1.70 (s, 1H), 1.65 - 1.55 (m, 4H), 1.40 (dd, J = 7.0, 3.7 Hz, 4H); 13 C10 NMR (100 MHz, CDCl3) δ 170.84, 170.29, 170.12, 169.93, 97.67, 69.79, 69.24, 68.53, 68.41, 66.35, 62.65, 51.46, 29.22, 28.85, 26.58, 25.83, 21.04, 20.88, 20.84. The 1H NMR spectrum of 2,3,4,6-tetraacetyl-1-O-(6-azidohexyl)-D-mannopyranose is shown below. Figure 8 The carbon NMR spectrum is shown below. Figure 9 .

[0037] Step 5, Synthesis of 1-O-(6-azidohexyl)-D-pyranomannose (compound 6): Compound 5 (2.8 g, 5.91 mmol) was dissolved in 50 mL of anhydrous methanol, and a catalytic amount of 30% sodium methoxide solution was added. The reaction was carried out at room temperature, and the reaction was monitored by thin-layer chromatography (dichloromethane:methanol = 10:1, V / V) until the reactants were completely reacted. The pH of the system was carefully adjusted to neutral with cation exchange resin, the cation exchange resin was removed by filtration, the filtrate was concentrated, and the residue was purified by rapid column chromatography to obtain a pale yellow solid (1.45 g, 80.30%), which is compound 6. 1 H NMR (400 MHz, MeOD) δ 4.77 (d, J= 1.7 Hz, 1H), 3.88- 3.80 (m, 2H), 3.80 - 3.68 (m, 3H), 3.64 (t, J =9.5 Hz, 1H), 3.54 (m, J =9.6, 5.6, 2.4 Hz, 1H), 3.44 (m, J = 9.7, 6.2 Hz, 1H), 3.30 (d, J = 6.9 Hz, 2H),1.69 - 1.55 (m, 4H), 1.43 (m, J = 6.0, 4.8, 2.5 Hz, 4H); 13 C10 NMR (100 MHz, MeOD) δ 101.41, 74.45, 72.59, 72.16, 68.49, 68.33, 62.79, 52.32, 30.40, 29.79, 27.55, 26.86. The 1H NMR spectrum of 1-O-(6-azidohexyl)-D-mannopyranose is shown below. Figure 10 The carbon NMR spectrum is shown below. Figure 11 .

[0038] Step 6, Synthesis of 1-O-(6-aminohexyl)-D-mannopyranose (compound 7): Compound 6 (220 mg) was dissolved in 20 mL of methanol, and 10% palladium on carbon (20 mg) was added. The reaction system was placed under hydrogen at room temperature for catalytic hydrogenation, and the reaction was monitored by thin-layer chromatography (dichloromethane:methanol = 10:1, V / V). After the reaction of the starting materials was complete, palladium on carbon was removed by diatomaceous earth filtration. The filtrate was concentrated to dryness to obtain a pale yellow solid, which is compound 7. 1 H NMR (400 MHz, MeOD) δ 4.74 (d, J = 1.7 Hz, 1H), 3.86 - 3.77 (m, 2H), 3.77 - 3.65 (m, 3H), 3.61 (t, J = 9.5 Hz, 1H), 3.51 (m, J = 9.6, 5.7, 2.4 Hz, 1H), 3.42 (m, J = 9.5, 6.2 Hz, 1H), 2.67 (t, J = 7.2 Hz, 2H), 1.60 (m, J = 9.1, 6.7, 2.7 Hz, 2H), 1.51 (m, J= 7.1 Hz, 2H), 1.46- 1.32 (m, 4H); 13 C10 NMR (100 MHz, MeOD) δ 101.49, 74.55, 72.61, 72.20, 68.53, 68.41, 62.81, 42.20, 32.98, 30.47, 27.68, 27.13. The 1H NMR spectrum of 1-O-(6-aminohexyl)-D-mannopyranose is shown below. Figure 12 The carbon NMR spectrum is shown below. Figure 13 .

[0039] Step 7, Synthesis of 1-O-{6-[(2,5-dioxopyrrolidone-1-yl)oxy]-6-oxohexanoylaminohexyl}-D-mannopyranose (Compound 8): Disuccinimidyl adipate (584.74 mg) was dissolved in 2 mL of dimethyl sulfoxide, and an appropriate amount of triethylamine was added. The mixture was stirred at room temperature for 10 min to obtain the linker solution. Compound 7 (40 mg) was dissolved in 2 mL of dimethyl sulfoxide and slowly added dropwise to the linker solution. The reaction was continued at room temperature for 2 h. The reaction solution was placed in an ice-water bath, 4 mL of PBS was added and stirred for 5 min, followed by the addition of 25 mL of chloroform and vigorous stirring for 10 min. The reaction solution was then centrifuged at 4000 r / min for 8 min. The supernatant after centrifugation was the aqueous solution of Compound 8.

[0040] Step 8, Synthesis of Bovine Serum Albumin-Silver Nanoclusters (BSA-AgNCs): Dissolve 250 mg of BSA in 10 mL of deionized water, add 5 mL of 10 mM silver nitrate solution, stir thoroughly at room temperature, then add 0.3 mL of 1 M sodium hydroxide solution to adjust the pH to approximately 8. Then, slowly add 10 mM sodium borohydride solution until the solution changes from colorless to reddish-brown and exhibits red fluorescence under UV light. Continue stirring for 1 h. Place the prepared BSA-AgNCs reaction solution into a dialysis bag (8000-12000 Da) and immerse it in ultrapure water for dialysis. Change the ultrapure water hourly until 8 hours have elapsed. Remove the dialysis bag, aliquot the liquid into 1.5 mL EP tubes, and freeze-dry to obtain the target silver clusters. See the MALDI-TOF-MS image of bovine serum albumin (BSA). Figure 14 The MALDI-TOF-MS image of bovine serum albumin-silver nanoclusters (BSA-AgNCs) is shown below. Figure 15 Calculations showed that 12 silver atoms were successfully coupled into each bovine serum albumin molecule.

[0041] Step 9, Synthesis of mannose-modified silver nanoclusters (MBAg): 100 mg of BSA-AgNCs was added to an aqueous solution of compound 8 and dissolved with a small amount of PBS. The reaction was carried out at room temperature for 20 h. The reaction solution was then dialyzed in a dialysis bag for 8 h. The dialyzed liquid was then aliquoted into 1.5 mL EP tubes and freeze-dried under vacuum to obtain the pale yellow flocculent target product MBAg. The MALDI-TOF-MS image of the mannose-modified silver nanocluster fluorescent probe (MBAg) is shown below. Figure 16 Calculations showed that 14 mannose molecules were successfully coupled through the connecting arm.

[0042] After obtaining mannose-modified silver nanoclusters (MBAg), their fluorescence properties were analyzed. Based on this, fluorescence performance experiments, fluorescence spectroscopy analysis experiments, and confocal experiments were used to verify the specific recognition and labeling ability of the MBAg probe for concanavalin A, Escherichia coli, and MCF-7 cells.

[0043] Example 2 Fluorescence performance analysis of BSA, BSA-AgNCs, and MBAG In this invention, the initial excitation wavelength was set to 340 nm, and the emission wavelength at this excitation wavelength was measured using a fluorescence spectrophotometer. Pure PBS buffer solution was used as a blank for background calibration.

[0044] BSA, BSA-AgNCs, and MBAg were prepared into 1 µM solutions using PBS buffer. The emission wavelength range was set between 500 nm and 700 nm, and the emission spectra of the three samples were measured at an excitation wavelength of 340 nm, yielding an emission wavelength of 680 nm. The fluorescence emission spectra of BSA, BSA-AgNCs, and MBAg are shown below. Figure 17 As shown: like Figure 17 As shown, the excitation and emission wavelengths of BSA-AgNCs are 340 nm and 680 nm, respectively. The half-width at half-maximum (FWHM) emission peak exhibited by BSA-AgNCs is only 30 nm, and the difference between the excitation and emission wavelengths is approximately 340 nm, indicating a large Stokes shift and a symmetrical single-peak emission. These fluorescence characteristics demonstrate that BSA-AgNCs possess high color purity and monochromaticity, ensuring the stability and reliability of their performance. The fluorescence properties of the mannose-modified silver nanoclusters MBAG remained unchanged, providing a solid foundation for subsequent biorecognition and targeted specific binding experiments.

[0045] Example 3 Fluorescent labeling experiment of mannose-modified silver nanoclusters MBAG as probes with concanavalin A: Concanavalin A (CPA) is low in toxicity, readily available naturally, and can specifically recognize mannose. This invention uses CPA as the research subject to conduct fluorescence spectroscopy analysis experiments to quantitatively characterize the interaction between mannose-modified silver nanoclusters (MBAg) and CPA. This experiment is conducted through the following steps: (1) Set the excitation wavelength of the fluorescence spectrophotometer to 340 nm, the emission wavelength to 680 nm, and the detection range to 620 nm-720 nm. Use PBS as a blank for baseline correction.

[0046] (2) Concanavalin A solutions with concentration gradients of 0.1 µM, 0.5 µM, 1.0 µM, 5.0 µM and 10 µM were prepared using PBS. They were incubated with MBAg at room temperature for 30 min, centrifuged, and the supernatant was taken to measure the fluorescence spectrum. The aggregation effect of concanavalin A on mannose was investigated by observing the change in the intensity of the characteristic peak at 680 nm.

[0047] (3) To verify the binding specificity of MBAG and concanavalin A, a concentration of 10 µM ConA was selected and 20 µM mannose (D-Mannose) was added to conduct a competitive inhibition experiment. The consistency of the binding site was evaluated by comparing the changes in fluorescence signal before and after the addition of mannose.

[0048] All measurements were calibrated using PBS buffer as a blank control, and three replicate experiments were performed to ensure data reliability. The fluorescence spectra of MBAg and concanavalin A are shown below. Figure 18 As shown, the fluorescence value gradually decreased with increasing concentration of concanavalin A in MBAG, indicating that concanavalin A bound and aggregated with MBAG (a white precipitate can be observed at the bottom of the EP tube, which shows red fluorescence under ultraviolet light). Figure 19 (As shown). After adding MBAg and 20 µM D-mannose to a 10 µM concanavalin A solution, the fluorescence value recovered to the fluorescence value of the original MBAg solution, demonstrating that the addition of the competitive ligand partially or completely inhibited the binding of the fluorescent probe to the target protein, leading to the recovery of fluorescence intensity.

[0049] Experiment Example 4 Fluorescence spectroscopy analysis of Escherichia coli with fluorescent probe (MBAg): This invention continues to focus on Escherichia coli as the research object, exploring the ability of fluorescent probes to specifically recognize and label Escherichia coli.

[0050] Escherichia coli was cultured in a shaker to OD. 600 The value reached between 0.6 and 0.8, indicating that *E. coli* was in the logarithmic growth phase. Subsequently, *E. coli* was prepared into test solutions with different concentration gradients (4 × 10⁻⁶) using sterile water. 7 CFU / mL, 8×10 7CFU / mL, 4×10 8 CFU / mL, 8×10 8 The *E. coli* test solution (CFU / mL) was incubated with MBAg at room temperature for 30 min. The aggregation effect of *E. coli* on MBAg was determined by measuring the fluorescence value of the supernatant after centrifugation. The fluorescence spectrum is shown in the figure below. Figure 20 As shown.

[0051] from Figure 20 It is known that when MBAG is co-incubated with E. coli, MBAG specifically binds to type I flagella on E. coli and aggregates, leading to a decrease in the concentration of MBAG in the supernatant and a subsequent decrease in fluorescence value. As the concentration of E. coli increases, more MBAG binds to E. coli, resulting in a gradual decrease in free MBAG in the supernatant and a gradual decrease in fluorescence value.

[0052] Select a concentration of 8×10 8 When CFU / mL of E. coli was incubated with MBAg and a high concentration of D-mannose (20 µM) was added, the fluorescence value of the MBAg-E. coli incubation solution was clearly restored, which was consistent with the fluorescence spectrum results of MBAg and concanavalin A in Experiment 3. This indicates that after MBAg binds to E. coli, it specifically labels and aggregates E. coli, demonstrating the sensitivity of MBAg to receptor binding.

[0053] Experimental Example 5 Confocal assay of E. coli and MBAg: Confocal microscopy can simultaneously detect multiple fluorescently labeled samples and achieve synchronous imaging of multiple labeled samples by exciting different fluorescent probes with lasers of different wavelengths. Experiment 4 demonstrated the binding of MBAg to the E. coli receptor protein. This example further uses fluorescence confocal microscopy to investigate the labeling ability of MBAg on E. coli. The simplified steps of the fluorescence confocal experiment are as follows: (1) Escherichia coli (OD) 600 The bacterial culture was incubated at a 1:1 ratio with MBAg (prepared in sterile water at a concentration of 2 μM) at room temperature for 30 min. The incubated mixed bacterial culture was then dropped onto a glass slide and an anti-quenching mounting medium was used to reduce photobleaching.

[0054] (2) Adjust the spatial overlap and temporal synchronization of the excitation light and the loss light, and adjust the field of view to focus on E. coli to optimize the pinhole size to match the super-resolution mode.

[0055] (3) Select an excitation wavelength of 340 nm and an emission wavelength of 680 nm respectively, and set the scanning speed (≤1 μs / pixel) and laser power.

[0056] (4) Obtain the original image by scanning point by point, and use the STED dedicated algorithm or SIM frequency domain reconstruction technology to eliminate background noise and reconstruct the super-resolution image.

[0057] (5) Use software for deconvolution processing, 3D rendering and quantitative analysis.

[0058] The results of the fluorescence confocal experiment are as follows: Figure 21 As shown. Figure 21 As shown in (a), when MBAg was added, E. coli exhibited an aggregation effect under bright field; further experiments were conducted at an emission wavelength of 680 nm, yielding images of MBAg-labeled E. coli, as shown in [image missing]. Figure 21 As shown in (b); Figure 21 (a) and Figure 21 The result obtained by superimposing (b) Figure 21 In image (c), aggregated red fluorescence can be observed on the surface of *E. coli*, indicating that MBAg fluorescently labeled *E. coli* and aggregated. (Summary) Figure 21 The changes in fluorescence signals in the three sets of images suggest that MBAg can label and induce E. coli aggregation by specifically binding to specific receptors or fimbriae on the surface of E. coli. These experimental results provide a solid theoretical basis for subsequent research.

[0059] Experimental Example 6 Confocal microscopy of human breast cancer cell line MCF-7 cells with MBAg: The results of MBAg labeling in *E. coli* and the specific binding experiments of MBAg to concanavalin A both indicate that MBAg has a significant binding ability to the mannose receptor. To further investigate the fluorescent labeling effect of MBAg, this study conducted cellular-level confocal fluorescence experiments.

[0060] The human breast cancer cell line MCF-7 possesses mannose receptors on its surface. Mannose fluorescent probes can specifically target and label breast cancer cells. Cells transport the mannose fluorescent probes to lysosomes via endocytosis, ultimately achieving co-localization labeling using lysosomal green fluorescent probes. This experiment aims to reveal the fluorescence confocalization phenomenon between fluorescent probes and the human breast cancer cell line MCF-7. The simplified experimental steps are as follows: (1) MCF-7 cells were passaged in confocal microplates and cultured in an incubator at 37°C and 5% CO2 until the cells covered 80% of the microplate area; (2) Add 1 μM mannose-silver nanoclusters MBAg prepared with MEM basal medium and incubate for 30 min; (3) Wash away the residue and add MEM basal medium to continue culturing in a cell culture incubator for 24 h; (4) After the cultured MCF-7 cells are removed, they are incubated with a certain concentration of lysosomal green fluorescent probe for 1 h for secondary labeling. Then, they are washed with PBS and subjected to fluorescence confocal microscopy. By observing the fluorescence pattern in the cells, it can be seen whether there is specific binding, and the red fluorescence wavelength at 680 nm ( Figure 22 (c) and the 520 nm green fluorescence wavelength ( Figure 22 The experiment was conducted in the middle (b) section.

[0061] The experimental results are as follows Figure 22 As shown, MBAg can achieve efficient fluorescent labeling of MCF-7 cells in a low concentration range, and the fluorescence signal observed under confocal microscopy is uniformly distributed, indicating that MBAg can specifically bind to the mannose receptor on the surface of MCF-7 cells, achieving precise labeling of cells.

[0062] from Figure 22 As can be seen in (c), under excitation at a red fluorescence wavelength of 680 nm, MCF-7 cells containing lysosomal green fluorescent probes can also exhibit the red fluorescence characteristic of MBAG, and from... Figure 22 In the overlay image (d), yellow fluorescence formed by the superposition of red and green fluorescence can be observed, indicating that MBAg, like the lysosomal green fluorescent probe, has also entered the MCF-7 cells and almost overlaps with the cell's lysosomes. This also shows that the mannose receptor on the surface of MCF-7 cells specifically binds to MBAg and is internalized into the cell, thereby entering the lysosomes of MCF-7 cells to generate fluorescent labels. MBAg can be used as a fluorescent probe for fluorescence imaging of breast cancer cells, providing important information for studying the metabolic characteristics of MCF-7 cells and drug delivery mechanisms.

[0063] The results of the above experimental examples show that the present invention has successfully developed a mannose-based silver nanocluster fluorescent probe, which has the advantages of simple synthesis, strong resistance to photobleaching, high stability, and low toxicity. It shows good application prospects in the fields of fluorescent labeling, bio-recognition, and targeted applications. The present invention provides new ideas and methods for sugar-modified nanofluorescent probes.

[0064] The above description is only a preferred embodiment of 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 principle 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 mannose-modified silver nanocluster, characterized in that, The modified mannose was obtained by coupling bovine serum albumin-silver nanoclusters and modified mannose via an acid-amine condensation reaction, wherein the structural formula of the modified mannose is: 。 2. The mannose-modified silver nanoclusters as described in claim 1, characterized in that, Each bovine serum albumin molecule is coupled with 14 modified mannose molecules.

3. The method for preparing mannose-modified silver nanoclusters as described in claim 1, characterized in that, Includes the following steps: Step 1, full acetylation: Mannose 1 undergoes a full acetylation reaction with acetic anhydride to obtain fully acetylated mannose 2; Step 2, selective removal of the 1-acetyl group: the 1-acetyl group of the fully acetylated mannose 2 is removed to obtain the product 3, which is deacetylated at the C-1-position; Step 3, trichloroacetylimine esterification: The product 3, which has been deacetylated at the C-1 position, reacts with trichloroacetonitrile to undergo trichloroacetylimine esterification, yielding the C-1-trichloroacetylimine esterified product 4; Step 4, Glycosylation: The C-1-trichloroacetylimine esterification product 4 reacts with 6-azido-1-hexanol under the action of a glycosylation promoter to obtain glycosylated product 5; Step 5, Deacetylation: Glycosylated product 5 undergoes deacetylation of all acetyl groups under the action of a catalyst to obtain deacetylated product 6; Step 6, reduction reaction: Deacetylated product 6 is reduced under the action of palladium on carbon and hydrogen, and the azide group is reduced to amino group to obtain amino product 7; Step 7, Connecting the connecting arm: Disuccinimid adipate reacts with amino product 7 to obtain mannose 8 with the connecting arm attached; Step 8: Prepare bovine serum albumin-silver nanoclusters (BSA-AgNCs); Step 9, Preparation of mannose-modified silver nanoclusters: Bovine serum albumin-silver nanoclusters BSA-AgNCs and mannose 8 with attached linker arms were stirred and reacted in PBS, dialyzed in a dialysis bag and freeze-dried under vacuum to obtain mannose-modified silver nanoclusters. The reaction pathway is as follows:

4. The preparation method according to claim 3, characterized in that, In step 1, mannose is dissolved in pyridine, 6-10 equivalents of acetic anhydride are added and reacted for 8-12 hours. The reaction system is diluted with dichloromethane and washed with dilute acid to remove pyridine. The organic phase is concentrated to obtain fully acetylated mannose 2. In step 2, fully acetylated mannose 2 is dissolved in tetrahydrofuran, and 1.0-1.5 equivalents of N,N-dimethylethylenediamine are added and reacted for 8-12 hours. The reaction system is diluted with an appropriate amount of dichloromethane and washed with dilute acid to remove organic amines. After the organic phase is concentrated, product 3 with deacetylated group at C-1-position is obtained. In step 3, the product from the previous step is reacted with 2-5 equivalents of trichloroacetonitrile in the presence of a catalytic amount of base for 4-8 hours. The reaction system is then concentrated to dryness and separated by rapid column chromatography to obtain C-1-position trichloroacetylimine esterified product 3. In step 4, the C-1-trichloroacetylimine esterification product 3 and 1.0-1.5 equivalents of 6-azido-1-hexanol are dissolved in anhydrous dichloromethane. A catalytic amount of glycosylation promoter is added at -40~-20℃, and the reaction is continued for 20-40 min. The reaction system is then washed with sodium bicarbonate solution, saturated brine, and dried with anhydrous sodium sulfate. After concentration, it is separated by rapid column chromatography to obtain glycosylated product 5. In step 5, glycosylated product 5 is dissolved in an appropriate amount of anhydrous methanol, and a catalytic amount of sodium methoxide solution is added. The mixture is stirred at room temperature for 2-6 hours. After the reaction is completed, the pH of the system is adjusted to neutral using a cation exchange resin. The cation exchange resin is removed by filtration. After the filtrate is concentrated, it is separated by rapid column chromatography to obtain deacetylated product 6. In step 6, the deacetylated product 6 is dissolved in methanol, a catalytic amount of palladium on carbon is added, and the mixture is reacted at room temperature under hydrogen atmosphere. After the reaction is complete, the palladium on carbon is removed, and the filtrate is dried by rotary evaporation to obtain the amino product 7. In step 7, disuccinimide adipate is dissolved in dimethyl sulfoxide, and then an organic base is added. Subsequently, the dimethyl sulfoxide solution of amino product 7 is slowly added dropwise, and the reaction is carried out at room temperature for 2-6 hours. The reaction solution is placed in an ice-water bath, PBS is added and stirred, then an appropriate amount of chloroform is added and stirred vigorously for 5-20 minutes. The reaction solution is then centrifuged, and the supernatant is mannose 8 with the connecting arm attached. In step 8, BSA is dissolved in deionized water, silver nitrate solution is added, and the mixture is stirred at room temperature. The pH of the solution is adjusted to 8.5-9.5 with 0.1-1.0M sodium hydroxide solution. Then, the color of the solution is adjusted from colorless to reddish-brown with 5-10mM sodium borohydride solution and red fluorescence is observed under ultraviolet light. After stirring for 0.5-1.5 hours, the reaction solution is placed in a dialysis bag with a molecular weight cutoff of 8000-12000 Da. Dialysis is performed with ultrapure water for 8-20 hours. The liquid in the dialysis bag is then freeze-dried to obtain bovine serum albumin-silver nanoclusters BSA-AgNCs. In step 9, mannose 8 with the connecting arm is stirred with BSA-AgNCs in PBS at room temperature for 20-40 hours. The reaction solution is then dialyzed in a dialysis bag for 8-20 hours. The dialysate is then freeze-dried under vacuum to finally obtain the mannose-modified silver nanocluster probe.

5. The preparation method according to claim 4, characterized in that, In steps 1 and 2, the dilute acid is dilute sulfuric acid, dilute hydrochloric acid, dilute nitric acid, or dilute phosphoric acid, preferably dilute phosphoric acid.

6. The preparation method according to claim 4, characterized in that, In step 3, the catalytic base is potassium carbonate, cesium carbonate, sodium carbonate, triethylamine, N,N-diisopropylethylamine, or DBU, preferably DBU.

7. The preparation method according to claim 4, characterized in that, In step 4, the glycosylation promoter is trifluoromethanesulfonic acid, trimethylsilyl trifluoromethanesulfonic acid, or boron trifluoride ethyl ether, preferably boron trifluoride ethyl ether.

8. The preparation method according to claim 4, characterized in that, In step 7, the organic base is N-methylmorpholine, triethylamine, N,N-diisopropylethylamine, DBU, etc., preferably triethylamine.

9. The application of the mannose-modified silver nanoclusters as described in claim 1 as a probe in biorecognition and targeted labeling.

10. The application of the mannose-modified silver nanoclusters as described in claim 1 as a probe for biorecognition or targeted labeling of concanavalin A, Escherichia coli, or human breast cancer cell line MCF-7.

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