Gold cluster fluorescent molecule, probe as well as preparation method and application of gold cluster fluorescent molecule

Abstract

The invention discloses a gold cluster fluorescent molecule, a probe and a preparation method and application thereof, and belongs to the technical field of nano-drugs. In order to solve the technical problem that in the prior art, visual imaging and accurate tracing of hematopoietic stem cells are difficult, the invention provides a gold cluster fluorescent molecule of a near-infrared two-region coupled antibody, a probe and a preparation method and application of the gold cluster fluorescent molecule. The gold cluster fluorescent molecule is composed of an Au atom, other metal atoms M and an outer layer ligand L, the gold cluster fluorescent molecule and an antibody are coupled through a chemical cross-linking method to prepare the gold cluster fluorescent probe of the near-infrared two-region coupled antibody. The gold cluster fluorescent probe provided by the invention has the advantages of strong photobleaching resistance, light stability, in-vivo stability, high sensitivity and high specificity, can realize the purpose of accurate tracing of in-vivo hematopoietic stem cells, and lays a foundation for imaging detection of hematopoietic stem cells, transplantation operation monitoring and postoperative evaluation.

Description

Technical Field

[0001] The invention belongs to the technical field of nano-drugs, and relates to a gold cluster fluorescent molecule, a probe, and a preparation method and application thereof. Background Art

[0002] Hematopoietic stem cells are a group of primitive hematopoietic cells existing in hematopoietic tissues. They have the ability to self-renew and differentiate into various blood cells. Therefore, they are considered to be the primitive cells of all blood cells (most of which are immune cells) and play a vital role in the development and maintenance of the human blood system. Hematopoietic stem cells can be divided into bone marrow hematopoietic stem cell transplantation, peripheral blood hematopoietic stem cell transplantation, and umbilical cord blood hematopoietic stem cells according to their source. They are mainly distributed in the bone marrow in adults. In 1957, Donal Thomas of the University of Washington in the United States discovered that transplanting normal bone marrow into patients could treat hematopoietic dysfunction. Subsequently, this technology was quickly recognized worldwide and became the main means of treating diseases such as leukemia. At present, hematopoietic stem cell transplantation has been used to treat a variety of fatal diseases such as malignant blood diseases, some malignant tumors, and some hereditary diseases; including acute leukemia, chronic leukemia, myelodysplastic syndrome, hematopoietic stem cell disease, myeloproliferative disease, lymphoproliferative disease, macrophage disease, hereditary metabolic disease, tissue cell disease, hereditary red blood cell disease, hereditary immune system disease, hereditary platelet disease, plasma cell disease, thalassemia, non-hematological malignancies, acute radiation sickness, etc.

[0003] The precise tracking of hematopoietic stem cells in the body is very important for understanding the disease status. For example, after bone marrow transplantation for acute radiation sickness, the precise tracking of hematopoietic stem cells in the body can be used to evaluate the postoperative condition, but the visualization of hematopoietic stem cells still faces difficulties, which limits the postoperative evaluation of hematopoietic stem cell transplantation. Fluorescent probes with strong targeting can promote the detection of small lesions or abnormal biomarkers in the early stages of the disease, achieve early intervention and treatment of the disease, and develop near-infrared zone II fluorescent probes with good targeting to improve the accuracy of disease detection, optimize treatment strategies, and promote medical research and clinical practice. Therefore, those skilled in the art are eager to develop a technology that can achieve precise tracking of hematopoietic stem cells in the body, providing a visual evaluation method for the medical application of hematopoietic stem cells. Summary of the invention

[0004] In order to solve the difficulties in visual imaging and accurate tracing of hematopoietic stem cells in the prior art, the present invention provides a gold cluster fluorescent molecule and probe coupled to a near-infrared second zone antibody, as well as a preparation method and application thereof.

[0005] One of the purposes of the present invention is to provide a gold cluster fluorescent molecule, the general formula of the gold cluster fluorescent molecule is Au xM y L z , x represents the number of Au atoms contained in the fluorescent molecule, M represents other metal atoms contained in the fluorescent molecule, y represents the number of other metal atoms, L represents the outer layer ligands contained in the fluorescent molecule, and z represents the number of outer layer ligands; the x:y:z atom number ratio is (25-n):n:18, and n represents a positive integer between 0 and 24.

[0006] In a preferred embodiment of the present invention, the other metal atoms M are one or more of calcium, scandium, titanium, vanadium, chromium, manganese, iron, cobalt, nickel, copper, zinc, gallium, germanium, strontium, yttrium, zirconium, niobium, molybdenum, technetium, ruthenium, rhodium, palladium, silver, cadmium, indium, tin, antimony, tellurium, barium, lanthanum, hafnium, tantalum, tungsten, rhenium, osmium, iridium, platinum, thallium, bismuth or polonium.

[0007] In a preferred embodiment of the present invention, the outer layer ligand L is one or more of glutathione, cysteine, captopril, 3-mercaptopropionic acid, p-mercaptobenzoic acid, 6-mercaptohexanoic acid, thioglycolic acid or lipoic acid.

[0008] The second purpose of the present invention is to provide a method for preparing the above-mentioned gold cluster fluorescent molecules, which specifically includes the following steps: preparing a HAuCl4 solution with a concentration range of 10-60mM, and then preparing an outer layer ligand L solution with a concentration range of 20-100mM, adding the HAuCl4 solution and the outer layer ligand L solution to deionized water at a volume ratio of 1:(1-3) at room temperature and mixing them evenly to obtain a mixed solution; under stirring, adding a 0.1-3M alkaline solution to adjust the pH, and then introducing a reducing gas for 2 minutes, stirring the above-mentioned mixed solution at room temperature for 12-36h, and then collecting the precipitate by a rotary evaporator, washing it with an organic solvent for 3 times, and vacuum drying it to obtain a gold cluster fluorescent molecule powder.

[0009] In a preferred embodiment of the present invention, the pH adjustment range is 9-12, the reducing gas is one or more of hydrogen, carbon monoxide, hydrogen sulfide or sulfur monoxide, and the organic solvent is one or more of methanol, ethanol, isopropanol, acetone, methyl butyl ketone, methyl isobutyl ketone, cyclohexane, cyclohexanone, toluene cyclohexanone, chlorobenzene, dichlorobenzene, dichloromethane, benzene, toluene, xylene, pentane, hexane, octane, ether, propylene oxide, methyl acetate, ethyl acetate, propyl acetate, ethylene glycol monomethyl ether, ethylene glycol monoethyl ether, ethylene glycol monobutyl ether, acetonitrile or pyridine.

[0010] The third object of the present invention is to provide a method for preparing a gold cluster fluorescent probe of a near-infrared region II coupled antibody, wherein the gold cluster fluorescent probe of the near-infrared region II coupled antibody contains the above-mentioned gold cluster fluorescent molecule, and the preparation method comprises the following steps: mixing a coupling activator and a gold cluster fluorescent molecule solution in a mass ratio of 1:(2-5), stirring at room temperature for 10 min-60 min, ultrafiltration twice, and then adding 10-80 μL of antibody, stirring at 4°C for 1-6 h, and ultrafiltration three times to obtain a gold cluster fluorescent probe of a near-infrared region II coupled antibody.

[0011] In a preferred embodiment of the present invention, the antibody is one or more of anti-CD3e, anti-CD43, anti-CD49 (integrin α6), anti-CD90 (Thy-1), anti-c-Kit / CD117, anti-Ly-6A / E (Sca-1), anti-CD105, anti-CD36, anti-CD44, anti-CD73, anti-Nestin, anti-STRO-1, anti-CD11b, anti-CD14, anti-CD19, anti-CD34, anti-CD79a or anti-HLA-DR antibodies.

[0012] In a preferred embodiment of the present invention, the coupling activator is one or more of Ynamides, EDC, NHS, HOBt, HBTU, HATU, PyBOP, DCC or DIC.

[0013] The fourth object of the present invention is to provide a gold cluster fluorescent probe of near-infrared region II coupled to an antibody, wherein the gold cluster fluorescent probe of near-infrared region II coupled to an antibody is obtained by the above-mentioned preparation method.

[0014] A fifth object of the present invention is to provide the use of the gold cluster fluorescent probe of the second near-infrared region coupled to an antibody in the tracing of hematopoietic stem cells.

[0015] Beneficial effects of the present invention: Near-infrared II region (NIR-II, 1000-3000nm) has submicron-level high resolution, which can realize continuous dynamic observation of deep tissues. The specific binding of near-infrared II region fluorescent probes is a key issue in improving imaging quality. After the fluorescent molecules are coupled with specific antibodies, they show targeting, which increases the concentration of fluorescent probes in specific tissues and significantly enhances the signal contrast between the target area and the background, so that the specific area has a higher fluorescent signal and a high signal-to-noise ratio, thereby providing clearer imaging results.

[0016] The present invention provides a gold cluster fluorescent molecule, a probe, a preparation method and an application thereof. The general formula of the gold cluster fluorescent molecule is Au x M y L z, M represents other metal atoms contained in the fluorescent molecule, L represents the outer ligand contained in the fluorescent molecule, the gold cluster fluorescent molecule has near-infrared second zone fluorescence characteristics and hematopoietic stem cell targeting, and the above-mentioned gold cluster fluorescent molecule is coupled with an antibody by a chemical cross-linking method to prepare a gold cluster fluorescent probe of near-infrared second zone coupled antibody. This method stably combines the fluorescent molecule with the antibody with the targeting characteristic of hematopoietic stem cells, thereby realizing the targeting of the fluorescent molecule to specific cells and tissues.

[0017] The gold cluster fluorescent probe of the near-infrared zone II coupled antibody provided by the present invention exhibits a very significant optical characteristic absorption peak at 670nm, the coupling yield can be maintained at 90%, and has excellent near-infrared zone II luminescence performance, strong resistance to photobleaching and good light stability, and can be combined with near-infrared zone II imaging technology for fluorescent image detection. Mouse experiments have proved that the gold cluster fluorescent probe of the near-infrared zone II coupled antibody provided by the present invention exhibits good in vivo stability, has the advantages of high sensitivity and high specificity, can achieve the purpose of accurate tracing of hematopoietic stem cells in vivo, and lays a material and technical foundation for hematopoietic stem cell imaging detection and transplantation surgery monitoring and postoperative evaluation. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 It is a comparison analysis diagram of the gold cluster fluorescent molecules and the gold cluster fluorescent probe coupled to the near-infrared second region antibody; a is the absorption spectrum diagram, and b is the statistical diagram of the coupling reaction yield and purity;

[0019] Figure 2 The fluorescence images of the gold cluster fluorescent molecules and the gold cluster fluorescent probe coupled with the near-infrared region II antibody, a is the near-infrared region II fluorescence image, and b is the fluorescence quantitative statistical graph;

[0020] Figure 3 This is a diagram for detecting the anti-photobleaching property of the gold cluster fluorescent probe coupled to an antibody in the near-infrared region II;

[0021] Figure 4 Figure 1 is a photostability detection diagram of the gold cluster fluorescent probe coupled to the antibody in the near-infrared region II; a is a fluorescence image, b is a fluorescence quantitative statistical diagram; ce are respectively the normalized results of three repeated infrared region II fluorescence quantitative analysis;

[0022] Figure 5 The diagram shows the tracing results of hematopoietic stem cells in vivo using a gold cluster fluorescent probe coupled to an antibody in the near-infrared region II; a is a fluorescence imaging image of the bladder of a C57 male mouse, and b is a fluorescence imaging image of the leg of a C57 male mouse. DETAILED DESCRIPTION

[0023] Those skilled in the art can refer to the content of this article and appropriately improve the process parameters. It should be particularly noted that all similar replacements and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The method and application of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications described herein without departing from the content and scope of the present invention to implement and apply the technology of the present invention.

[0024] In order to make the purpose, technical scheme and advantages of the present invention more clear, the present invention is further described in detail below in conjunction with specific embodiments and drawings. The experimental methods used in the following examples are conventional methods unless otherwise specified, and the materials, reagents, methods and instruments used are conventional materials, reagents, methods and instruments in the art unless otherwise specified, and can be obtained by those skilled in the art through commercial channels.

[0025] The sources of raw materials and instrument models used in the following examples are described:

[0026] Tetrachloroauric acid trihydrate (HAuCl4·3H2O, 1 g, 99%) was purchased from Sigma and Tianjin Xiensiopude Technology Co., Ltd.;

[0027] Reduced glutathione and sodium hydroxide were purchased from Shanghai Aladdin Reagent Co., Ltd.;

[0028] Organic solvents such as methanol and ethanol were purchased from Tianjin Yuanli Chemical Co., Ltd.;

[0029] The UV spectrophotometer was a UV-2600 UV-visible spectrophotometer purchased from Shimadzu Corporation;

[0030] The near-infrared zone II optical imaging light path is a self-built imaging system, in which the near-infrared zone II camera model is NIRvana:640ST, manufactured by Princeton, USA, and the laser model used is FLC300-0808-8W-F200, manufactured by Frankfurt Laser Company, Germany.

[0031] Example 1: Preparation of gold cluster fluorescent molecules

[0032] In this example, an aqueous solution of HAuCl4 (3.75 mL, 40 mM) and glutathione GSH (4.5 mL, 50 mM) was added to 141.75 mL of ultrapure water, and 0.1-3 M NaOH was added to adjust the pH to 11 under stirring, and then CO was introduced for 2 min. The mixture was stirred at room temperature for 24 h (500 rpm), and the precipitate was collected by rotary evaporation, washed with methanol 3 times (3500 rpm, 10 min), and vacuum dried to finally obtain 40 mg of gold cluster fluorescent molecules with GSH ligands.

[0033] In this example, an ultraviolet spectrophotometer was used to detect the optical characteristic absorption peaks of the gold cluster fluorescent molecules. The results are as follows: Figure 1 As shown in a, the gold cluster fluorescent molecules prepared in this example show a very obvious optical characteristic absorption peak at 670nm.

[0034] Example 2: Preparation of gold cluster fluorescent probes coupled to antibodies in the near-infrared region II

[0035] In this example, 12 mg of EDC coupling activator and 12 mg of NHS coupling activator were added to 2 mL of the gold cluster fluorescent molecule (2 mg / mL) solution prepared in Example 1, stirred at room temperature for 20 min (500 rpm), ultrafiltered (10K, 3000 rpm, 15 min), and repeated twice; then, 40 μL of Thy1 / CD90 antibody was added to the above solution, stirred at 4°C for 2 h (500 rpm), ultrafiltered (30K, 3000 rpm, 10 min), and washed 3 times, finally obtaining a gold cluster fluorescent probe of near-infrared region II coupled antibody, which was stored at 4°C for future use.

[0036] In this example, the gold cluster fluorescent probe coupled to the antibody in the near-infrared region II was subjected to near-infrared region II optical imaging detection. The results are as follows: Figure 1 As shown in a, the gold cluster fluorescent probe coupled to the antibody still exhibits a very significant optical characteristic absorption peak at 670nm, which is consistent with the optical characteristic absorption peak of the gold cluster fluorescent molecule. This example quantitatively analyzes the fluorescent molecules before and after the 670nm characteristic peak reaction of the gold cluster fluorescent probe coupled to the antibody in the near-infrared region II. The results are as follows Figure 1 As shown in b, the coupling yield of the gold cluster fluorescent probe coupled to the antibody in the near-infrared region II can be maintained at 90%.

[0037] Embodiment 3:

[0038] (1) Fluorescence image detection of gold cluster fluorescent probes coupled to antibodies in the near-infrared region II:

[0039] The gold cluster fluorescent molecules obtained in Example 1 and the gold cluster fluorescent probe of the near-infrared zone II coupled antibody obtained in Example 2 were dissolved in 2.5 mL of deionized water respectively. The absorption values ​​of the aqueous solutions of the two solutions at 808 nm were determined to be 0.1 using a UV-visible spectrophotometer. The near-infrared zone II camera built by the self-built system was excited at 808 nm with a power of 25 mW / cm 2 The fluorescence intensity was observed with an exposure time of 100 ms.

[0040] The results are as follows Figure 2 As shown, under 808nm excitation, the fluorescence intensity of the gold cluster fluorescent molecules obtained in Example 1 and the gold cluster fluorescent probe coupled to the antibody obtained in Example 2 showed almost no attenuation, indicating that both have excellent near-infrared second-region luminescence performance.

[0041] (2) Detection of the anti-photobleaching property of gold cluster fluorescent probes coupled to antibodies in the near-infrared region II:

[0042] Using an 808 nm laser and setting an exposure time of 100 ms, the gold cluster fluorescent probe of the near-infrared second region coupled antibody obtained in Example 2 was tested for its anti-photobleaching property. The above 0.59 mg / mL gold cluster fluorescent probe was dissolved in H2O, PBS and FBS respectively and exposed to a power of 25 mW / cm 2 808nm laser and continued irradiation.

[0043] The results are as follows Figure 3 As shown in the figure, after continuous irradiation for 30 minutes, the fluorescence quenching of the gold clusters coupled to antibodies in H2O, PBS and FBS in the near-infrared second zone was slow, and the attenuation was within an acceptable range, showing good anti-photobleaching performance, which was significantly better than the anti-photobleaching ability of organic small molecule dyes such as ICG used in clinical practice.

[0044] (3) Photostability detection of gold cluster fluorescent probes coupled to antibodies in the near-infrared region II:

[0045] The gold cluster fluorescent probe of the near-infrared zone II conjugated antibody obtained in Example 2 was dissolved in deionized water, PBS and plasma, respectively. The absorption value at 808 nm was determined to be 0.1 by a UV-visible spectrophotometer. The near-infrared zone II camera built by the manufacturer was excited at 808 nm with a power of 25 mW / cm 2 , with an exposure time of 100 ms, the fluorescence intensity of the gold cluster fluorescent probe was observed at 25°C for 2h, 4h, 6h, 8h, 12h, and 24h respectively.

[0046] The results are as follows Figure 4As shown in a, at 0h, the gold cluster fluorescent probe coupled to the antibody in the near-infrared zone II has a strong fluorescence intensity, and its fluorescence value can reach about 20,000 in water, PBS, and plasma; it is placed in an environment with a temperature of 25°C for incubation, and fluorescence imaging is performed after 2h. The results show that the fluorescent probe still exhibits a strong fluorescence intensity, and the fluorescence value can be maintained at about 20,000.

[0047] In this example, fluorescence images were collected after incubation for 4 h, 8 h, 12 h, and 24 h, and the photostability of the gold cluster fluorescent probe was quantitatively analyzed by fluorescence intensity. Figure 4 As shown in be, the gold cluster fluorescent probe coupled to the near-infrared region II antibody is known to maintain a strong fluorescence intensity. After 24 hours, its fluorescence value is maintained at around 16,000, with no obvious fluorescence attenuation. Each group of experiments was repeated 3 times. It can be seen that the gold cluster fluorescent probe coupled to the near-infrared region II antibody maintains good photostability within 24 hours in water, PBS and plasma at 25°C.

[0048] Example 4: Application of gold cluster fluorescent probes coupled to antibodies in the second near-infrared region in hematopoietic stem cell tracing

[0049] In this example, the gold cluster fluorescent probe of the near-infrared zone II coupled antibody obtained in Example 2 was dissolved in deionized water to obtain a 10 mg / mL gold cluster fluorescent probe solution; C57 male mice purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd. were taken as experimental subjects, and their hair was processed. The C57 male mice were anesthetized using 2% tribromoethanol, and the above 200 μL gold cluster fluorescent probe solution was injected into the C57 male mice through the tail vein; then the C57 male mice were fixed on the storage table of the near-infrared zone II camera, and the self-built near-infrared zone II camera was used to excite at 808 nm with a power of 25 mW / cm 2 , with an exposure time of 500ms, the mice were imaged in vivo at different times to observe the fluorescence intensity of their legs.

[0050] like Figure 5As shown, in this embodiment, 200 μL of the gold cluster fluorescent probe of the near-infrared zone II coupled antibody obtained in Example 2 was injected into the body through the tail vein at different time points, and near-infrared zone II fluorescence imaging was performed on the whole body and femoral imaging agent enrichment of C57 male mice. 5 minutes after injection, obvious fluorescence signals can be seen in the bladder of C57 male mice. As time increases, the fluorescence signal in the bladder gradually increases, and its signal can be maintained to 120 minutes, indicating that its main excretion route is urinary system excretion. 2 hours after injection, strong fluorescence signals can be observed in the legs of C57 male mice. These fluorescence signals are all in the bone marrow of the legs, which is consistent with the location of hematopoietic stem cells; at 4h, 8h, 12h and 24h after injection, the mouse legs were continuously imaged with fluorescence, and it was found that 24h after injection, the legs of C57 male mice still showed strong fluorescence signals, indicating that the gold cluster fluorescent probe of the near-infrared zone II coupled antibody obtained in Example 2 is enriched in the femur, and shows good in vivo stability, which can achieve the tracing effect of hematopoietic stem cells in vivo.

[0051] The contents not described in detail in the specification of the present invention are well-known technologies for those skilled in the art. Although the present invention has been disclosed as above with preferred embodiments, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention should be based on the definition of the claims.

Claims

1. A gold cluster fluorescent molecule, characterized in that: The general formula of the gold cluster fluorescent molecule is Au x M y L z , x represents the number of Au atoms contained in the fluorescent molecule, M represents other metal atoms contained in the fluorescent molecule, y represents the number of other metal atoms, L represents the outer layer ligands contained in the fluorescent molecule, and z represents the number of outer layer ligands; the x:y:z atom number ratio is (25-n):n:18, and n represents a positive integer between 0 and 24.

2. The gold cluster fluorescent molecule according to claim 1, characterized in that: The other metal atoms M are one or more of calcium, scandium, titanium, vanadium, chromium, manganese, iron, cobalt, nickel, copper, zinc, gallium, germanium, strontium, yttrium, zirconium, niobium, molybdenum, technetium, ruthenium, rhodium, palladium, silver, cadmium, indium, tin, antimony, tellurium, barium, lanthanum, hafnium, tantalum, tungsten, rhenium, osmium, iridium, platinum, thallium, bismuth or polonium.

3. The gold cluster fluorescent molecule according to claim 1, characterized in that: The outer layer ligand L is one or more of glutathione, cysteine, captopril, 3-mercaptopropionic acid, p-mercaptobenzoic acid, 6-mercaptohexanoic acid, thioglycolic acid or lipoic acid.

4. The method for preparing the gold cluster fluorescent molecule according to any one of claims 1 to 3, characterized in that: The preparation method specifically comprises the following steps: preparing a HAuCl4 solution with a concentration range of 10-60 mM, and then preparing an outer layer ligand L solution with a concentration range of 20-100 mM, adding the HAuCl4 solution and the outer layer ligand L solution into deionized water at a volume ratio of 1:(1-3) at room temperature and mixing them evenly to obtain a mixed solution; under stirring conditions, adding a 0.1-3 M alkaline solution to adjust the pH, and then introducing a reducing gas for 2 minutes, stirring the mixed solution at room temperature for 12-36 hours, and then collecting the precipitate by a rotary evaporator, washing it with an organic solvent for 3 times, and vacuum drying it to obtain a gold cluster fluorescent molecular powder.

5. The preparation method according to claim 4, characterized in that: The pH adjustment range is 9-12, the reducing gas is one or more of hydrogen, carbon monoxide, hydrogen sulfide or sulfur monoxide, and the organic solvent is one or more of methanol, ethanol, isopropanol, acetone, methyl butyl ketone, methyl isobutyl ketone, cyclohexane, cyclohexanone, toluene cyclohexanone, chlorobenzene, dichlorobenzene, dichloromethane, benzene, toluene, xylene, pentane, hexane, octane, ether, propylene oxide, methyl acetate, ethyl acetate, propyl acetate, ethylene glycol monomethyl ether, ethylene glycol monoethyl ether, ethylene glycol monobutyl ether, acetonitrile or pyridine.

6. A method for preparing a gold cluster fluorescent probe coupled to an antibody in the second near-infrared region, characterized in that: The gold cluster fluorescent probe of the near-infrared region II antibody coupling contains the gold cluster fluorescent molecule according to any one of claims 1 to 3, and the preparation method comprises the following steps: mixing the coupling activator and the gold cluster fluorescent molecule solution in a mass ratio of 1:(2-5), stirring at room temperature for 10 min-60 min, ultrafiltration twice, and then adding 10-80 μL of antibody, stirring at 4° C. for 1-6 h, and ultrafiltration three times to obtain the gold cluster fluorescent probe of the near-infrared region II antibody coupling.

7. The preparation method according to claim 6, characterized in that: The antibody is one or more of anti-CD3e, anti-CD43, anti-CD49 (integrin α6), anti-CD90 (Thy-1), anti-c-Kit / CD117, anti-Ly-6A / E (Sca-1), anti-CD105, anti-CD36, anti-CD44, anti-CD73, anti-Nestin, anti-STRO-1, anti-CD11b, anti-CD14, anti-CD19, anti-CD34, anti-CD79a or anti-HLA-DR antibodies.

8. The preparation method according to claim 6, characterized in that: The coupling activator is one or more of Ynamides, EDC, NHS, HOBt, HBTU, HATU, PyBOP, DCC or DIC.

9. A gold cluster fluorescent probe coupled to an antibody in the near-infrared region II, characterized in that: The gold cluster fluorescent probe of the near-infrared second region coupled antibody is obtained by the preparation method described in any one of claims 6 to 8.

10. Use of the gold cluster fluorescent probe of near-infrared second region coupled to an antibody as claimed in claim 9 in tracing hematopoietic stem cells.

Images