Hematopoietic stem cell sensing method based on proximity click labeling

Abstract

A hematopoietic stem cell sensing method based on proximity click labeling first captures hematopoietic stem cells with CD45-functionalized magnetic beads, then adds a secondary antibody coupled to horseradish peroxidase to bind to the CD34 antibody retained on the stem cell membrane. Then, in the presence of hydrogen peroxide, the added alkyne-tyramine is catalyzed by horseradish peroxidase to form a highly active tyramide free radical intermediate, which then labels a large number of electron-rich amino acid residues on proximal membrane proteins and exposes the terminal alkynyl group. Then, a DNA probe modified with an N3 group at the 5' end and carrying silver nanoparticles at the 3' end is added. Under the catalysis of monovalent copper ions, the probe undergoes a click chemistry reaction with the alkynyl groups on the cell membrane surface, thereby coupling the DNA probe to the cell membrane surface. Finally, the silver nanoparticles are hydrolyzed with nitric acid to obtain a large amount of silver ions, generating a significant electrochemical signal, which can achieve efficient detection of hematopoietic stem cells.

Description

Technical Field

[0001] The present invention relates to a method for detecting stem cells, and in particular to a method for implementing qualitative and quantitative detection of hematopoietic stem cells using an electrochemical method. Background Art

[0002] Hematopoietic stem cells (HSCs), also known as pluripotent stem cells, are highly undifferentiated cells with the potential for self-renewal and multidirectional differentiation. They are widely found in adult bone marrow, umbilical cord blood, and to a lesser extent, peripheral blood. They are the longest-studied and most intensive type of adult stem cell, providing valuable insights into other stem cell research. They also serve as a model system and tool for studying human hematopoiesis and leukemia. Their key biomarkers include CD34, CD38, and Lys-6A / E (Sca-1), and are currently widely used in bone marrow transplantation, tolerance induction, and gene therapy.

[0003] Proximity oxidation labeling is an enzyme-mediated in situ labeling detection method that uses horseradish peroxidase in the presence of hydrogen peroxide to activate the substrate tyramine into a short-lived intermediate. This intermediate then rapidly deposits on electron-rich residues on adjacent protein molecules and covalently binds, thereby labeling a large number of available binding sites. This method is currently commonly used for fluorescent detection of bacteria, electrochemiluminescence sensitive detection of viral antigens, and electrochemical detection of tumor cells.

[0004] CuCCA is a copper-catalyzed propargyl-azo cycloaddition reaction. This reaction involves the cycloaddition of an alkyne (propargyl) and an azo compound (typically an alkynyl azo compound) using a copper catalyst to form a 1,2,3-triazole product. Due to its rapidity, high efficiency, high selectivity, and broad substrate compatibility, this reaction has become a popular method in fields such as organic synthesis, medicinal chemistry, biology, and materials science. Furthermore, the reaction can be performed in solution at room temperature and does not require any special conditions or reagents.

[0005] The application of electrochemical methods in the field of molecular recognition and biosensing is becoming increasingly mature, especially in the detection of disease-related biomarkers such as proteins, nucleic acids, and cells. Commonly used electrochemical methods include cyclic voltammetry (CV), differential pulse voltammetry (DPV), square wave voltammetry (SWV), linear sweep voltammetry (LSV), and electrochemical impedance spectroscopy (EIS). Among them, differential pulse voltammetry adds voltage pulses to linear sweep voltammetry and step sweep voltammetry. By measuring the current before the potential changes, the influence of the charging current is reduced, thereby greatly improving the detection sensitivity. Only a lower concentration of supporting electrolyte is required to detect trace substances. Summary of the Invention

[0006] One object of the present invention is to provide a hematopoietic stem cell sensing method based on proximity click labeling to realize the detection of hematopoietic stem cells by electrochemical method.

[0007] Another object of the present invention is to provide a hematopoietic stem cell sensing method based on proximity click labeling to efficiently detect hematopoietic stem cells and improve detection sensitivity.

[0008] Another object of the present invention is to provide a hematopoietic stem cell sensing method based on proximity click labeling to implement quantitative detection of hematopoietic stem cells in an electrochemical manner.

[0009] A hematopoietic stem cell sensing method based on proximity click labeling, comprising:

[0010] First, use CD45 functionalized magnetic beads to capture hematopoietic stem cells.

[0011] Then add the secondary antibody coupled with horseradish peroxidase to bind to the CD34 antibody retained on the stem cell membrane.

[0012] Then, in the presence of hydrogen peroxide, the added alkyne-tyramine is catalyzed by horseradish peroxidase to form a highly active tyramine free radical intermediate, which then labels a large number of electron-rich amino acid residues on proximal membrane proteins and exposes the terminal alkyne group.

[0013] Then, a DNA probe (N3-DNA@AgNPs) with an N3 group modified at the 5' end and silver nanoparticles (AgNPs) at the 3' end was added. Under the catalysis of monovalent copper ions (e.g., obtained by reducing copper sulfate with sodium ascorbate), a click chemistry reaction occurred with the alkynyl groups on the cell membrane surface, thereby coupling the DNA probe to the cell membrane surface.

[0014] Finally, the silver nanoparticles are hydrolyzed with nitric acid (eg, adding 100-120 μL of 1-2 M nitric acid solution and reacting at room temperature for 0.5-1 h) to obtain a large amount of silver ions, thereby generating a significant electrochemical signal.

[0015] In the method of the present invention, the nucleic acid sequence contained in the sequence of the DNA probe is:

[0016] 5'-TTTTTTACCGATTTGACACTAAACTTTGTAATTTTTTTTCCCCCCCCCCCCCCC CCCCCC-3',

[0017] According to the method of the present invention, the average particle size of the silver nanoparticles is 30 to 40 nm.

[0018] The method of the present invention is a method for producing monovalent copper ions, such as: copper sulfate pentahydrate (CuSO4·5H2O), tris(3-hydroxypropyltriazolemethyl)amine (THPTA) and sodium ascorbate, until the final concentration ratio is 0.1mM:0.5mM:5mM, and the reaction is carried out at room temperature in the dark for 1-2 hours.

[0019] After CD34-functionalized magnetic beads capture hematopoietic stem cells, they are separated and enriched by magnetic attraction.

[0020] The magnetic beads were activated with NHS / EDC.

[0021] The specific parameters of the electrochemical deposition process of the method of the present invention are: deposition at a potential of -1.2V for 8 minutes; the specific parameters of the differential pulse voltammetry used are: potential scanning range of -0.1V to 0.4V, amplitude of 25mV, and frequency of 15Hz.

[0022] Beneficial effects achieved by the technical solution of the present invention:

[0023] Hematopoietic stem cells are a type of adult stem cells with the potential for self-renewal and differentiation. They can form all cells in the blood system during the hematopoietic process, and can even differentiate into other non-hematopoietic cell types when properly stimulated. Currently, the main methods for detecting and collecting hematopoietic stem cells include colony culture, flow cytometer detection, etc., but these methods are susceptible to interference, and the experimental technology requirements and costs are relatively high. The present invention targets hematopoietic stem cells and is an electrochemical method for detecting biomarkers. After hematopoietic stem cells are efficiently labeled by proximity oxidation labeling, signal amplification is performed by click chemistry reaction, and then electrochemical detection of stem cells is performed. The presence of stem cells can be detected more sensitively through the signal response and changes of the current, and it also shows good stability in complex environments, and is expected to be efficiently captured and detected in clinical practice.

[0024] Compared with the known methods, the advantages of the technical solution of the present invention are:

[0025] (1) Antibody-modified functional magnetic beads have good selectivity and high capture efficiency, and can easily and efficiently enrich stem cells even in complex serum environments.

[0026] (2) Proximity oxidation labeling technology can quickly and efficiently label highly active tyramide free radical intermediates on electron-rich protein residues on the cell surface. Since it is independent of specific proteins, it can provide more signal probe coupling sites on the cell surface, thereby improving signal coupling efficiency and detection sensitivity.

[0027] (3) The copper-catalyzed azide-alkyne cycloaddition reaction is an excellent bioorthogonal click chemistry reaction with the advantages of high efficiency, high selectivity, and wide substrate adaptability. It can quickly form a stable triazole moiety similar to an amide bond under mild conditions. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 Schematic diagram of the technical scheme for detecting hematopoietic stem cells;

[0029] Figure 2 This is a graph of electrochemical signal results obtained by using the method of the present invention to detect hematopoietic stem cells;

[0030] Figure 3 The graph shows the electrochemical quantitative results of hematopoietic stem cell concentrations at various levels;

[0031] Figure 4 Figure 2 shows the electrochemical analysis results of several cell types. DETAILED DESCRIPTION

[0032] The technical solution of the present invention is described in detail below with reference to the accompanying drawings. The embodiments of the present invention are intended only to illustrate the technical solution of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that the technical solution of the invention may be modified or replaced with equivalents without departing from the spirit and scope of the technical solution of the present invention, and all such modifications or equivalents should be included in the scope of the claims of the present invention.

[0033] Figure 1 A roadmap for the technical solution for detecting hematopoietic stem cells. Specific methods include:

[0034] (a) Preparation of capture magnetic beads: 50-60 μL of carboxylated magnetic beads were rinsed thoroughly with 1 mL of 10 mM PBS (pH 7.5), followed by magnetic separation and discarding the solution. This was repeated three times. The beads were then resuspended in 450-500 μL of a 0.22 M EDC / NHS mixture for activation. After incubation at room temperature for 30-40 minutes, the beads were magnetically rinsed three times to obtain carboxylated magnetic beads with coupling / covalent binding capabilities. The beads were then resuspended in 1 mL of PBS for later use. The activated beads were then incubated with 10-15 μL of CD34 antibody at room temperature for 2-2.5 hours to obtain functionalized capture magnetic beads.

[0035] (b) Hematopoietic stem cell capture: The cell suspension was collected by centrifugation at 1000 rpm, rinsed 2-3 times with PBS, and resuspended. The capture magnetic beads (100 μL) prepared in step (a) were mixed with the hematopoietic stem cell solution and incubated at room temperature for 2-2.5 hours.

[0036] (c) The specific process of antibody binding is as follows: 200 μL of CD45 antibody dilution (1:1000) was added to the hematopoietic stem cell solution captured by the magnetic beads, and the reaction was carried out at 25°C for 2-2.5 hours for antigen-antibody binding. After washing twice with PBS, 200 μL of HRP-conjugated secondary antibody dilution (1:1000) was added and incubated for 1 hour for binding of the secondary antibody.

[0037] (d) The specific process of proximity oxidation labeling is as follows: after magnetic rinsing twice, the sample is resuspended in 100 μL PBS, and then 200 μL of alkyne tyramine solution (1 mM) is added. Then, hydrogen peroxide (H2O2) is added to make the final concentration reach 1 mM. The reaction is carried out at room temperature in the dark for 15-30 minutes to perform signal labeling. Finally, the sample is washed twice with PBS to remove unbound alkyne tyramine.

[0038] (e) The specific process of click chemistry coupling is as follows: 200 μL of N3-DNA@AgNPs solution was added to (d), and then copper sulfate pentahydrate (CuSO4·5H2O), tris(3-hydroxypropyltriazolemethyl)amine (THPTA) and sodium ascorbate were added in sequence until the final concentration ratio was 0.1 mM:0.5 mM:5 mM. The reaction was carried out at room temperature in the dark for 1-2 hours, and then the supernatant was washed twice.

[0039] (f) Electrochemical detection: The specific process is as follows: 100-120 μL of 1-2 M nitric acid solution is added to (e) and reacted at room temperature for 0.5-1 h to fully acidify the silver nanoparticles, thereby releasing a large amount of silver ions. Finally, the acid-hydrolyzed reaction solution is mixed with 3.8-3.9 mL of 0.5-1 M sodium acetate solution as the electrolyte. Electrochemical measurements are performed using a traditional three-electrode system. The three-electrode system includes a graphite electrode as the working electrode, a saturated calomel electrode as the reference electrode, and a platinum wire as the counter electrode. After silver ions are enriched on the graphite electrode through an electrochemical deposition process, the electrochemical signal is measured by differential pulse voltammetry.

[0040] Wherein: the DNA chain sequence used in step (e) is: 5'-N3-TTTTTACCGATTTGACACTAAA CTTTGTAATTTTTTTTCCCCCCCCCCCCCCCCCCCC-3', denoted as: N3-DNA.

[0041] The specific process of N3-DNA@AgNPs preparation is as follows: 3-5 μL of 100-105 μM N3-DNA is added to 1200-1500 μL of silver nanoparticle solution and incubated at room temperature for 1-1.5 hours to allow DNA to adsorb on the silver nanoparticles; then 10-15 μL of 500-510 mM trisodium citrate buffer (pH = 3) is added to make its final concentration reach 5-10 mM, and the reaction is again carried out at room temperature for 30-45 minutes; then 150-200 μL of 200 mM PB buffer (pH = 7.4) is added to neutralize the pH of the silver nanoparticle solution, incubated at room temperature for 10-15 minutes, and then centrifuged at 14000 rpm at 4°C for 20-30 minutes to remove unbound DNA chains; 1-2 mL Resuspend the solution in 10-15mM PB buffer (pH 7.4) and repeat centrifugation three times. After washing, disperse the N3-DNA-coupled silver nanoparticle complex in 1-1.5mL of 10mM PB buffer (pH 7.4) and store at 4°C for subsequent use. Protect from light throughout the reaction. To prepare 200mM PB buffer (pH 7.4), prepare Solution A by dissolving 3.5-3.6g of disodium hydrogen phosphate dihydrate in 100-101mL of deionized water and Solution B by dissolving 3.1-3.2g of sodium dihydrogen phosphate dihydrate in 100-101mL of deionized water. Before use, thoroughly mix Solution A and Solution B in a certain ratio and store the prepared PB buffer at 4°C for use.

[0042] The specific parameters of the electrochemical deposition process used in step (f) are: deposition at a potential of -1.2 V for 8 minutes; the specific parameters of the differential pulse voltammetry used are: potential scanning range of -0.1 V to 0.4 V, amplitude of 25 mV, and frequency of 15 Hz.

[0043] Example 1 Electrochemical Analysis and Detection of Hematopoietic Stem Cells

[0044] The steps are as follows:

[0045] (a) Preparation of capture magnetic beads: 50-60 μL of carboxylated magnetic beads were rinsed thoroughly with 1 mL of 10 mM PBS (pH 7.5), followed by magnetic separation and discarding the solution. This was repeated three times. The beads were then resuspended in 450-500 μL of a 0.22 M EDC / NHS mixture for activation. After incubation at room temperature for 30-40 minutes, the beads were magnetically rinsed three times to obtain carboxylated magnetic beads with coupling / covalent binding capabilities. The beads were then resuspended in 1 mL of PBS for later use. The activated beads were then incubated with 10-15 μL of CD34 antibody at room temperature for 2-2.5 hours to obtain functionalized capture magnetic beads.

[0046] (b) Hematopoietic stem cell capture: The cell suspension was collected by centrifugation at 1000 rpm, rinsed 2-3 times with PBS, and resuspended. The capture magnetic beads (100 μL) prepared in step (a) were mixed with the hematopoietic stem cell solution and incubated at room temperature for 2-2.5 hours.

[0047] (c) The specific process of antibody binding is as follows: 200 μL of CD45 antibody dilution (1:1000) is added to the hematopoietic stem cell solution captured by the magnetic beads, and the reaction is carried out at 25°C for 2-2.5 hours for antigen-antibody binding. After washing twice with PBS, 200 μL of HRP-conjugated secondary antibody dilution (1:1000) is added and incubated for 1 hour for binding of the secondary antibody.

[0048] (d) Proximity oxidation labeling: After magnetic rinsing twice, the sample was resuspended in 100 μL PBS, 200 μL of alkyne tyramine solution (1 mM) was added, and then H2O2 was added to reach a final concentration of 1 mM. The reaction was incubated at room temperature in the dark for 15–30 min for signal labeling, and the sample was washed twice with PBS to remove unbound tyramine.

[0049] (e) The specific process of click chemistry is as follows: add 200 μL N3-DNA@AgNPs solution to (d), and then add copper sulfate pentahydrate (CuSO4·5H2O), tris(3-hydroxypropyltriazolemethyl)amine (THPTA) and sodium ascorbate in sequence until the final concentration ratio is 0.1 mM:0.5 mM:5 mM. React at room temperature in the dark for 1-2 hours, then wash twice and remove the supernatant.

[0050] (f) Electrochemical detection: 100–120 μL of 1–2 M nitric acid solution was added to (e), and the reaction was allowed to proceed at room temperature for 0.5–1 h. The acid-hydrolyzed solution was then mixed with 0.5–1 M sodium acetate solution. Silver ions were concentrated onto the graphite electrode by stripping voltammetry, and the electrochemical signal of the silver nanoparticles was detected by differential pulse voltammetry in the range of -0.1 V to 0.4 V.

[0051] Figure 2 shows that when used to detect 1×10 7 The electrochemical signal obtained when a hematopoietic stem cell is generated. Figure 2 As shown, when there are 1×10 7 When there are hematopoietic stem cells, the solution has an obvious current peak near the voltage of 0.15V (curve a); however, when there are no hematopoietic stem cells (curve b) or no alkyned tyramine (curve c), the solution has only a very small background signal near this voltage, which verifies that the method of this embodiment can be used to analyze and detect hematopoietic stem cells.

[0052] Example 2 Electrochemical Quantitative Detection of Hematopoietic Stem Cells

[0053] The steps are as follows:

[0054] (a) Preparation of capture magnetic beads: 50-60 μL of carboxylated magnetic beads were rinsed thoroughly with 1 mL of 10 mM PBS (pH 7.5), followed by magnetic separation and discarding the solution. This was repeated three times. The beads were then resuspended in 450-500 μL of a 0.22 M EDC / NHS mixture for activation. After incubation at room temperature for 30-40 minutes, the beads were magnetically rinsed three times to obtain carboxylated magnetic beads with coupling / covalent binding capabilities. The beads were then resuspended in 1 mL of PBS for later use. The activated beads were then incubated with 10-15 μL of CD34 antibody at room temperature for 2-2.5 hours to obtain functionalized capture magnetic beads.

[0055] (b) Hematopoietic stem cell capture: Different amounts of cell suspension were collected by centrifugation at 1000 rpm, washed 2-3 times with PBS, and then resuspended. The capture magnetic beads (100 μL) prepared in step (a) were mixed with different amounts of hematopoietic stem cell solution and incubated at room temperature for 2-2.5 hours.

[0056] (c) The specific process of antibody binding is as follows: 200 μL of CD45 antibody dilution (1:1000) is added to the hematopoietic stem cell solution captured by the magnetic beads, and the reaction is carried out at 25°C for 2-2.5 hours for antigen-antibody binding. After washing twice with PBS, 200 μL of HRP-conjugated secondary antibody dilution (1:1000) is added and incubated for 1 hour for binding of the secondary antibody.

[0057] (d) Proximity oxidation labeling: After magnetic rinsing twice, the sample was resuspended in 100–200 μL PBS, to which 100–200 μL of alkyne tyramine solution (1 mM) was added, followed by addition of H 2 O 2 to a final concentration of 1 mM. The sample was reacted at room temperature in the dark for 15–30 min for signal labeling, and finally washed twice with PBS to remove unbound tyramine.

[0058] (e) The specific process of click chemistry is as follows: add 200-250 μL N3-DNA@AgNPs solution to (d), and then add copper sulfate pentahydrate (CuSO4·5H2O), tris(3-hydroxypropyltriazolemethyl)amine (THPTA) and sodium ascorbate in sequence until the final concentration ratio is 0.1 mM:0.5 mM:5 mM. React at room temperature in the dark for 1-2 hours, then wash twice and remove the supernatant.

[0059] (f) Electrochemical detection: 100–120 μL of 1–2 M nitric acid solution was added to (e), and the mixture was reacted at room temperature for 0.5–1 h. The acid-hydrolyzed solution was then mixed with 0.5–1 M sodium acetate solution. Silver ions were concentrated onto the graphite electrode by stripping voltammetry, and the electrochemical signal of the silver nanoparticles was detected by differential pulse voltammetry in the range of -0.1 V to 0.4 V.

[0060] Subsequently, a series of electrochemical analyses were performed on hematopoietic stem cells at different concentrations. The electrochemical quantitative results of different numbers of stem cells are shown in Figure 2. Figure 3 As shown in Figure 2, the peak current increases with the increase in the number of hematopoietic stem cells. When the number of cells reaches 1×10 7 The current value tends to saturate when the number of stem cells increases, which is consistent with the expected result. The increase in the number of stem cells increases the content of alkyne tyramine labeled on their surface, increasing the number of N3-DNA@AgNPs clicked on, thereby enhancing the electrochemical signal.

[0061] Example 3 Electrochemical Specificity Analysis of Hematopoietic Stem Cells

[0062] (a) Preparation of capture magnetic beads: 50-60 μL of carboxylated magnetic beads were rinsed thoroughly with 1 mL of 10 mM PBS (pH 7.5), followed by magnetic separation and discarding the solution. This was repeated three times. The beads were then resuspended in 450-500 μL of a 0.22 M EDC / NHS mixture for activation. After incubation at room temperature for 30-40 minutes, the beads were magnetically rinsed three times to obtain carboxylated magnetic beads with coupling / covalent binding capabilities. The beads were then resuspended in 1 mL of PBS for later use. The activated beads were then incubated with 10-15 μL of CD34 antibody at room temperature for 2-2.5 hours to obtain functionalized capture magnetic beads.

[0063] (b) Hematopoietic stem cell capture: Cell suspensions of different types were collected by centrifugation at 1000 rpm, washed 2-3 times with PBS, and then resuspended. The capture magnetic beads (100 μL) prepared in step (a) were mixed with the hematopoietic stem cell solution and incubated at room temperature for 2-2.5 hours.

[0064] (c) The specific process of antibody binding is as follows: 200 μL of CD45 antibody dilution (1:1000) is added to the hematopoietic stem cell solution captured by the magnetic beads, and the reaction is carried out at 25°C for 2-2.5 hours for antigen-antibody binding. After washing twice with PBS, 200 μL of HRP-conjugated secondary antibody dilution (1:1000) is added and incubated for 1 hour for binding of the secondary antibody.

[0065] (d) Proximity oxidation labeling: After magnetic rinsing twice, the sample was resuspended in 100 μL PBS, 200 μL of alkyne tyramine solution (1 mM) was added, and then a certain amount of H2O2 was added to make the final concentration reach 1 mM. The reaction was incubated at room temperature in the dark for 15-30 minutes for signal labeling, and finally washed twice with PBS to remove unbound tyramine.

[0066] (e) The specific process of click chemistry is as follows: add 200 μL N3-DNA@AgNPs solution to (d), and then add copper sulfate pentahydrate (CuSO4·5H2O), tris(3-hydroxypropyltriazolemethyl)amine (THPTA) and sodium ascorbate in sequence until the final concentration ratio is 0.1:mM:0.5mM:5mM. React at room temperature in the dark for 1-2 hours, then wash twice and remove the supernatant.

[0067] (f) Electrochemical detection: 100–120 μL of 1–2 M nitric acid solution was added to (e), and the reaction was allowed to proceed at room temperature for 0.5–1 h. The acid-hydrolyzed solution was then mixed with 0.5–1 M sodium acetate solution. Silver ions were concentrated onto the graphite electrode by stripping voltammetry, and the electrochemical signal of the silver nanoparticles was detected by differential pulse voltammetry in the range of -0.1 V to 0.4 V.

[0068] The specific detection of the method of this embodiment was verified. The electrochemical analysis results of HK-2, MCF-10A and several hematopoietic stem cell types are shown in Figure 2. Figure 4 As shown in Figure 3, a high electrochemical signal was detected when the detection system contained hematopoietic stem cells, while only a low electrochemical signal was detected when the detection system contained normal kidney cells (HK-2) or breast epithelial cells (MCF-10A). This indicates that our method has excellent detection specificity.

Claims

1. A hematopoietic stem cell detection method based on proximity click labeling, characterized in that: include: First, use CD45 functionalized magnetic beads to capture hematopoietic stem cells. Then add the secondary antibody coupled with horseradish peroxidase to bind to the CD34 antibody retained on the stem cell membrane. Then, in the presence of hydrogen peroxide, the added alkyne-tyramine is catalyzed by horseradish peroxidase to form a highly active tyramine free radical intermediate, which then labels a large number of electron-rich amino acid residues on proximal membrane proteins and exposes the terminal alkyne group. Then, a DNA probe with an N3 group modified at the 5' end and silver nanoparticles at the 3' end was added. Under the catalysis of monovalent copper ions, the DNA probe reacted with the alkynyl groups on the cell membrane surface to couple the DNA probe to the cell membrane surface. Finally, a large amount of silver ions were obtained after the silver nanoparticles were hydrolyzed in nitric acid. After the silver ions were enriched on the graphite electrode through the electrochemical deposition process, the electrochemical signal was measured by differential pulse voltammetry.

2. The hematopoietic stem cell detection method based on proximity click labeling according to claim 1, characterized in that: The nucleic acid sequence contained in the sequence of the DNA probe is: 5'-TTTTTACCGATTTGACACTAAACTT TGTAATTTTTTTTCCCCCCCCCCCCCCCCCCCC-3'.

3. The hematopoietic stem cell detection method based on proximity click labeling according to claim 1, characterized in that: The average particle size of the silver nanoparticles is 30-40 nm.

4. The hematopoietic stem cell detection method based on proximity click labeling according to claim 1, characterized in that: The method for generating monovalent copper ions is as follows: copper sulfate pentahydrate, tris (3-hydroxypropyltriazolemethyl) amine and sodium ascorbate are reacted at room temperature in the dark for 1-2 hours.

5. The hematopoietic stem cell detection method based on proximity click labeling according to claim 4, characterized in that: The final concentration ratio of copper sulfate pentahydrate, tris(3-hydroxypropyltriazolylmethyl)amine and sodium ascorbate is 0.1 mM:0.5 mM:5 mM.

6. The hematopoietic stem cell detection method based on proximity click labeling according to claim 1, characterized in that: The magnetic beads are activated by NHS / EDC.

7. The hematopoietic stem cell detection method based on proximity click labeling according to claim 1, characterized in that: The specific parameters of the electrochemical deposition process are: deposition at a potential of -1.2 V for 8 minutes.

8. The hematopoietic stem cell detection method based on proximity click labeling according to claim 1, characterized in that: The potential scan range was -0.1 V to 0.4 V, with an amplitude of 25 mV and a frequency of 15 Hz.

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