Electrochemical detection of hematopoietic stem cells based on tyramide signal amplification

Abstract

A method for electrochemical detection of hematopoietic stem cells based on tyramide signal amplification comprises the following steps: first, a gold electrode functionalized with a CD45 antibody is formed into CD45@GE, and then CD45@GE is co-incubated with hematopoietic stem cells. CD45-positive hematopoietic stem cells are captured on the surface of the functionalized gold electrode through immune recognition. Next, G-quadruplex / heme functionalized with a CD34 antibody is anchored to the electrode surface by recognizing the CD34 protein on the surface of the hematopoietic stem cells captured on the electrode. Then, the G-quadruplex / heme catalyzes the enrichment of a large number of tyramide-coated gold nanoparticles on the cell membranes of the hematopoietic stem cells. These gold nanoparticles can then serve as seeds for the deposition of silver ions. Finally, by measuring the electrochemical signal of the silver deposited on the electrode surface, efficient detection of hematopoietic stem cells can be achieved.

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 are multipotent, self-renewing cells that can give rise to all cell types found in the blood, including red blood cells, white blood cells, and platelets. The levels of these stem cells play a role in blood cancers, autoimmune diseases, hematopoiesis, and cell therapy. They are the longest-studied and most intensive type of adult stem cell, providing valuable insights into various stem cell types, including cancer stem cells. Commonly used hematopoietic stem cell markers include CD34, CD38, and CD45, which are highly expressed in over 95% of hematopoietic stem cells.

[0003] Currently, conventional quantitative methods for hematopoietic stem cell detection, such as enzyme-linked immunosorbent assay (ELISA) and flow cytometry, require complex sample pretreatment and specialized analytical instruments and are highly dependent on operator experience. Furthermore, flow cytometry has extremely high requirements for cell samples and cumbersome data processing, while ELISA is susceptible to interference, resulting in false positives and numerous interfering factors.

[0004] G-quadruplex is a DNA secondary structure with four nucleotide chains connected by one or more G-rich sequences through intramolecular or intermolecular Hoogsteen hydrogen bonds. + 、Na + and NH4 + The G-quadruplex-heme complex exhibits peroxidase catalytic activity and is therefore known as a G-quadruplex / heme deoxyribozyme (G4 / hemin DNAzyme), also known as a G-quadruplex peroxidase. Currently, as an artificial enzyme or catalyst, G-quadruplex peroxidase is used in a variety of fields, including bioanalysis, molecular machines, and DNA sensors. Compared to traditional catalytic enzymes, it offers advantages such as low cost, ease of operation, and high stability.

[0005] Electrochemical methods are becoming increasingly mature in molecular recognition, detection, and analysis, as well as in biosensing applications. This is particularly true in the life sciences for the detection of disease-related biomarkers such as proteins, nucleic acids, and cells. Common electrochemical methods include cyclic voltammetry (CV), differential pulse voltammetry (DPV), square wave voltammetry (SWV), linear sweep voltammetry (LSV), and electrochemical impedance spectroscopy (EIS). DPV, which measures the current before the potential change to minimize the effects of charging current, offers high sensitivity and a very low detection limit. SWV, on the other hand, boasts high sensitivity and high efficiency. Currently, it is widely used in industries such as industry, agriculture, the environment, medicine, food science, and the life sciences. Electrochemical methods offer advantages such as high sensitivity, strong specificity, and easy instrumentation and learning. Combined with the emergence of new functional nanomaterials and evolving scientific theories, they can be applied to a wide range of fields, including life science research, health monitoring, and environmental monitoring. Summary of the Invention

[0006] One object of the present invention is to provide a method for electrochemical detection of hematopoietic stem cells based on tyramide signal amplification, thereby realizing the detection of hematopoietic stem cells by electrochemical method.

[0007] Another object of the present invention is to provide a method for electrochemical detection of hematopoietic stem cells based on tyramide signal amplification, so as to efficiently detect hematopoietic stem cells and improve detection sensitivity.

[0008] Another object of the present invention is to provide a method for electrochemical detection of hematopoietic stem cells based on tyramide signal amplification, which implements quantitative detection of hematopoietic stem cells in an electrochemical manner.

[0009] A method for electrochemical detection of hematopoietic stem cells based on tyramide signal amplification, comprising:

[0010] First, CD45 antibody was functionalized into gold electrode (GE), which was recorded as: CD45@GE.

[0011] Then, CD45@GE was co-incubated with hematopoietic stem cells, and CD45-positive hematopoietic stem cells were captured on the surface of the functionalized gold electrode through immune recognition.

[0012] Then, the CD34 antibody-functionalized G-quadruplex / heme is anchored to the electrode surface by recognizing the CD34 protein on the surface of hematopoietic stem cells captured on the electrode.

[0013] Then, G-quadruplex / heme catalyzes the accumulation of a large number of tyramine-coated gold nanoparticles on the cell membrane of hematopoietic stem cells. These gold nanoparticles can then serve as seeds for the deposition of silver ions.

[0014] Finally, quantitative detection of hematopoietic stem cells can be achieved by measuring the electrochemical signal of silver deposited on the electrode surface.

[0015] The method of the present invention first modifies a gold electrode with a calix[4]arene (pSC4) having an electron-rich cavity structure. The specific method is as follows: the surface of the gold electrode is modified through gold-thiol interaction.

[0016] The two nucleic acid sequences of the G-quadruplex are as follows:

[0017] 5'-SH-AAAAACCGTGAGTGTAGCTTGAGATCTGTGAGGGAAGGGA-3', and 5'-AGGGAAGGGACACAGATCTCAAGCTACA-3'.

[0018] According to the method of the present invention, the average particle size of gold nanoparticles (AuNPs) is 30 to 40 nm.

[0019] The method of the present invention prepares tyramide-coated gold nanoparticles through electrostatic binding. The specific process is: a tyramide solution (10-15 μL, 100 μM) is added to a 5 mL solution of AuNPs, the mixture is gently shaken for 12-14 hours, and the solution is then centrifuged twice (2000×g, 10-15 minutes) to obtain tyramide-coated gold nanoparticles.

[0020] The method of the present invention specifically involves the following steps for detecting silver electrochemical signals: first, a gold electrode is incubated with a heme solution (2 mM, 150 to 200 μL) at room temperature in the dark for 20 to 30 minutes; then, the electrode is incubated with the prepared tyramine-coated gold nanoparticles at room temperature in the dark for 1 to 1.5 hours; finally, a mixed solution (150 to 200 μL) of AgNO3 (0.5 mM) and ascorbic acid (0.25 mM) is added and incubated at room temperature in the dark for 7 to 10 minutes; and then, the electrochemical signal is collected.

[0021] In the method of the present invention, electrochemical signals are collected using differential pulse voltammetry, with specific parameters as follows: potential scanning range of -0.2V to 0.5V, amplitude of 25mV, and frequency of 15Hz.

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

[0023] This method proposes an electrochemical method based on the recognition of hematopoietic stem cell surface markers CD34 and CD45, combined with tyramide signal amplification technology, for highly sensitive detection of hematopoietic stem cells. Compared to these methods, the electrochemical technology used in this method is more convenient and sensitive, and it achieves precise identification and highly sensitive electrochemical analysis of hematopoietic stem cells without the need for complex sample processing or sophisticated instrumentation.

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

[0025] (1) The antibody-functionalized gold electrode constructed based on host-guest recognition has good selectivity and high capture efficiency, and can simply and efficiently enrich hematopoietic stem cells in a complex serum environment.

[0026] (2) An improved tyramide signal amplification strategy was created, in which horseradish peroxidase, the main component of signal amplification, was replaced by the biomimetic enzyme G-quadruplex / heme, thus avoiding the drawbacks caused by the use of natural enzymes in traditional methods.

[0027] (3) Thanks to its effective signal enhancement capability, it can detect hematopoietic stem cells in samples with high sensitivity, thereby meeting the research needs of its related mechanisms and biological applications. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0029] Figure 2 Graphs showing impedance detection results after the electrode of the present invention is co-incubated with various substances;

[0030] Figure 3 This is a graph of electrochemical signal results obtained by using the method of the present invention to detect hematopoietic stem cells;

[0031] Figure 4 The graph shows the electrochemical detection results of hematopoietic stem cell concentrations at various orders of magnitude. 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 antibody-functionalized gold electrodes. The following steps were performed: First, the unmodified gold electrode was polished with 3000-grit and then 5000-grit sandpaper until the surface scratches were uniform and smooth. The electrode surface was then polished with 1μm and 0.03μm aluminum powders, respectively. The gold electrode was then ultrasonically cleaned in anhydrous ethanol and then in ultrapure water for 3–5 minutes. After drying with nitrogen, 40–50μL of freshly prepared piranha solution (98% H₂SO₄:30% H₂O₂ = 3:1) was added dropwise to the electrode surface. The solution was allowed to stand for 3–5 minutes before being rinsed with ultrapure water. Finally, the gold electrode was activated and subjected to cyclic voltammetry for 20 cycles in the potential range of 0–1.6 V using 0.5 M H₂SO₄ as the electrolyte. Then, the electrodes were incubated with 1 mM pSC4 overnight, followed by treatment in 1 mM MCH solution for 1 hour to block nonspecific sites, and finally incubated with CD45 antibody solution at 4°C for 2 hours.

[0035] (b) Preparation of CD34 antibody-functionalized G-quadruplex / heme: First, two single chains (1 μL, 100 μM) of the G-quadruplex were simultaneously added to a solution of the thiol reducing agent TCEP (90-100 μL, 1 mM) to break the disulfide bonds in the G-quadruplex. The two chains were then incubated at 37°C for 2 hours. Simultaneously, the CD34 antibody solution was mixed with the coupling agent SMCC solution and incubated at room temperature for 2-3 hours. Finally, the CD34 antibody solution was added to the G-quadruplex solution and incubated at 4°C for 12-14 hours.

[0036] (c) Tyramide-coated gold nanoparticles (AuNPs) were prepared by electrostatic binding. The following steps were performed: 10–15 μL of 100 μM tyramide solution was added to a 5 mL solution of AuNPs. The mixture was gently shaken for 12–14 hours, and the solution was centrifuged twice (2000 × g for 10–15 minutes) to obtain the tyramide-coated gold nanoparticle solution, which was stored at 4°C for later use.

[0037] (d) The process of cell capture by antibody-functionalized gold electrode is as follows: first, the antibody-functionalized gold electrode is treated with BSA solution (2%, 100-200 μL) for 30 minutes to avoid nonspecific adsorption, then the electrode is incubated with 50 μL of hematopoietic stem cells of different concentrations at 37°C for 2-2.5 hours, and then incubated with the prepared CD34 antibody-functionalized G-quadruplex at 37°C for 1-2 hours.

[0038] (e) Detection of silver electrochemical signals. The specific process is as follows: first, the gold electrode is incubated with a heme solution (2 mM, 150-200 μL) at room temperature in the dark for 20-30 minutes, then incubated with the prepared tyramide-coated gold nanoparticles at room temperature in the dark for 1-1.5 hours, and finally, a mixed solution of AgNO3 (0.5 mM) and ascorbic acid (0.25 mM) (150-200 μL) is added and incubated at room temperature in the dark for 7-10 minutes. The electrochemical signals are then collected by differential pulse voltammetry.

[0039] The sequence of the G-quadruplex used in step (b) is:

[0040] 5'-SH-AAAAACCGTGAGTGTAGCTTGAGATCTGTGAGGGAAGGGA-3' and 5'-AGGGAAGGGACACAGATCTCAAGCTACA-3'

[0041] The experimental conditions used in the differential pulse voltammetry (DPV) measurement in step (e) are: potential scanning range, -0.2 to 0.5 V; amplitude 25 mV; frequency 15 Hz.

[0042] Example 1 Assembly of Antibody-Functionalized Gold Electrodes and Verification of Hematopoietic Stem Cell Capture

[0043] The steps are as follows:

[0044] (a) Preparation of antibody-functionalized gold electrodes. The following steps were performed: First, the unmodified gold electrode was polished with 3000-grit and then 5000-grit sandpaper until the surface scratches were uniform and smooth. The electrode surface was then polished with 1μm and 0.03μm aluminum powders, respectively. The gold electrode was then ultrasonically cleaned in anhydrous ethanol and then in ultrapure water for 3–5 minutes. After drying with nitrogen, 40–50μL of freshly prepared piranha solution (98% H₂SO₄:30% H₂O₂ = 3:1) was added dropwise to the electrode surface. The solution was allowed to stand for 3–5 minutes before being rinsed with ultrapure water. Finally, the gold electrode was activated and subjected to cyclic voltammetry for 20 cycles in the potential range of 0–1.6 V using 0.5 M H₂SO₄ as the electrolyte. Then, the electrodes were incubated with 1 mM pSC4 overnight, followed by treatment in 1 mM MCH solution for 1 hour to block nonspecific sites, and finally incubated with CD45 antibody solution at 4°C for 2 hours.

[0045] (b) Preparation of CD34 antibody-functionalized G-quadruplex / heme: First, two single chains (1 μL, 100 μM) of the G-quadruplex were simultaneously added to a solution of the thiol reducing agent TCEP (90-100 μL, 1 mM) to break the disulfide bonds in the G-quadruplex. The two chains were then incubated at 37°C for 2 hours. Simultaneously, the CD34 antibody solution was mixed with the SMCC solution and incubated at room temperature for 2-3 hours. Finally, the CD34 antibody solution was added to the G-quadruplex solution and incubated at 4°C for 12-14 hours.

[0046] (c) Tyramide-coated gold nanoparticles were prepared by electrostatic binding. The following steps were performed: 10–15 μL of 100 μM tyramide solution was added to a 5 mL solution of AuNPs. The mixture was gently shaken for 12–14 h, and the solution was centrifuged twice (2000 × g for 10–15 min) to obtain a tyramide-coated gold nanoparticle solution, which was stored at 4°C for later use.

[0047] (d) The process of cell capture by antibody-functionalized gold electrode is as follows: first, the antibody-functionalized gold electrode is treated with BSA solution (2%, 100-200 μL) for 30 minutes to avoid nonspecific adsorption, then the electrode is incubated with 50 μL of hematopoietic stem cells of different concentrations at 37°C for 2-2.5 hours, and then incubated with the prepared CD34 antibody-functionalized G-quadruplex at 37°C for 1-2 hours.

[0048] (e) Detection of silver electrochemical signals. The specific process is as follows: first, the gold electrode is incubated with a heme solution (2 mM, 150-200 μL) at room temperature in the dark for 20-30 minutes, then incubated with the prepared tyramide-coated gold nanoparticles at room temperature in the dark for 1-1.5 hours, and finally, a mixed solution of AgNO3 (0.5 mM) and ascorbic acid (0.25 mM) (150-200 μL) is added and incubated at room temperature in the dark for 7-10 minutes. The electrochemical signals are then collected by differential pulse voltammetry.

[0049] The results are as follows Figure 2 The impedance of the electrode modified with pSC4 (curve b), MCH (curve c), CD45 antibody (curve d), and BSA (curve e) is greater than that of the bare gold electrode (a) and shows a gradually increasing trend, which proves the successful assembly of the antibody-functionalized gold electrode. After co-incubation with hematopoietic stem cells (curve f), the impedance increase proves that our functionalized electrode can successfully capture hematopoietic stem cells. The impedance increase after adding CD34 antibody proves that CD34 antibody can capture and identify hematopoietic stem cells enriched on the electrode.

[0050] Example 2 Electrochemical Quantitative Detection of Hematopoietic Stem Cells

[0051] The steps are as follows:

[0052] (a) Preparation of antibody-functionalized gold electrodes. The following steps were performed: First, the unmodified gold electrode was polished with 3000-grit and then 5000-grit sandpaper until the surface scratches were uniform and smooth. The electrode surface was then polished with 1μm and 0.03μm aluminum powders, respectively. The gold electrode was then ultrasonically cleaned in anhydrous ethanol and then in ultrapure water for 3–5 minutes. After drying with nitrogen, 40–50μL of freshly prepared piranha solution (98% H₂SO₄:30% H₂O₂ = 3:1) was added dropwise to the electrode surface. The solution was allowed to stand for 3–5 minutes before being rinsed with ultrapure water. Finally, the gold electrode was activated and subjected to cyclic voltammetry for 20 cycles in the potential range of 0–1.6 V using 0.5 M H₂SO₄ as the electrolyte. Then, the electrodes were incubated with 1 mM pSC4 overnight, followed by treatment in 1 mM MCH solution for 1 hour to block nonspecific sites, and finally incubated with CD45 antibody solution at 4°C for 2 hours.

[0053] (b) Preparation of CD34 antibody-functionalized G-quadruplex / heme: First, two single chains (1 μL, 100 μM) of the G-quadruplex were simultaneously added to a solution of the thiol reducing agent TCEP (90-100 μL, 1 mM) to break the disulfide bonds in the G-quadruplex. The two chains were then incubated at 37°C for 2 hours. Simultaneously, the CD34 antibody solution was mixed with the SMCC solution and incubated at room temperature for 2-3 hours. Finally, the CD34 antibody solution was added to the G-quadruplex solution and incubated at 4°C for 12-14 hours.

[0054] (c) Tyramide-coated gold nanoparticles were prepared by electrostatic binding. The following steps were performed: 10–15 μL of 100 μM tyramide solution was added to a 5 mL solution of AuNPs. The mixture was gently shaken for 12–14 h, and the solution was centrifuged twice (2000 × g for 10–15 min) to obtain a tyramide-coated gold nanoparticle solution, which was stored at 4°C for later use.

[0055] (d) The process of cell capture by antibody-functionalized gold electrode is as follows: first, the antibody-functionalized gold electrode is treated with BSA solution (2%, 100-200 μL) for 30 minutes to avoid nonspecific adsorption, then the electrode is incubated with 50 μL of hematopoietic stem cells of different concentrations at 37°C for 2-2.5 hours, and then incubated with the prepared CD34 antibody-functionalized G-quadruplex at 37°C for 1-2 hours.

[0056] (e) Detection of silver electrochemical signals. The specific process is as follows: first, the gold electrode is incubated with a heme solution (2 mM, 150-200 μL) at room temperature in the dark for 20-30 minutes, then incubated with the prepared tyramide-coated gold nanoparticles at room temperature in the dark for 1-1.5 hours, and finally, a mixed solution of AgNO3 (0.5 mM) and ascorbic acid (0.25 mM) (150-200 μL) is added and incubated at room temperature in the dark for 7-10 minutes. The electrochemical signals are then collected by differential pulse voltammetry.

[0057] Figure 3 The present method was shown to be useful for detecting 5×10 6 As shown in the figure, when hematopoietic stem cells are present in the system, the solution has a clear current peak near 0.18V (curve a); however, in the blank control group, i.e., when no hematopoietic stem cells are present, the solution has only a small background emission peak near 0.18V (curve b).

[0058] Example 3 Electrochemical Specificity Analysis of Hematopoietic Stem Cells

[0059] (a) Preparation of antibody-functionalized gold electrodes. The following steps were performed: First, the unmodified gold electrode was polished with 3000-grit and then 5000-grit sandpaper until the surface scratches were uniform and smooth. The electrode surface was then polished with 1μm and 0.03μm aluminum powders, respectively. The gold electrode was then ultrasonically cleaned in anhydrous ethanol and then in ultrapure water for 3–5 minutes. After drying with nitrogen, 40–50μL of freshly prepared piranha solution (98% H₂SO₄:30% H₂O₂ = 3:1) was added dropwise to the electrode surface. The solution was allowed to stand for 3–5 minutes before being rinsed with ultrapure water. Finally, the gold electrode was activated and subjected to cyclic voltammetry for 20 cycles in the potential range of 0–1.6 V using 0.5 M H₂SO₄ as the electrolyte. Then, the electrodes were incubated with 1 mM pSC4 overnight, followed by treatment in 1 mM MCH solution for 1 hour to block nonspecific sites, and finally incubated with CD45 antibody solution at 4°C for 2 hours.

[0060] (b) Preparation of CD34 antibody-functionalized G-quadruplex / heme: First, two single chains (1 μL, 100 μM) of the G-quadruplex were simultaneously added to a solution of the thiol reducing agent TCEP (90-100 μL, 1 mM) to break the disulfide bonds in the G-quadruplex. The two chains were then incubated at 37°C for 2 hours. Simultaneously, the CD34 antibody solution was mixed with the SMCC solution and incubated at room temperature for 2-3 hours. Finally, the CD34 antibody solution was added to the G-quadruplex solution and incubated at 4°C for 12-14 hours.

[0061] (c) Tyramide-coated gold nanoparticles were prepared by electrostatic binding. The following steps were performed: 10–15 μL of 100 μM tyramide solution was added to a 5 mL solution of AuNPs. The mixture was gently shaken for 12–14 h, and the solution was centrifuged twice (2000 × g for 10–15 min) to obtain a tyramide-coated gold nanoparticle solution, which was stored at 4°C for later use.

[0062] (d) The process of cell capture by antibody-functionalized gold electrode is as follows: first, the antibody-functionalized gold electrode is treated with BSA solution (2%, 100-200 μL) for 30 minutes to avoid nonspecific adsorption, then the electrode is incubated with 50 μL of hematopoietic stem cells of different concentrations at 37°C for 2-2.5 hours, and then incubated with the prepared CD34 antibody-functionalized G-quadruplex at 37°C for 1-2 hours.

[0063] (e) Detection of silver electrochemical signals. The specific process is as follows: first, the gold electrode is incubated with a heme solution (2 mM, 150-200 μL) at room temperature in the dark for 20-30 minutes, then incubated with the prepared tyramide-coated gold nanoparticles at room temperature in the dark for 1-1.5 hours, and finally, a mixed solution of AgNO3 (0.5 mM) and ascorbic acid (0.25 mM) (150-200 μL) is added and incubated at room temperature in the dark for 7-10 minutes. The electrochemical signals are then collected by differential pulse voltammetry.

[0064] Figure 4 The figure shows how the final peak current of the solution changes with the concentration of hematopoietic stem cells. As can be seen from the figure, the electrochemical response detected increases with the number of hematopoietic stem cells. This is because as the number of hematopoietic stem cells captured on the electrode surface increases, more G-quadruplexes / heme bind to the cells, which in turn catalyzes the accumulation of more tyramine-coated gold nanoparticles on the hematopoietic stem cell membrane, allowing more silver ions to deposit on the gold electrode surface, generating a significant electrochemical signal.

Claims

1. A method for electrochemical detection of hematopoietic stem cells based on tyramide signal amplification, characterized in that: include: First, CD45 antibody was functionalized on gold electrode to form CD45@GE. Then, CD45@GE was co-incubated with hematopoietic stem cells, and CD45-positive hematopoietic stem cells were captured on the surface of the functionalized gold electrode through immune recognition. Then, the CD34 antibody-functionalized G-quadruplex / heme is anchored to the electrode surface by recognizing the CD34 protein on the surface of hematopoietic stem cells captured on the electrode. Then, G-quadruplex / heme catalyzes the accumulation of a large number of tyramine-coated gold nanoparticles on the cell membrane of hematopoietic stem cells. These gold nanoparticles then serve as seeds for the deposition of silver ions. Finally, the quantitative detection of hematopoietic stem cells was achieved by measuring the electrochemical signal of silver deposited on the electrode surface; Modifying the gold electrode with calix[4]arene having an electron-rich cavity structure; The specific process of detecting the electrochemical signal of silver is as follows: The gold electrode was first incubated with 2 mM, 150-200 μL hemoglobin solution at room temperature in the dark for 20-30 minutes, then incubated with the prepared tyramide-coated gold nanoparticles at room temperature in the dark for 1-1.5 hours, and finally, 150-200 μL of a mixed solution of 0.5 mM AgNO3 and 0.25 mM ascorbic acid was added and incubated at room temperature in the dark for 7-10 minutes, and then the electrochemical signals were collected.

2. The method for electrochemical detection of hematopoietic stem cells based on tyramide signal amplification according to claim 1, characterized in that: The gold electrode surface was modified with calix[4]arene having an electron-rich cavity structure through gold-thiol interaction.

3. The method for electrochemical detection of hematopoietic stem cells based on tyramide signal amplification according to claim 1, characterized in that: The two nucleic acid sequences of the G-quadruplex are as follows: 5'-SH-AAAAACCGTGAGTGTAGCTTGAGATCTGTGAGGGAAGGGA-3', and 5'-AG GGAAGGGACACAGATCTCAAGCTACA-3'.

4. The method for electrochemical detection of hematopoietic stem cells based on tyramide signal amplification according to claim 1, characterized in that: The preparation process of tyramine-coated gold nanoparticles is as follows: Add 10-15 μL of 100 μM tyramide solution to 5 mL of gold nanoparticle solution, gently shake for 12-14 hours, and then centrifuge the solution twice at 2000g for 10-15 minutes to obtain tyramide-coated gold nanoparticles.

5. The method for electrochemical detection of hematopoietic stem cells based on tyramide signal amplification according to claim 1, characterized in that: The average particle size of gold nanoparticles is 30~40 nm.

6. The method for electrochemical detection of hematopoietic stem cells based on tyramide signal amplification according to claim 1, characterized in that: Electrochemical detection was performed by differential pulse voltammetry.

7. The method for electrochemical detection of hematopoietic stem cells based on tyramide signal amplification according to claim 6, characterized in that: The potential scan range was -0.2 V to 0.5 V, with an amplitude of 25 mV and a frequency of 15 Hz.

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