Hematopoietic stem cell detection method based on e-Y-Click marker

Abstract

A method for detecting hematopoietic stem cells comprises the steps that a CD133 nucleic acid aptamer functionalized ITO electrode is used as a substrate, e-Y-Click labeling of hematopoietic stem cell surface protein is achieved through in-situ electrochemical activation of PTAD-N3, N3 groups combined on the surface and DBCO modified nucleic acid chains T are subjected to SPAAC reaction, and the hematopoietic stem cells are obtained. The method comprises the following steps of: triggering a self-replication cycle reaction of DNA (Deoxyribose Nucleic Acid), enriching electrochemical signal substances on the surfaces of the hematopoietic stem cells, qualitatively judging the hematopoietic stem cells by detecting electrochemical signals, and correspondingly judging the quantity of the hematopoietic stem cells contained in a sample to be detected according to the established relationship between the intensity of the electrochemical signals and the quantity of the hematopoietic stem cells, so as to realize quantitative detection.

Description

Technical Field

[0001] The present invention relates to a method for detection, and in particular to a method for achieving qualitative and quantitative detection of hematopoietic stem cells using eY-Click labeling. Background Art

[0002] Hematopoietic stem cells, as adult stem cells, are located at the top of the hematopoietic system cascade. Found in umbilical cord blood, peripheral blood, and adult bone marrow, they are small in number. They are a self-renewing and totipotent group of cells, capable of differentiating into various types of hematopoietic progenitor cells, which in turn give rise to different lineages of myeloid and lymphoid blood cells. Hematopoietic stem cells persist throughout human life, constantly adapting to meet the needs of different life stages. During the fetal period, hematopoietic stem cells focus on proliferation, establishing the basic hematopoietic system. In adulthood, they remain quiescent, supporting health and immune function through a balance of self-renewal and differentiation. In old age, due to aging-related metabolic and environmental changes, the function of hematopoietic stem cells begins to decline.

[0003] In recent years, site-selective chemical modification of proteins has become a highly sought-after protein labeling strategy. By targeting specific amino acid residues on the protein surface (such as tryptophan and tyrosine), site-specific modification of native proteins can be achieved without genetic manipulation. Tyrosine, due to its polar phenolic hydroxyl group, is one of the most prominent sites for protein chemical modification. Consequently, various protein surface tyrosine-click modification (Y-Click) methods have been developed. 4-Phenyl-1,2,4-triazolin-3,5-dione (PTAD) and luminol derivatives, which possess a cyclic diazodicarbonamide structure, can be activated by various catalysts, such as heme, HRP, and photocatalysts, for labeling tyrosine residues. eY-Click, a Y-Click-based electrochemical oxidation activation method, can target protein surface tyrosines for site-selective modification while avoiding side reactions and maintaining protein activity.

[0004] 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). SWV, among others, is characterized by high sensitivity and high efficiency. Currently, it is widely used in industries such as industry, agriculture, the environment, medicine, food, 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

[0005] One object of the present invention is to provide a method for detecting hematopoietic stem cells, which uses eY-Click labeling site selective labeling combined with the high efficiency of SPAAC reaction to perform efficient qualitative detection of hematopoietic stem cells.

[0006] Another object of the present invention is to provide a method for detecting hematopoietic stem cells, which uses eY-Click labeling site selective labeling combined with the high efficiency of SPAAC reaction to perform efficient quantitative detection of hematopoietic stem cells.

[0007] The method of the present invention uses an ITO electrode functionalized with a CD133 nucleic acid aptamer (e.g., amino-modified) as a substrate, and achieves eY-Click labeling of hematopoietic stem cell surface proteins through in situ electrochemical activation of 4-(4-(2-azidoethoxy)phenyl)-1,2,4-triazolidine-3,5-dione (PTAD-N3). The surface-bound N3 group then reacts with a dibutyltin oxide (DBCO)-modified nucleic acid chain T (DBCO-T) through a strain-promoted azide-alkyne cycloaddition (SPAAC) reaction, triggering a DNA self-replication cycle reaction and enriching electrochemical signal substances on the hematopoietic stem cell surface. The hematopoietic stem cells are qualitatively determined by detecting the electrochemical signal, and the number of hematopoietic stem cells contained in the test sample is then determined based on the established relationship between the electrochemical signal intensity and the number of hematopoietic stem cells (standard curve), thereby achieving quantitative detection.

[0008] Electrochemical measurements were performed using a three-electrode system on a CHI-660C electrochemical workstation. The auxiliary electrode was a platinum wire, the reference electrode was a saturated calomel electrode, and the working electrode was an ITO electrode. The buffer was 20 mM Tris-HCl. The solution was thoroughly deoxygenated with high-purity nitrogen gas, and nitrogen flow was used throughout the electrochemical measurements to maintain anaerobic conditions. Square wave voltammetry (SWV) parameters were as follows: potential scan range, -0.6 to 0.2 V; potential step, 4 mV; amplitude, 25 mV; frequency, 15 Hz.

[0009] A specific embodiment of detecting hematopoietic stem cells includes: First, fix the hematopoietic stem cells on the working electrode. Then, PTAD-N3 is used to activate hematopoietic stem cells in situ, so that the surface of hematopoietic stem cells has a large number of azide groups as signal binding sites. The DBCO-T probe binds to the azide groups on the cell surface through the SPAAC reaction, thereby enriching on hematopoietic stem cells. Adding a nucleic acid chain with a hairpin structure, a DNA self-replication cycle occurs, forming a tree-like DNA product. Qualitative and quantitative detection of hematopoietic stem cells is achieved by measuring the electrochemical signals on the ITO surface.

[0010] The method of the present invention, the site-selective labeling of nucleic acid aptamers and eY-Click ensures the accuracy and high specificity of the method, while the high efficiency of the SPAAC reaction significantly improves the sensitivity of the detection method. This method can meet the needs of different cell detection by replacing nucleic acid aptamers.

[0011] Another specific embodiment for detecting hematopoietic stem cells is that DBCO-T is a nucleic acid T strand whose 3' end is modified by DBCO, and its sequence is as follows: 5'-GCTTCATCTTCATCTCCGACACTC-3'.

[0012] Another specific embodiment of detecting hematopoietic stem cells uses four hairpin-structured nucleic acid chains to achieve a DNA self-replication cycle. The specific sequence is as follows: H1: 5'-GCTTCATCTTCGAGTGTCGGAGATGAAGATGAAGCCATCGTGCTTCAT CTTCATCTCCG-3'; H2: 5'-GCTTCATCTTCATCTCCGATCTCGTTTTGCGGAGATGAAGATGAAGCA CGATG-3'; H3: 5'-CAAAACGAGATCGGAGATGAAGATGAAGCTTGCCTGCTTCATCTTCATCTCCGATCTCCGACACTC-3'; H4: 5'-GCTTCATCTTCATCTCCGACACTCCGGAGATGAAGATGAAGCAGGCA A-3'.

[0013] A chemical group is bound to the end of H4 to respond to electrochemical detection, for example, its 5' end is modified with methylene blue (MB), denoted as H4-MB.

[0014] Another specific implementation method for detecting hematopoietic stem cells is to covalently bind the CD133 nucleic acid aptamer to the working electrode, such as through an amide bond, to immobilize the hematopoietic stem cells on the working electrode.

[0015] The sequence of the CD133 nucleic acid aptamer is: 5'- CCCUCCUACAUAGGG-3', and its 5' end is modified with an amino group (NH2-). The specific process is as follows: the ITO electrode is immersed in a solution (28% NH3·H2O:30% H2O2:H2O = 1:1:6, v / v / v) and heated in an 85°C water bath for 25-30 minutes to first obtain a hydroxylated surface. The electrode is then placed in a mixed solution of the silane coupling agent Glymo and toluene in a 1:1 volume ratio and heated at 90°C for 1-1.5 hours to form an epoxy monolayer on the electrode surface. Finally, the electrode is placed in a 0.75 M iminodiacetic acid (IDA) solution to obtain a carboxyl-functionalized ITO electrode.

[0016] The carboxyl-functionalized ITO electrode was incubated in an EDC / NHS solution (0.22 M) at room temperature to activate the carboxyl groups. After rinsing with water, it was incubated with an amino-modified CD133 nucleic acid aptamer and blocked with 0.1% Tween 20. A solution containing hematopoietic stem cells or a sample was added and reacted at room temperature for 1-1.5 hours. The CD133 nucleic acid aptamer captured the hematopoietic stem cells and ultimately fixed them to the working electrode surface.

[0017] Compared with the prior art, the technical solution of the present invention has the following beneficial effects: This method uses CD133 on the surface of hematopoietic stem cells as a target. Leveraging eY-Click and the tree-like DNA structure formed by the DNA self-replication cycle, this method establishes an electrochemical detection method for the precise identification and quantitative analysis of hematopoietic stem cells, enabling sensitive detection of hematopoietic stem cells. Compared to existing technologies, this method offers the following advantages: ITO electrodes have good chemical stability and mechanical durability, and can remain stable during long-term use and are not easily damaged. Aptamer-functionalized ITO electrodes have the advantages of good selectivity and high capture efficiency for the specific capture of hematopoietic stem cells. eY-Click is a novel protein labeling technology that uses low-potential electrochemical in situ activation of PTAD and luminol derivatives with a cyclic diazodicarbonamide structure. It can target tyrosine residues on protein surfaces for site-selective modification while avoiding side reactions and not affecting the activity of the protein itself. SPAAC is an azide-alkyne cycloaddition reaction triggered by the ring tension between azide and cyclooctyne. It does not require catalysts such as metal ions, has a fast reaction and mild conditions, and does not interfere with the biochemical processes in the body, greatly promoting the application scope of click chemistry.

[0018] The non-enzymatic self-replicating DNA circulation system can spontaneously form a tree-like DNA structure through a self-stacking design of only a basic cascade hybridization reaction. It does not require the participation of enzymes and has high signal amplification efficiency and fast speed. With simple DNA configuration and low reactant complexity, the self-replicating DNA circulation system can achieve efficient biosensing on living cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is a schematic diagram of the principle of detecting hematopoietic stem cells according to the present invention; Figure 2 This is the fluorescence response result diagram of the DNA self-replication cycle; Figure 3 The electrochemical signal response results in each experimental group are shown; Figure 4 The graph shows the electrochemical signal intensity response results for various cell concentrations. DETAILED DESCRIPTION

[0020] 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.

[0021] Figure 1 This is a schematic diagram of the principle of detecting hematopoietic stem cells according to the present invention. The specific method includes: (1) Preparation of aptamer-functionalized electrodes and specific capture of hematopoietic stem cells: The surface of the ITO electrode is gradually modified to obtain a carboxylated electrode surface. After activation, the carboxyl group can react with the amino-modified CD133 aptamer to form a stable amide bond, which can then be used for the specific capture of hematopoietic stem cells.

[0022] (2) In situ activation of PTAD-N3: The ITO electrode used to capture hematopoietic stem cells was used as the working electrode, and the substrate PTAD-N3 in the buffer solution was activated in situ using a complete three-electrode system. It was labeled on the tyrosine residues of the hematopoietic stem cell membrane protein through eY-click. After activation, the surface of the hematopoietic stem cell will have a large number of azide groups as signal binding sites; and the DBCO-T probe will bind to the azide groups on the cell surface through copper-free click chemistry SPAAC, thereby enriching on the hematopoietic stem cells; then, when H1, H2, H3 and H4-MB with hairpin structures are added to the reaction system, a DNA self-replication cycle will occur to form a tree-like DNA product.

[0023] (3) The specific process of the DNA self-replication cycle reaction is as follows: hairpin nucleic acids H1, H2, H3 and methylene blue-modified H4-MB are added to PTAD-N3-activated hematopoietic stem cells. The DBCO-T probe coupled to the hematopoietic stem cells can open the hairpin structure of H1, the opened H1 can open the hairpin structure of H2, the opened H2 can open the hairpin structure of H3, and the opened H3 can open the hairpin structure of H4-MB. Therefore, the DBCO-T probe can make the four nucleic acids with hairpin structures H1, H2, H3, and H4-MB form a cycle unit through base complementary pairing. At the same time, the opened H4-MB can act as a DBCO-T probe to continue opening the hairpin structure of H1, so that the loop unit can gradually extend into a longer DNA chain. At the same time, H1 and H3 each contain a part of the T sequence. When the two loop units are connected together, H3 and H1 of the two loop units can also act as DBCO-T probes, further extending to form new loop units. Therefore, the hairpin nucleic acid continues to extend outward in the shape of a tree under the triggering of the DBCO-T probe, and eventually forms a tree-like DNA structure.

[0024] (4) When hematopoietic stem cells are present in the reaction system, they can be captured by the aptamer-functionalized ITO electrode. Activated PTAD-N3 can couple with the DBCO-T probe, triggering a self-replication cycle to form a tree-like DNA structure. H4-MB is enriched on the cell surface, generating a significant electrochemical signal. Therefore, sensitive detection of hematopoietic stem cells can be achieved by measuring the electrochemical signal of methylene blue on the ITO surface.

[0025] (5) When there are no hematopoietic stem cells in the reaction system, the added DBCO-T probe is free in the detection solution and cannot activate the subsequent DNA self-replication cycle after being removed by washing. The H4-MB with electrochemical signals will not be enriched on the ITO electrode, so no electrochemical signal can be detected in the final reaction system.

[0026] Example 1 The steps for verifying the DNA self-replication cycle are as follows: To prepare and verify the dendrimer DNA, four single DNA strands were annealed. Specifically, the DNA strands (H1, H2, H3, and FAM-H4-TAMRA) were placed in a 95°C metal bath for 5–10 minutes and then slowly cooled to room temperature. A mixed solution of T, H1, H2, H3, and FAM-H4-TAMRA was then placed in a 37°C metal bath for 120–130 minutes. After the reaction, the final fluorescence emission intensity of the solution was measured. The experimental conditions used for fluorescence emission intensity were an excitation wavelength of 480 nm and an emission wavelength scan range of 500–600 nm.

[0027] The sequences of the DNA used in the steps are: T: 5'-GCTTCATCTTCATCTCCGACACTC-3'; H1: 5'-GCTTCATCTTCGAGTGTCGGAGATGAAGATGAAGCCATCGTGCTTCAT CTTCATCTCCG-3'; H2: 5'-GCTTCATCTTCATCTCCGATCTCGTTTTGCGGAGATGAAGATGAAGCA CGATG-3'; H3: 5'-CAAAACGAGATCGGAGATGAAGATGAAGCTTGCCTGCTTCATCTTCATCTCCGATCTCCGACACTC-3'; H4: 5'-GCTTCATCTTCATCTCCGACACTCCGGAGATGAAGATGAAGCAGGC A A-3', its 5' end is modified with the fluorescent group FAM, and its 3' end is modified with the quenching group TAMRA, denoted as: FAM-H4-TAMRA.

[0028] The fluorescence verification results of DNA self-replication cycle are as follows Figure 2As shown, the results show that only when T exists can the four nucleic acids containing hairpin structures undergo DNA self-replication cycle reaction and restore FAM fluorescence, thereby being able to monitor a strong fluorescence signal. The mixed samples of H1, H2, H3 and FAM-H4-TAMRA cannot trigger the DNA self-replication cycle reaction due to the lack of T, so the fluorescence signal of the control group drops significantly, thereby verifying the occurrence of DNA self-replication cycle in the presence of T chain.

[0029] Example 2 (a) Preparation of functionalized ITO electrodes. First, the ITO electrodes were cleaned with ethanol and then deionized water, followed by immersion in a solution (28% NH₃·H₂O:30% H₂O₂:H₂O = 1:1:6, v / v / v) and heating in an 85°C waterbath for 25–30 minutes to achieve a hydroxylated surface. The electrodes were then rinsed with deionized water and dried with nitrogen. The dried ITO electrodes were then immersed in a 1:1 (volume ratio) mixture of the silane coupling agent Glymo and toluene and heated at 90°C for 1–1.5 hours to form an epoxy monolayer on the electrode surface. After rinsing with acetone, the electrodes were then immersed in a 0.75 M IDA solution (containing 0.34 M NaCl and 2 M Na₂CO₃, pH = 11) and heated at 70°C for 0.5–1 hour to achieve surface carboxylation. Finally, the carboxyl-functionalized ITO electrodes were rinsed with copious amounts of deionized water to obtain the resulting surfaces.

[0030] (b) Specific capture of hematopoietic stem cells: The prepared carboxyl-functionalized ITO electrode was immersed in a 0.22 M EDC / NHS solution at room temperature for 25–30 minutes to activate the carboxyl groups. After rinsing with water, the electrode was incubated with 100 μL of an amino-modified CD133 aptamer for 1–1.5 hours. The plate was then sealed with 0.1% Tween 20 for 30–40 minutes and incubated with 100–150 μL of various amounts of hematopoietic stem cell dilutions at room temperature for 1–1.5 hours.

[0031] (c) In situ activation of PTAD-N3. The ITO electrode with hematopoietic stem cells captured in (b) served as the working electrode, along with a platinum electrode (auxiliary electrode) and an Ag / AgCl electrode (reference electrode) to form a three-electrode system. Electrodeposition was performed in a 40-50 μM PTAD-N3 buffer at a voltage of 750 mV for 10-15 minutes to achieve in situ activation and site-selective modification of PTAD-N3. After rinsing with deionized water, the ITO electrode was reacted with a DBCO-T probe at 37°C for 1-1.5 hours to couple DBCO-T to the hematopoietic stem cell surface. The ITO electrode was then reacted with a 100-150 μL mixed solution of H1, H2, H3, and H4-MB (600 nM) in a metal bath at 37°C for 120-130 minutes to initiate the DNA self-replication cycle.

[0032] (d) Electrochemical signal detection. Electrochemical measurements were performed using a three-electrode system on a CHI-660C electrochemical workstation. The auxiliary electrode was a platinum wire, the reference electrode was a saturated calomel electrode, and the working electrode was the ITO electrode obtained in (d). The buffer was 20 mM Tris-HCl. The solution was thoroughly deoxygenated with high-purity nitrogen gas and maintained anaerobic using a nitrogen flow throughout the electrochemical detection. Square wave voltammetry (SWV) parameters were as follows: potential scan range, -0.6 to 0.2 V; potential step, 4 mV; amplitude, 25 mV; frequency, 15 Hz.

[0033] in: The amino-modified CD133 aptamer used in step (b) has a sequence of 5'-NH2-CCCUCCUACAUAGGG-3', which is a known sequence for recognizing hematopoietic stem cells and can specifically recognize the CD133 receptor on the outer membrane of hematopoietic stem cells.

[0034] The sequences of the DNA used in step (c) are: T: 5'-GCTTCATCTTCATCTCCGACACTC-3', whose 3' end is modified by DBCO, denoted as: DBCO-T.

[0035] H1: 5'-GCTTCATCTTCGAGTGTCGGAGATGAAGATGAAGCCATCGTGCTTCAT CTTCATCTCCG-3'; H2: 5'-GCTTCATCTTCATCTCCGATCTCGTTTTGCGGAGATGAAGATGAAGCA CGATG-3'; H3: 5'-CAAAACGAGATCGGAGATGAAGATGAAGCTTGCCTGCTTCATCTTCATCTCCGATCTCCGACACTC-3'; H4: 5′-GCTTCATCTTCATCTCCGACACTCCGGAGATGAAGATGAAGCAGGCA A -3′, whose 5′ end was modified with methylene blue (MB), denoted as: H4-MB.

[0036] Figure 3 The electrochemical signal obtained when this method was used to detect hematopoietic stem cells is shown. When hematopoietic stem cells are present in the system, DBCO-T can couple to the stem cell surface and trigger the DNA self-replication cycle. The formation of a dendritic DNA structure allows the methylene blue-modified H4 to be enriched on the stem cell surface, resulting in a distinct current peak during electrochemical detection. In the blank control group, which lacks hematopoietic stem cells, the DBCO-T is removed after washing and cannot activate the DNA self-replication cycle, resulting in only a small background emission peak.

[0037] Example 3 The steps for the fluorescence quantitative detection and verification of hematopoietic stem cells are as follows: (a) Preparation of functionalized ITO electrodes. First, the ITO electrodes were cleaned with ethanol and then deionized water, followed by immersion in a solution (28% NH₃·H₂O:30% H₂O₂:H₂O = 1:1:6, v / v / v) and heating in an 85°C waterbath for 25–30 minutes to achieve a hydroxylated surface. The electrodes were then rinsed with deionized water and dried with nitrogen. The dried ITO electrodes were then immersed in a 1:1 (volume ratio) mixture of Glymo and toluene and heated at 90°C for 1–1.5 hours to form an epoxy monolayer on the electrode surface. After rinsing with acetone, the electrodes were then immersed in a 0.75 M IDA solution (containing 0.34 M NaCl and 2 M Na₂CO₃, pH = 11) and heated at 70°C for 0.5–1 hour to achieve surface carboxylation. Finally, the carboxyl-functionalized ITO electrodes were rinsed with copious amounts of deionized water to obtain the resulting surfaces.

[0038] (b) Specific capture of hematopoietic stem cells: The prepared carboxyl-functionalized ITO electrode was immersed in a 0.22 M EDC / NHS solution at room temperature for 25–30 minutes to activate the carboxyl groups. After rinsing with water, the electrode was incubated with 100 μL of an amino-modified CD133 aptamer for 1–1.5 hours. The plate was then sealed with 0.1% Tween 20 for 30–40 minutes and incubated with 100–150 μL of various amounts of hematopoietic stem cell dilutions at room temperature for 1–1.5 hours.

[0039] (c) In situ activation of PTAD-N3. The ITO electrode with hematopoietic stem cells captured in (b) served as the working electrode, along with a platinum electrode (auxiliary electrode) and an Ag / AgCl electrode (reference electrode) to form a three-electrode system. Electrodeposition was performed in a 40-50 μM PTAD-N3 buffer at a voltage of 750 mV for 10-15 minutes to achieve in situ activation and site-selective modification of PTAD-N3. After rinsing with deionized water, the ITO electrode was reacted with a DBCO-T probe at 37°C for 1-1.5 hours to couple DBCO-T to the hematopoietic stem cell surface. The ITO electrode was then reacted with a 100-150 μL mixed solution of H1, H2, H3, and H4-MB (600 nM) in a metal bath at 37°C for 120-130 minutes to initiate the DNA self-replication cycle.

[0040] (d) Electrochemical signal detection: Electrochemical measurements were performed using a three-electrode system on a CHI-660C electrochemical workstation. The auxiliary electrode was a platinum wire, the reference electrode was a saturated calomel electrode, and the working electrode was an ITO electrode. The buffer was 20 mM Tris-HCl. The solution was thoroughly deoxygenated with high-purity nitrogen gas, and nitrogen flow was used throughout the electrochemical measurement to maintain an anaerobic state. Square wave voltammetry (SWV) parameters were as follows: potential scan range, -0.6 to 0.2 V; potential step, 4 mV; amplitude, 25 mV; frequency, 15 Hz.

[0041] in: The sequence of the CD133 nucleic acid aptamer used in step (b) is: 5'-CCCUCCUACAUAGGG-3', and its 5' end is modified with an amino group (NH2-) to specifically recognize the CD133 receptor on the outer membrane of hematopoietic stem cells.

[0042] The sequences of the DNA used in step (c) are: DBCO-T: 5'-GCTTCATCTTCATCTCCGACACTC-DBCO-3'; H1: 5'-GCTTCATCTTCGAGTGTCGGAGATGAAGATGAAGCCATCGTGCTTCAT CTTCATCTCCG-3'; H2: 5'-GCTTCATCTTCATCTCCGATCTCGTTTTGCGGAGATGAAGATGAAGCA CGATG-3'; H3: 5'-CAAAACGAGATCGGAGATGAAGATGAAGCTTGCCTGCTTCATCTTCATCTCCGATCTCCGACACTC-3'; H4-MB: 5'-MB-GCTTCATCTTCATCTCCGACACTCCGGAGATGAAGATGAAGC AGGCAA -3'.

[0043] On the basis of verifying the feasibility of this method for detecting hematopoietic stem cells, this example also conducted electrochemical analysis on a series of hematopoietic stem cells. The results are as follows: Figure 4 As predicted, the measured peak current increases with the increase in the concentration of added hematopoietic stem cells. This is due to the coupling of more DBCO-T probes, which can stimulate more DNA self-replication cycles, allowing more methylene blue to accumulate on the surface of hematopoietic stem cells, and the resulting peak current also increases.

Claims

1. A method for detecting hematopoietic stem cells, comprising: Using the CD133 nucleic acid aptamer-functionalized ITO electrode as the substrate, eY-Click labeling of hematopoietic stem cell surface proteins was achieved through in situ electrochemical activation of PTAD-N3. The surface-bound N3 group then underwent SPAAC reaction with the DBCO-modified nucleic acid chain T, triggering the DNA self-replication cycle reaction and enriching the electrochemical signal substance on the hematopoietic stem cell surface. The hematopoietic stem cells were qualitatively identified by detecting the electrochemical signal.

2. The method according to claim 1, characterized in that Based on the established relationship between the electrochemical signal intensity and the number of hematopoietic stem cells, the number of hematopoietic stem cells contained in the sample to be tested is judged accordingly to achieve quantitative detection.

3. The method according to claim 1, characterized in that Electrochemical measurements were performed using a three-electrode system with a platinum wire as the auxiliary electrode, a saturated calomel electrode as the reference electrode, and an ITO electrode as the working electrode.

4. The method according to claim 3, characterized in that Square-wave voltammetry was used for detection with a potential scan range of -0.6 V to 0.2 V, a potential step of 4 mV, an amplitude of 25 mV, and a frequency of 15 Hz.

5. The method according to claim 1, characterized in that DBCO-T is a nucleic acid T strand whose 3' end is modified by DBCO. Its sequence is as follows: 5'-GCTTCATCTTCATCTCCGACACTC-3'.

6. The method according to claim 1, characterized in that The DNA self-replication cycle forms a tree-like DNA product.

7. The method according to claim 1, characterized in that The four hairpin nucleic acid chains together complete the DNA self-replication cycle reaction.

8. The method according to claim 6, characterized in that The 4 hairpin nucleic acid chains are as follows: H1: 5'-GCTTCATCTTCGAGTGTCGGAGATGAAGATGAAGCCATCGTGCTTCAT CTTCATCTCCG-3'; H2: 5'-GCTTCATCTTCATCTCCGATCTCGTTTTGCGGAGATGAAGATGAAGCA CGATG-3'; H3: 5'-CAAAACGAGATCGGAGATGAAGATGAAGCTTGCCTGCTTCATCTTCATCTCCGATCTCCGACACTC-3'; and H4: 5'-GCTTCATCTTCATCTCCGACACTCCGGAGATGAAGATGAAGCAGGCA A -3', A chemical group is bound to the end of H4 to respond to electrochemical detection.

9. The method according to claim 8, characterized in that The chemical group is methylene blue, which is modified at the 5' end of the H4 nucleic acid chain.

10. The method according to claim 1, characterized in that The sequence of the CD133 aptamer is as follows: 5′- CCCUCCUACAUAGGG-3′.

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