Compositions and methods for detecting hematopoietic stem cells

Abstract

A composition for detecting hematopoietic stem cells includes hematopoietic stem cell-binding peptide-functionalized magnetic beads, crRNA, maleimide-modified activating nucleic acid chains, and methylene blue-modified signaling nucleic acid chains. Using a CRISPR / Cas signal amplification reaction, the activating nucleic acid chains of the CRISPR / Cas system are labeled onto the surface of hematopoietic stem cells via a mild reduction reaction. Through complementary binding of the labeled chains with crRNA, the trans-cleavage activity of Cas12a protein is activated, randomly cleaving the long DNA signal chain and releasing terminally modified methylene blue (MB). MB molecules are incubated with a graphite electrode co-functionalized with CB[7], AuNPs, and PDDA. The MB molecules are captured by the hydrophobic cavity of CB[7], forming a stable host-guest complex that generates an electrochemical signal. The detection of hematopoietic stem cells can be achieved by measuring the methylene blue electrochemical signal enriched on the surface of the functionalized graphite electrode.

Description

Technical Field

[0001] This invention relates to a composition for detecting cells, and more particularly to a composition applied to an electrochemical method for qualitative and quantitative detection of hematopoietic stem cells, thereby improving detection sensitivity. Background Technology

[0002] Aptamers are oligonucleotide sequences or oligopeptides produced through systematic evolutionary techniques that use exponential enrichment of ligands, enabling them to specifically bind to a variety of target molecules. Since aptamers were first reported in 1990, aptamer-based biotechnology has developed rapidly and is widely used in fields such as biosensing, clinical therapy, and targeted drug delivery. Compared to antibodies, aptamers not only possess stronger binding affinity and specificity but also offer advantages such as in vitro production, the ability to be modified at both ends with different chemical molecules for easy detection, lower preparation costs, and high stability. These characteristics hold great potential for therapeutic, diagnostic, and analytical applications.

[0003] Stem cells possess unique self-renewal capabilities and the ability to differentiate into various cells, making them unique among all cells in the body. Hematopoietic stem cells (HSCs) are among the most distinctive stem cell types. They are adult stem cells in the blood system, a heterogeneous population with long-term self-renewal capacity and the potential to differentiate into various mature blood cells. They are the oldest and most extensively studied type of adult stem cell, providing important guidance for research on various stem cell types. Related studies have shown that most leukemias, especially acute marrow leukemia (AML) and chronic marrow leukemia (CML), are directly or indirectly related to abnormalities in hematopoietic stem cells. Hematopoietic stem cells also play a role in regulating the microenvironment of solid tumors. More importantly, hematopoietic stem cell transplantation is widely used in the clinical treatment of hematological diseases and autoimmune diseases. The function and potential therapeutic importance of HSCs have attracted in-depth research on these cells. Therefore, establishing an efficient and sensitive analytical method for hematopoietic stem cells is particularly important. Summary of the Invention

[0004] One object of the present invention is to provide a composition for detecting hematopoietic stem cells, enabling the detection of hematopoietic stem cells by an electrochemical method.

[0005] Another object of the present invention is to provide a composition for detecting hematopoietic stem cells, so as to efficiently detect hematopoietic stem cells and improve detection sensitivity.

[0006] Another object of the present invention is to provide a method for detecting hematopoietic stem cells by performing the detection of hematopoietic stem cells in an electrochemical manner.

[0007] Another objective of this invention is to provide a method for detecting hematopoietic stem cells, thereby achieving quantitative detection of hematopoietic stem cells.

[0008] A composition for detecting hematopoietic stem cells, comprising:

[0009] Hematopoietic stem cell-binding peptide-functionalized magnetic beads

[0010] crRNA,

[0011] Maleimide-modified activated nucleic acid chains, and

[0012] Methylene blue-modified signal nucleic acid strand.

[0013] The composition for detecting hematopoietic stem cells of the present invention has the hematopoietic stem cell binding peptide sequence as: GGGPFSSTKTE.

[0014] The composition for detecting hematopoietic stem cells of the present invention has the following crRNA sequence:

[0015] 5'-UAAUUUCUACUAAGUGUAGAUUAUGAUGUGGAGAUACUCGGUAUAA-3'.

[0016] The composition for detecting hematopoietic stem cells of the present invention has the following signal nucleic acid chain sequence:

[0017] 5'-ATGCCGATCCCCAAAAGTGTACACTTAATCGAAGCTTT-MB-3'.

[0018] The composition for detecting hematopoietic stem cells of the present invention has the following sequence for activating the nucleic acid chain:

[0019] 5'-MI-TTATACCGAGTATCTCCACATCATA-3'

[0020] The composition for detecting hematopoietic stem cells of the present invention utilizes a CRISPR / Cas signal amplification reaction. Through a mild reduction reaction, the activated nucleic acid strand of the CRISPR / Cas system is labeled onto the surface of hematopoietic stem cells. By the complementary binding of the labeled strand with crRNA, the trans-cleavage activity of the Cas12a protein is activated, which randomly cleaves the long DNA signal chain and releases terminally modified methylene blue (MB).

[0021] The free methylene blue molecules generated by cutting are incubated with a graphite electrode co-functionalized with cucurbit[7]urea (CB[7]), gold nanoparticles (AuNPs) and polydiallyldimethylammonium chloride (PDDA). The CB[7] molecules can be captured by the hydrophobic cavity to form a stable host-guest complex and generate an electrochemical signal. By measuring the methylene blue electrochemical signal enriched on the surface of the functionalized graphite electrode, the (quantitative) detection of hematopoietic stem cells can be achieved.

[0022] The composition provided by this invention is used to detect hematopoietic stem cells using an electrochemical method, thereby improving signal labeling efficiency and detection sensitivity.

[0023] A method for detecting hematopoietic stem cells, comprising:

[0024] Mix the test solution with magnetic beads functionalized with hematopoietic stem cell binding peptides and incubate at room temperature (e.g., 2-3 hours);

[0025] Then, the activation nucleic acid chain of the CRISPR / Cas system was labeled onto the surface of hematopoietic stem cells through a mild reduction reaction. The trans-cleavage activity of the Cas12a protein was activated by the complementary binding of the signal nucleic acid chain to crRNA, which randomly cleaved the signal nucleic acid chain and released terminally modified methylene blue.

[0026] The generated free methylene blue molecules are incubated with a graphite electrode co-functionalized with cucurbita[7]urea (CB[7]), gold nanoparticles (AuNPs) and polydiallyldimethylammonium chloride (PDDA). The CB[7] molecules can be captured by the hydrophobic cavity to form a stable host-guest complex and generate an electrochemical signal. The detection of hematopoietic stem cells can be achieved by measuring the methylene blue electrochemical signal enriched on the surface of the functionalized graphite electrode.

[0027] Another method for detecting hematopoietic stem cells includes:

[0028] Mix the test solution with magnetic beads functionalized with hematopoietic stem cell binding peptides and incubate at room temperature (e.g., 2-3 hours);

[0029] Then, maleimide-modified activated nucleic acid strands (e.g., 1–1.5 μM, 100–150 μL) are added, and the mixture is reacted at 37 °C ± 0.5 °C (e.g., 30–60 min) for signal labeling, followed by washing twice.

[0030] Next, crRNA (e.g., 200–300 nM), methylene blue-labeled signal nucleic acid strand (e.g., 600–800 nM), and Cas12a protein (e.g., 200–300 nM) were added, and DEPC water was added to bring the final solution volume to 100–150 μL. The reaction was carried out at 37 °C ± 0.5 °C (e.g., 10–15 minutes) and heated at 65 °C–70 °C (e.g., 10–20 minutes) to inactivate the enzyme. The supernatant was obtained by magnetic separation.

[0031] The supernatant was incubated on a functionalized graphite electrode at room temperature for 1–2 hours. Finally, the modified electrode was thoroughly cleaned for electrochemical measurements. The experimental conditions used for square wave voltammetry (SWV) were: potential scan range 0–-0.6 V, potential step 4 mV, amplitude 25 mV, and frequency 15 Hz.

[0032] The beneficial effects of the technical solution of this invention are as follows:

[0033] Compared with traditional labeling methods, mild reduction reaction-mediated labeling does not depend on specific proteins on the cell surface, and therefore can bind to more cell surface sites, improving signal labeling efficiency and detection sensitivity.

[0034] The enrichment and separation of hematopoietic stem cells by peptide-modified magnetic beads can improve the anti-interference ability of detection and has strong selectivity even in complex serum environments, which is helpful for detection and analysis in real samples.

[0035] The CRISPR / Cas12a system has non-specific cleavage activity for single-stranded DNA, and its signal amplification effect in solution is significant. Its nucleic acid detection sensitivity can reach the pM level, achieving a cell detection limit of 7 cells / mL. Attached Figure Description

[0036] Figure 1 A schematic diagram of a hematopoietic stem cell testing protocol;

[0037] Figure 2 This is a graph of electrochemical analysis data obtained by performing hematopoietic stem cell detection using the method of the present invention;

[0038] Figure 3 Graph showing the electrochemical quantitative results for different concentrations of hematopoietic stem cells;

[0039] Figure 4 The graph shows the electrochemical analysis results for different cell types. Detailed Implementation

[0040] The technical solution of the present invention will be described in detail below with reference to the accompanying drawings. The embodiments of the present invention are only used to illustrate the technical solution of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solution of the invention without departing from the spirit and scope of the technical solution of the present invention, and all such modifications and substitutions should be covered within the scope of the claims of the present invention.

[0041] Figure 1 This is a roadmap for testing hematopoietic stem cell technology protocols. Specific methods include:

[0042] (1) Preparation of hematopoietic stem cell capture magnetic beads: Carboxylated magnetic beads and hematopoietic stem cell binding peptides with amino groups can be coupled through the interaction of carboxyl and amino groups to capture hematopoietic stem cells.

[0043] (2) In situ labeling of hematopoietic stem cells: This method utilizes a mild reduction reaction to perform in situ labeling of enriched and isolated hematopoietic stem cells. The thiol reducing agent TCEP can reduce disulfide bonds in proteins to free active thiol groups, and then perform signal labeling of hematopoietic stem cells through a click chemistry reaction of thiol-maleimide groups. On the one hand, cell surface membrane proteins are rich in disulfide bonds, and after TCEP treatment, they can provide abundant active thiol sites that are easy to label; on the other hand, membrane proteins containing disulfide bonds are widely available, avoiding the influence of specific protein labeling on signal acquisition efficiency. Therefore, the signal labeling method can reduce the influence of cell differences.

[0044] (3) CRISPR / Cas signal amplification reaction: The activation DNA strand of the CRISPR / Cas system is labeled on the surface of hematopoietic stem cells through a mild reduction reaction. The signal nucleic acid strand is complementary to the crRNA, which activates the trans-cleavage activity of the Cas12a protein, randomly cleaves the signal nucleic acid strand, and releases terminally modified methylene blue (MB).

[0045] (4) Electrochemical signal acquisition: The free methylene blue molecules generated by cutting are incubated with cucurbit[7]urea (CB[7]), gold nanoparticles (AuNPs) and polydiallyldimethylammonium chloride (PDDA) co-functionalized graphite electrodes. They can be captured by the hydrophobic cavity of CB[7] to form a stable host-guest complex and generate an electrochemical signal. By measuring the methylene blue electrochemical signal enriched on the surface of the functionalized graphite electrode, the (quantitative) detection of hematopoietic stem cells can be realized.

[0046] (5) When the detection system does not contain hematopoietic stem cells, the activation DNA strand of the CRISPR / Cas system cannot be labeled on the surface of the magnetic beads, and therefore cannot activate the trans-cleavage activity of the Cas12a protein. There is obvious electrostatic repulsion between the long signal DNA strand in the solution and CB[7], and the MB modified at its end cannot approach the electrode, so no electrochemical response signal is generated.

[0047] The solution adopted in the following embodiments of the present invention mainly includes the following steps:

[0048] (a) The specific process for preparing the capture magnetic beads is as follows: 50–70 μL of carboxylated magnetic beads are placed in a centrifuge tube, and 100–200 μL of PBS (10 mM, pH 7.5) is added. After thorough mixing and washing, magnetic separation is performed, and the solution is discarded. The above washing process is repeated three times. Subsequently, the beads are resuspended in 450–500 μL of buffer containing 77.6 mg / mL EDC and 11.5 mg / mL NHS, and reacted at 25°C for 30–40 min. After reaction, magnetic washing is performed three times, and the obtained activated carboxylated functionalized magnetic beads are resuspended in 1 mL of PBS for later use. The above activated magnetic beads are incubated with 10 μL of hematopoietic stem cell binding peptide at room temperature for 2–2.5 hours to obtain functionalized capture magnetic beads.

[0049] (b) The specific procedure for capturing hematopoietic stem cells is as follows: Collect the cell suspension into a 1.5 mL centrifuge tube, centrifuge at 500 rpm to collect the cells, and wash with PBS 2–3 times. Then, resuspend the cells in 1–2 mL of PBS solution containing 1–1.5 mM TCEP and incubate at 37°C for 20–30 min. Subsequently, wash 2–3 times and then use a solution containing 2–3 mM Mg... 2+ Resuspend the cells in PBS solution. Mix the capture magnetic beads (100 μL) prepared according to step (a) above with the hematopoietic stem cell solution and incubate at room temperature for 2–3 hours.

[0050] (c) The specific process of mild reduction labeling is as follows: 100-150 μL of activated DNA strands modified with 1-1.5 μM maleimide is added to the TCEP-pretreated hematopoietic stem cell solution captured by the above magnetic beads, and the reaction is carried out at 37°C for 30-60 min to perform signal labeling. Finally, the cells are washed twice with PBS.

[0051] (d) The specific activation process of the CRISPR / Cas system is as follows: Add 200–300 nM crRNA, 600–800 nM methylene blue-labeled DNA (MB-SS), and 200–300 nM Cas12a protein to the above solution, and add DEPC water to make the final solution volume 100–150 μL. Finally, the reaction conditions for Cas12a catalytic degradation are: react at 37°C for 10–15 minutes, and then heat at 65–70°C for 10–20 minutes to inactivate the enzyme.

[0052] (e) The specific process of preparing the functionalized graphite electrode is as follows: First, the graphite electrode is polished on fine sandpaper and then polished with 0.05μm aluminum powder. Then, the electrode is ultrasonically cleaned in double-distilled water and ethanol for 3-5 minutes respectively. Then, the electrode is immersed in PDDA solution (3.5mg / mL, containing 0.05M NaCl) for 20-30 minutes to form a positively charged PDDA film on the electrode surface. After washing with double-distilled water, 13nm AuNPs prepared by HAuCl4 (0.01%) reduced by citric acid are incubated on the electrode at room temperature for 1-1.5 hours, which can be fixed on the electrode surface by electrostatic interaction with PDDA. Finally, the electrode is incubated with 1mMCB[7] at room temperature for 1-1.5 hours, so that CB[7] can be firmly attached to AuNPs through the interaction between carbonyl and gold. After thorough washing with double-distilled water and drying with nitrogen, the CB[7] / AuNPs / PDDA functionalized electrode is finally prepared for subsequent use.

[0053] (f) The specific procedure for electrochemical detection is as follows: The supernatant from (d) is incubated on a functionalized graphite electrode at room temperature for 1–2 hours. Finally, the modified electrode is thoroughly cleaned for electrochemical measurement. The experimental conditions used for square wave voltammetry (SWV) are: potential scan range 0–-0.6 V; potential step 4 mV; amplitude 25 mV; frequency 15 Hz. Wherein:

[0054] The sequence of the hematopoietic stem cell binding peptide used in step (a) is: GGGPFSSTKTE.

[0055] The activation chain used in step (c) has its 5' end modified with maleimide (MI), and the sequence is as follows:

[0056] 5'-MI-TTATACCGAGTATCTCCACATCATA-3'.

[0057] The sequence of the crRNA used in step (d) is as follows:

[0058] 5'-UAAUUUCUACUAAGUGUAGAUUAUGAUGUGGAGAUACUCGGUAUAA-3';

[0059] The 3' end of the signal chain is labeled with methylene blue (MB), and its sequence is as follows:

[0060] 5'-ATGCCGATCCCCAAAAGTGTACACTTAATCGAAGCTTT-MB-3'. .

[0061] Example 1: Electrochemical Analysis and Detection of Hematopoietic Stem Cells

[0062] The steps are as follows:

[0063] (a) The specific process for preparing the capture magnetic beads is as follows: 50–70 μL of carboxylated magnetic beads are placed in a centrifuge tube, and 100–200 μL of PBS (10 mM, pH 7.5) is added. After thorough mixing and washing, magnetic separation is performed, and the solution is discarded. The above washing process is repeated three times. Subsequently, the beads are resuspended in 450–500 μL of buffer containing 77.6 mg / mL EDC and 11.5 mg / mL NHS, and reacted at 25°C for 30–40 min. After reaction, magnetic washing is performed three times, and the obtained activated carboxylated functionalized magnetic beads are resuspended in 1 mL of PBS for later use. The above activated magnetic beads are incubated with 10 μL of hematopoietic stem cell binding peptide at room temperature for 2–2.5 hours to obtain functionalized capture magnetic beads.

[0064] (b) The specific procedure for capturing hematopoietic stem cells is as follows: Collect the cell suspension into a 1.5 mL centrifuge tube, centrifuge at 500 rpm to collect the cells, and wash with PBS 2–3 times. Then, resuspend the cells in 1–2 mL of PBS solution containing 1–1.5 mM TCEP and incubate at 37°C for 20–30 min. Subsequently, wash 2–3 times and then use a solution containing 2–3 mM Mg... 2+ Resuspend the cells in PBS solution. Mix the capture magnetic beads (100 μL) prepared according to step (a) above with the hematopoietic stem cell solution and incubate at room temperature for 2–3 hours.

[0065] (c) The specific process of mild reduction labeling is as follows: 100-150 μL of activated DNA strands modified with 1-1.5 μM maleimide is added to the TCEP-pretreated hematopoietic stem cell solution captured by the above magnetic beads, and the reaction is carried out at 37°C for 30-60 min to perform signal labeling. Finally, the cells are washed twice with PBS.

[0066] (d) The specific activation process of the CRISPR / Cas system is as follows: Add 200–300 nM crRNA, 600–800 nM methylene blue-labeled DNA (MB-SS), and 200–300 nM Cas12a protein to the above solution, and add DEPC water to make the final solution volume 100–150 μL. Finally, the reaction conditions for Cas12a catalytic degradation are: react at 37°C for 10–15 minutes, and then heat at 65–70°C for 10–20 minutes to inactivate the enzyme.

[0067] (e) The specific procedure for electrochemical detection is as follows: the supernatant from (d) is incubated on a functionalized graphite electrode at room temperature for 1–2 hours. Finally, the modified electrode is thoroughly cleaned for electrochemical measurement. The experimental conditions used for square wave voltammetry (SWV) determination are: potential scan range 0–-0.6 V; potential step 4 mV; amplitude 25 mV; frequency 15 Hz.

[0068] Figure 2 For use in detecting 1×10 6 The image shows the electrochemical signal detection results obtained when 1 hematopoietic stem cell is extracted. (See figure.) Figure 2 As shown in curve a, when hematopoietic stem cells are present in the system, the solution exhibits a distinct current peak near a voltage of -0.35V. Figure 2 medium curve b and Figure 2 In curve c, the solution in the blank control group (without hematopoietic stem cells or without TCEP treatment) has only a small background emission peak near -0.35V, showing a significant difference in electrical signal compared to curve a.

[0069] Example 2: Electrochemical Quantitative Detection of Hematopoietic Stem Cells

[0070] The steps are as follows:

[0071] (a) The specific process for preparing the capture magnetic beads is as follows: 50–70 μL of carboxylated magnetic beads are placed in a centrifuge tube, and 100–200 μL of PBS (10 mM, pH 7.5) is added. After thorough mixing and washing, magnetic separation is performed, and the solution is discarded. The above washing process is repeated three times. Subsequently, the beads are resuspended in 450–500 μL of buffer containing 77.6 mg / mL EDC and 11.5 mg / mL NHS, and reacted at 25 °C for 30–40 min. After reaction, magnetic washing is performed three times, and the obtained activated carboxylated functionalized magnetic beads are resuspended in 1 mL of PBS for later use. The above activated magnetic beads are incubated with 10 μL of hematopoietic stem cell binding peptide at room temperature for 2–2.5 hours to obtain functionalized capture magnetic beads.

[0072] (b) The specific process for capturing hematopoietic stem cells is as follows: Collect different quantities of cell suspension into 1.5 mL centrifuge tubes, centrifuge at 500 rpm to collect cells, and wash with PBS 2-3 times. Then, resuspend the cells in 1-2 mL of PBS solution containing 1-1.5 mM TCEP and incubate at 37°C for 20-30 min. Subsequently, wash 2-3 times and then use a solution containing 2-3 mM Mg... 2+ Resuspend the cells in PBS solution. Mix the capture magnetic beads (100 μL) prepared according to step (a) above with the hematopoietic stem cell solution and incubate at room temperature for 2–3 hours.

[0073] (c) The specific process of mild reduction labeling is as follows: 100-150 μL of activated DNA strands modified with 1-1.5 μM maleimide is added to the TCEP-pretreated hematopoietic stem cell solution captured by the above magnetic beads, and the reaction is carried out at 37°C for 30-60 min to perform signal labeling. Finally, the cells are washed twice with PBS.

[0074] (d) The specific activation process of the CRISPR / Cas system is as follows: Add 200–300 nM crRNA, 600–800 nM methylene blue-labeled DNA (MB-SS), and 200–300 nM Cas12a protein to the above solution, and add DEPC water to make the final solution volume 100–150 μL. Finally, the reaction conditions for Cas12a catalytic degradation are: react at 37°C for 10–15 minutes, and then heat at 65–70°C for 10–20 minutes to inactivate the enzyme.

[0075] (e) The specific procedure for electrochemical detection is as follows: the supernatant from (d) is incubated on a functionalized graphite electrode at room temperature for 1–2 hours. Finally, the modified electrode is thoroughly cleaned for electrochemical measurement. The experimental conditions used for square wave voltammetry (SWV) determination are: potential scan range 0–-0.6 V; potential step 4 mV; amplitude 25 mV; frequency 15 Hz.

[0076] Electrochemical analysis was then performed on a series of hematopoietic stem cells at different concentrations. The quantitative electrochemical results for different numbers of hematopoietic stem cells are as follows: Figure 3 As shown, the peak current increases with the increase in the number of hematopoietic stem cells, reaching a peak when the cell count reaches 1×10⁻⁶. 6 At this point, the current value tends to saturate. This is consistent with the expected result. The increase in the number of hematopoietic stem cells leads to an increase in the number of activated chains labeled on their surface, which enhances the trans-cleavage activity of the Cas12a protein and further strengthens the response value of the electrochemical signal.

[0077] Example 3: Electrochemical Specificity Analysis of Hematopoietic Stem Cells

[0078] (a) The specific process for preparing the capture magnetic beads is as follows: 50–70 μL of carboxylated magnetic beads are placed in a centrifuge tube, and 100–200 μL of PBS (10 mM, pH 7.5) is added. After thorough mixing and washing, magnetic separation is performed, and the solution is discarded. The above washing process is repeated three times. Subsequently, the beads are resuspended in 450–500 μL of buffer containing 77.6 mg / mL EDC and 11.5 mg / mL NHS, and reacted at 25°C for 30–40 min. After reaction, magnetic washing is performed three times, and the obtained activated carboxylated functionalized magnetic beads are resuspended in 1 mL of PBS for later use. The above activated magnetic beads are incubated with 10 μL of hematopoietic stem cell binding peptide at room temperature for 2–2.5 hours to obtain functionalized capture magnetic beads.

[0079] (b) Hematopoietic stem cell capture: Different types of cell suspensions were collected into 1.5 mL centrifuge tubes and centrifuged at 500 rpm. The cells were then washed 2-3 times with PBS, and resuspended in 1 mL of PBS containing 1 mM TCEP and incubated at 37°C for 20-30 min. Afterward, the cells were washed 2-3 times with PBS containing 2 mM Mg...2+ Resuspend the cells in PBS solution. Mix the capture magnetic beads (100 μL) prepared according to step (a) above with the hematopoietic stem cell solution and incubate at room temperature for 2–3 hours.

[0080] (c) Mild reduction of the labeling of the activation strand, the specific process is as follows: 100 μL of the activation DNA strand modified with 1 μM maleimide is added to the TCEP pretreated hematopoietic stem cell solution captured by the above magnetic beads, and the reaction is carried out at 37°C for 30 to 60 min to perform signal labeling. Finally, the sample is washed twice with PBS.

[0081] (d) Activation of the CRISPR / Cas system: 200 nM crRNA, 600 nM methylene blue-labeled DNA (MB-SS), and 200 nM Cas12a protein were added to the above solution, and DEPC water was added to bring the final solution volume to 100 μL. Finally, the Cas12a-catalyzed degradation reaction was carried out at 37°C for 10 minutes, followed by heating at 65°C for 10 minutes to inactivate the enzyme.

[0082] (e) The specific procedure for electrochemical detection is as follows: the supernatant from (d) is incubated on a functionalized graphite electrode at room temperature for 1–2 hours. Finally, the modified electrode is thoroughly cleaned for electrochemical measurement. The experimental conditions used for square wave voltammetry (SWV) determination are: potential scan range 0–-0.6 V; potential step 4 mV; amplitude 25 mV; frequency 15 Hz.

[0083] Electrochemical analysis results of different cell types, such as Figure 4 As shown in the figure. Verification showed that a high electrochemical signal could be detected when the detection system contained hematopoietic stem cells, while a very low electrochemical signal was detected in the control group (including breast epithelial cells MCF-10A and normal lung epithelial cells BEAS-2B). This indicates that the method has excellent detection specificity.

[0084] This paper compares our proposed method with existing methods for detecting hematopoietic stem cells. Current methods include colony culture, automated blood cell counting, and molecular biological assays. These methods are complex and cumbersome, susceptible to interference from immature cells, leading to poor specificity. Furthermore, they require sophisticated experimental conditions and advanced techniques, making clinical implementation difficult. The technical solution provided in this embodiment offers higher sensitivity and stability, and exhibits strong selectivity even in complex serum environments, promising efficient capture and detection of hematopoietic stem cells in clinical practice.

Claims

1. The application of a composition in the electrochemical detection of hematopoietic stem cells, characterized in that... By using a mild reduction reaction, the activated nucleic acid chain of the CRISPR / Cas system was labeled onto the surface of hematopoietic stem cells. By the complementary binding of the labeled nucleic acid chain with crRNA, the trans-cleavage activity of Cas12a protein was activated, which randomly cleaved the DNA signal chain and released the terminal-modified methylene blue. The resulting free methylene blue molecules were incubated with a graphite electrode co-functionalized with cucurbita[7]urea, gold nanoparticles and polydiallyldimethylammonium chloride. The graphite electrode was captured by the hydrophobic cavity of cucurbita[7]urea, forming a stable host-guest complex and generating an electrochemical signal. The detection of hematopoietic stem cells was achieved by measuring the electrochemical signal of methylene blue enriched on the surface of the functionalized graphite electrode. The composition comprises: Magnetic beads functionalized with hematopoietic stem cell binding peptides, wherein the sequence of the hematopoietic stem cell binding peptides is: GGGPFSSTKTE; crRNA, sequence: 5'-UAAUUUCUACUAAGUGUAGAUUAUGAUGUGGAGAUACUCGGUAUAA-3'; The maleimide-modified activating nucleic acid chain has the sequence: 5'-MI-TTATACCGAGTATCTCCACATCA TA-3', and The methylene blue-modified signal nucleic acid chain has the following sequence: 5'-ATGCCGATCCCCAAAAGTGTACACTTAATCGAAGCTTT-MB-3'.

2. A method for detecting hematopoietic stem cells, characterized in that... include: The test solution was mixed with magnetic beads functionalized with hematopoietic stem cell binding peptides and incubated at room temperature. Then, the maleimide-modified activation nucleic acid strand of the CRISPR / Cas system was labeled onto the surface of hematopoietic stem cells through a mild reduction reaction. The trans-cleavage activity of the Cas12a protein was activated by the complementary binding of the methylene blue-modified signal nucleic acid strand to crRNA, which randomly cleaved the signal nucleic acid strand and released the terminally modified methylene blue. The generated free methylene blue molecules are incubated with cucurbit[7]urea, gold nanoparticles and polydiallyldimethylammonium chloride co-functionalized graphite electrodes. They can be captured by the hydrophobic cavity of cucurbit[7]urea to form a stable host-guest complex and generate an electrochemical signal. By measuring the methylene blue electrochemical signal enriched on the surface of the functionalized graphite electrode, the detection of hematopoietic stem cells can be realized. The hematopoietic stem cell binding peptide sequence is: GGGPFSSTKTE; The crRNA sequence is as follows: 5'-UAAUUUCUACUAAGUGUAGAUUAUGAUGUGGAGAUACUCGGUAUAA-3'; The sequence of the signal nucleic acid strand is as follows: 5'-ATGCCGATCCCCAAAAGTGTACACTTAATCGAAGCTTT-MB-3'; The sequence for activating the nucleic acid chain is as follows: 5'-MI-TTATACCGAGTATCTCCACATCATA-3'.

3. The method for detecting hematopoietic stem cells according to claim 2, characterized in that... The electrochemical detection and analysis were performed using square wave voltammetry, with a scanning range of 0V to -0.6V, an amplitude of 25mV, a potential step of 4mM, and a frequency of 15Hz.

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