Composition and method for electrochemical detection of hematopoietic stem cells

Abstract

A composition for detecting hematopoietic stem cells comprises magnetic beads functionalized with CD34 antibodies, nucleic acid probes bound to CD31 antibodies, nucleic acid probes bound to CD45 antibodies, H1 chains, H2 chains, crRNA and signal nucleic acid chains. When the two probes are fixed to the surface of hematopoietic stem cells, a long chain S is formed. The long chain S acts as an activation chain to open the H1 chain, generating a T / H1 double-stranded structure, thereby exposing the terminal sequence and opening the H2 chain, generating an H1 / H2 double-stranded structure and releasing T, and then proceeding to the next round of hybridization reaction. The end of the H1 / H2 double-stranded structure contains a partial single-stranded structure, which acts as an activation chain to activate the trans-cleavage activity of the CRISPR / cas system, randomly cutting the DNA chain, thereby releasing the terminal MB. The MB molecule can be captured by the hydrophobic cavity of CB[7] to form a stable host-guest complex, generate an electrochemical signal, and realize the detection of hematopoietic stem cells.

Description

Technical Field

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

[0002] Stem cells, unique among all other cells in the body, possess the ability to self-renew and give rise to differentiated cells. Hematopoietic stem cells (HSCs) are one of the most distinctive stem cell types. They are the adult stem cells of the blood system, a heterogeneous population with the capacity for long-term self-renewal and the potential to differentiate into various mature blood cell types. They are the longest-studied and most intensively studied type of adult stem cell, and they hold important guiding significance for the study of various stem cell types. Studies have shown that the development of most leukemias, particularly acute myeloid leukemia (AML) and chronic myeloid leukemia (CML), is directly or indirectly linked to abnormalities in HSCs. HSCs also play a role in regulating the microenvironment of solid tumors. Most importantly, HSC transplantation is widely used in the clinical treatment of hematologic and autoimmune diseases. The functions and potential therapeutic importance of HSCs have attracted intensive research on these cells. Therefore, the development of efficient and sensitive methods for analyzing HSCs is of paramount importance.

[0003] Currently, existing methods for detecting hematopoietic stem cells include colony culture, fully automatic blood cell counting, enzyme-linked immunosorbent assay (ELISA), and flow cytometry. These conventional methods require complex sample pretreatment and dedicated analytical instruments. Flow cytometry is highly dependent on the operator's experience. In addition, flow cytometry has limitations such as expensive instruments, cumbersome data processing, and the fact that the analysis object must be a single cell suspension, which makes the results easily affected by cell pretreatment. The enzyme-linked immunosorbent assay (ELISA) has poor reproducibility and is subject to interference from autoantibodies, heterophilic antibodies, and is prone to false positives. Regardless of the instrument or manual operation, there are many interfering factors.

[0004] Catalytic hairpin assembly (CHA) is an isothermal, enzyme-free nucleic acid amplification technology that overcomes the drawbacks of protease dependence. Importantly, the CHA process enables promoter-loop signal amplification, requiring only two metastable DNA hairpins and requiring no human intervention. Therefore, CHA boasts simple design, strong adaptability, and high signaling conversion efficiency. Therefore, CHA is a powerful molecular biotechnology tool, showing promising prospects in biosensing and bioimaging applications. CRISPR / Cas is an RNA-mediated, heritable adaptive immune defense system in bacteria and archaea, currently being used as an effective tool for genome editing and biosensing. CRISPR / Cas12a exhibits nonspecific cleavage activity against single-stranded DNA, with sensitivity reaching the picoM level. This reaction can be used to detect DNA, microRNA, and non-nucleic acid small molecules. Square wave voltammetry (SWV), also known as modern square wave voltammetry, is a versatile, rapid, highly sensitive, and efficient electroanalytical method, a type of linear sweep voltage, with widespread applications in various fields, including medical clinical communications. Summary of the Invention

[0005] One object of the present invention is to provide a composition for electrochemical detection of hematopoietic stem cells, thereby improving the accuracy of hematopoietic stem cell detection and effectively avoiding false positive results of the detection.

[0006] Another object of the present invention is to provide a composition for electrochemical detection of hematopoietic stem cells, so as to efficiently detect hematopoietic stem cells, efficiently capture hematopoietic stem cells, and improve detection sensitivity.

[0007] Another object of the present invention is to provide a method for electrochemically detecting hematopoietic stem cells, which implements the detection of hematopoietic stem cells in an electrochemical manner.

[0008] Another object of the present invention is to provide a method for electrochemical detection of hematopoietic stem cells to achieve quantitative detection of hematopoietic stem cells.

[0009] A composition for electrochemical detection of hematopoietic stem cells, comprising:

[0010] CD34 antibody functionalized magnetic beads,

[0011] CD31 antibody-conjugated nucleic acid probe,

[0012] CD45 antibody-conjugated nucleic acid probe,

[0013] Catalyzes the hairpin assembly reaction H1 chain,

[0014] Catalyzes the hairpin assembly reaction H2 chain,

[0015] crRNA, and

[0016] Methylene blue modified signal nucleic acid chain.

[0017] In the composition for detecting hematopoietic stem cells of the present invention, the nucleic acid probe sequence to which the CD31 antibody binds is:

[0018] 5′-TTTTTTTTTAAGTG-3′.

[0019] In the composition for detecting hematopoietic stem cells of the present invention, the nucleic acid probe sequence to which the CD45 antibody binds is:

[0020] 5′-TCTCTATCATTATTT-3′.

[0021] The crRNA sequence of the composition for detecting hematopoietic stem cells of the present invention is:

[0022] 5'-UAAUUUCUACUAAGUGUAGAUAAGUGUGAAGAU-3'.

[0023] The sequence of the signal nucleic acid chain of the composition for detecting hematopoietic stem cells of the present invention is:

[0024] 5'-ATGCCGATCCCCAAAAGTGTACACTTAATCGAAGCTTT-MB-3'.

[0025] The composition for detecting hematopoietic stem cells of the present invention has a sequence of the H1 chain that catalyzes the hairpin assembly reaction:

[0026] 5'-TCTCTATCATTAATCTTCATAATGATAGAGACACTT-3'.

[0027] The composition for detecting hematopoietic stem cells of the present invention has a sequence of the H2 chain that catalyzes the hairpin assembly reaction:

[0028] 5'-ATTCTCATCTCTATCATTATGAAGATTAATG-3'.

[0029] The composition for detecting hematopoietic stem cells of the present invention is used to capture hematopoietic stem cells in combination with immunomagnetic beads of CD34 antibodies. S1 with terminal modification N3 and S2 with terminal modification DBCO are respectively combined with CD31 antibody and CD45 antibody to form a specific antibody-nucleic acid composite probe. When the two probes are fixed to the surface of hematopoietic stem cells by identifying surface markers CD31 and CD45, the distance between S1 and S2 is shortened, and then the ends of S1 and S2 are connected together by click chemistry to form a long chain S. The partial sequence (T) of long chain S serves as an activation chain to open the probe chain H1 with a hairpin loop structure, produce a T / H1 double-stranded structure, and then expose the terminal sequence to open another nucleic acid probe H2 of the hairpin loop structure, produce H1 / H2 double-stranded and release T, and carry out the next round of hybridization reaction. The end of the H1 / H2 double-stranded structure contains a partial single-stranded structure, which can be used as the activation chain of the Cas12a system to activate the trans-cutting activity of the CRISPR / cas system, randomly cut the DNA chain, thereby releasing the terminal methylene blue molecule and generating an electrochemical signal.

[0030] When hematopoietic stem cells are present in the detection system, the capture probe (MBS@CD34) specifically recognizes hematopoietic stem cells. Simultaneously, the CD31 and CD45 proteins on the surface of hematopoietic stem cells bind to the corresponding antibody-modified S1 and S2 nucleic acid chains through an antibody-antigen reaction. Click chemistry then connects adjacent S1 and S2 chains into a long chain S, activating the catalytic hairpin assembly reaction and further activating the CRISPR / Cas system, generating an electrochemical signal.

[0031] The composition provided by the present invention is used to detect hematopoietic stem cells using an electrochemical method, thereby improving signal labeling efficiency and detection sensitivity.

[0032] A method for electrochemically detecting hematopoietic stem cells, comprising:

[0033] The test solution is mixed with CD34 antibody-functionalized magnetic beads and incubated overnight. After that, the magnetic beads bound to hematopoietic stem cells are extracted.

[0034] Then, a nucleic acid probe bound to a CD31 antibody and a nucleic acid probe bound to a CD45 antibody are added, and after reaction, magnetic separation is performed;

[0035] Then, the catalytic hairpin assembly reaction H1 chain of the hairpin loop structure and the catalytic hairpin assembly reaction H2 chain of the hairpin loop structure were added, and after the reaction, the supernatant was magnetically aspirated;

[0036] After that, crRNA and signal nucleic acid chain are added and CRISPR / cas reaction is carried out;

[0037] Finally, the generated free methylene blue molecules were incubated with a graphite electrode functionalized with cucurbit[7]uril (CB[7]), gold nanoparticles (AuNPs) and polydiallyldimethylammonium chloride (PDDA). They were captured by the hydrophobic cavity of CB[7] to form a stable host-guest complex and generate electrochemical signals. The detection of hematopoietic stem cells was achieved by measuring the electrochemical signals of methylene blue enriched on the surface of the functionalized graphite electrode.

[0038] Another method for electrochemically detecting hematopoietic stem cells includes:

[0039] Mix the test solution with CD34 antibody-functionalized magnetic beads and incubate them (e.g., 4°C ± 0.2°C). After overnight, extract the magnetic beads bound to hematopoietic stem cells.

[0040] Then, a nucleic acid probe bound to a CD31 antibody and a nucleic acid probe bound to a CD45 antibody are added, reacted (e.g., 37°C ± 0.2°C, 1-1.5 hours), and then magnetically separated;

[0041] Next, add the hairpin loop structure catalytic hairpin assembly reaction H1 chain (8-10 μL, 20-30 μM) and the hairpin loop structure catalytic hairpin assembly reaction H2 chain (8-10 μL, 20-30 μM), react (e.g., 37°C ± 0.2°C, 2 hours), and magnetically aspirate the supernatant;

[0042] After that, DEPC water, 10× Buffer, crRNA (20-25 mM, 2-5 μL), cas12a protein (20-30 mM, 4-6 μL), and signal nucleic acid chain (100-150 mM, 2-5 μL) were added, and the CRISPR / cas reaction was performed (e.g., 37°C ± 0.2°C, 1-1.5 hours);

[0043] Finally, the generated free methylene blue molecules were incubated with a graphite electrode functionalized with cucurbit[7]uril (CB[7]), gold nanoparticles (AuNPs) and polydiallyldimethylammonium chloride (PDDA). They were captured by the hydrophobic cavity of CB[7] to form a stable host-guest complex and generate electrochemical signals. The detection of hematopoietic stem cells was achieved by measuring the electrochemical signals of methylene blue enriched on the surface of the functionalized graphite electrode.

[0044] The supernatant was incubated on a functionalized graphite electrode at room temperature for 1–2 hours. The modified electrode was then thoroughly rinsed for electrochemical measurements. The solution was thoroughly deoxygenated with high-purity nitrogen and maintained anaerobic using a nitrogen flow throughout the electrochemical measurements. Square wave voltammetry (SWV) measurements were performed using a potential scan range of 0–0.6 V, a potential step of 4 mV, an amplitude of 25 mV, and a frequency of 15 Hz.

[0045] Beneficial effects achieved by the technical solution of the present invention:

[0046] When using a single marker to detect hematopoietic stem cells, the missed detection rate is usually high. Commonly used hematopoietic stem cell markers include CD31, CD45, and CD34. The present invention uses the surface markers CD31 and CD45 to construct an electrochemical analysis system based on surface dual recognition, which can effectively avoid false positive and false negative results caused by a single target, meeting the needs of higher specificity recognition analysis.

[0047] The present invention utilizes the characteristics of catalytic hairpin assembly (CHA) with low cost, easy operation and excellent signal amplification performance. At the same time, the catalytic hairpin assembly reaction is combined with the CRISPR / Cas system to further enhance the sensitivity of hematopoietic stem cell analysis.

[0048] The present invention takes advantage of the mild, simple and efficient characteristics of click chemistry reactions. At the same time, combined with electrochemical technology with the advantages of simple operation, sensitive signals and economical price, it can meet the needs of more convenient hematopoietic stem cell analysis.

[0049] The electrochemical technology used in the present invention is more convenient and sensitive, does not require complex sample processing and analysis by sophisticated instruments, and is expected to be realized in clinical applications and detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] Figure 1 Schematic diagram of the technical scheme for detecting hematopoietic stem cells;

[0051] Figure 2 This is a diagram showing the electrochemical signal amplification results of hematopoietic stem cell detection using the method of the present invention;

[0052] Figure 3 This is a graph showing the results of electrochemical specific detection of hematopoietic stem cell concentration using the method of the present invention;

[0053] Figure 4 The graph shows the electrochemical quantitative results of different numbers of hematopoietic stem cells. DETAILED DESCRIPTION

[0054] 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.

[0055] Figure 1 A roadmap for the technical solution for detecting hematopoietic stem cells. Specific methods include:

[0056] (1) Description of the reaction principle. Carboxylated magnetic beads are combined with CD34 antibodies through condensation to prepare immunomagnetic beads for capturing hematopoietic stem cells. S1 with N3 terminal modification and S2 with DBCO terminal modification bind to CD31 antibody and CD45 antibody respectively to form specific antibody-nucleic acid composite probes. When the two probes are fixed to the surface of hematopoietic stem cells by recognizing surface markers CD31 and CD45, the distance between S1 and S2 is shortened, and then the ends of S1 and S2 are connected together through click chemistry reaction to form a long chain S. Part of the sequence (T) of the long chain S can serve as an activation chain, which can open the probe chain H1 with a hairpin loop structure to produce a T / H1 double-stranded structure, thereby exposing the terminal sequence, which can open another nucleic acid probe H2 with a hairpin loop structure to produce an H1 / H2 double-stranded structure and release T for the next round of hybridization reaction. The end of the H1 / H2 double-stranded structure contains a partial single-stranded structure, which can serve as the activation chain of the Cas12a system, activate the trans-cutting activity of the CRISPR / cas system, randomly cut the DNA chain, thereby releasing the terminal methylene blue molecule and generating an electrochemical signal.

[0057] (2) 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 a CB[7] / AuNP / PDDA functionalized graphite electrode. The solution was thoroughly deoxygenated using high-purity nitrogen gas and nitrogen flow was used throughout the electrochemical detection process to maintain an oxygen-free environment for the solution. The parameters of square wave voltammetry (SWV) were as follows: potential scan range, Potential step, 4 mV; amplitude, 25 mV; frequency, 15 Hz.

[0058] (3) When hematopoietic stem cells are present in the detection system, the capture probe (MB S @CD34) specifically recognizes hematopoietic stem cells. Meanwhile, the CD31 and CD45 proteins on the surface of hematopoietic stem cells bind to the corresponding antibody-modified S1 and S2 nucleic acid chains through an antibody-antigen reaction. Click chemistry then connects adjacent S1 and S2 chains into a long S chain, activating the catalytic hairpin assembly reaction and further activating the CRISPR / Cas system, generating an electrochemical signal.

[0059] (4) When there are no hematopoietic stem cells in the detection system, the capture probe (MB S @CD34) cannot fix hematopoietic stem cells on the magnetic bead interface, and the free nucleic acids S1 and S2 cannot bind to the magnetic bead surface, inhibiting the occurrence of downstream reactions. Therefore, in the absence of hematopoietic stem cells, due to the electrostatic repulsion between the long DNA chain and cucurbit[7]uril, the terminally modified MB molecules cannot approach the electrode, inhibiting the electrochemical signal.

[0060] The solution adopted in the following embodiment of the present invention mainly includes the following steps:

[0061] (a) Prepare capture probes by mixing 8-10 μL of CD34 antibody and 80-100 μL of activated carboxyl-functionalized magnetic beads. Incubate at 25°C for 2-3 hours, then magnetically separate the beads and discard the solution. Add 45-100 μL of 10-15 mM PBS (pH 7.5), mix thoroughly again, and then magnetically separate the beads. Resuspend the CD34 antibody-functionalized beads in 1-2 mL of 10 mM PBS (pH 7.5) for later use.

[0062] (b) Antibody-functionalized magnetic beads capture hematopoietic stem cells. Take 80-100 μL of antibody-functionalized magnetic beads, add 150-200 μL of hematopoietic stem cells, and incubate at 4°C overnight. The next day, remove the beads and vortex to remove unbound cells.

[0063] (c) Antibody-nucleic acid probe synthesis. First, 10-15 μL of CD31 and CD45 were mixed with 10-15 μL of 10-15 mM SMCC and 80-100 μL of PBS, respectively, and reacted at room temperature for 2-3 hours. The activated antibodies were then ultrafiltered at 10,000-12,000 rpm for 10-15 minutes using a 30K ultrafiltration tube. The activated DNA strands were then reacted with the ultrafiltered antibodies at room temperature for 2-3 hours to produce antibody-nucleic acid probes S1 and S2.

[0064] (d) Click chemistry reaction: 80-100 μL of the hematopoietic stem cells captured in step (b) were taken and added to the prepared antibody-nucleic acid composite probes S1 and S2, respectively. The mixture was reacted at 37°C for 1-1.5 hours to allow S1 and S2 to fully bind to the hematopoietic stem cells. The mixture was then magnetically separated to remove unbound S1 and S2 in the supernatant.

[0065] (e) Catalytic hairpin assembly reaction: First, H1 (8-10 μL, 20-30 μM) and H2 chains (8-10 μL, 20-30 μM) were separately annealed at 95°C for 5 minutes and then naturally cooled to room temperature to form a stable hairpin loop structure. Then, 8-10 μL and 20-30 μM of H1 and H2, respectively, were added to the system in (d) and reacted at 37°C for 2 hours. The supernatant was then magnetically aspirated.

[0066] (f) CRISPR / cas reaction: DEPC water (42-45 μL), 10× Buffer (10-15 μL), crRNA (20-25 mM, 2-5 μL), cas12a protein (20-30 mM, 4-6 μL) and long-chain SS connected with MB signal (100-150 mM, 2-5 μL) were added to the system in (e) and reacted at 37°C for 1-1.5 hours.

[0067] (g) Modification of graphite electrode: The specific steps are as follows: PDDA solution (3.5-5 mg / ml) is applied to the polished graphite electrode and incubated for 20-30 minutes; after gently rinsing with ultrapure water, the gold nanoparticle solution is applied to the surface of the graphite electrode and incubated for 1-1.5 hours; finally, CB[7] (1.1-2.0 mg / ml) is applied to the surface of the graphite electrode and incubated for 1-1.5 hours.

[0068] (h) 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 a CB[7] / AuNP / PDDA functionalized graphite electrode. The solution was thoroughly deoxygenated by high-purity nitrogen gas, and nitrogen flow was used throughout the electrochemical detection process to maintain the solution anaerobic. The parameters of square wave voltammetry (SWV) were as follows: potential scan range, Potential step, 4 mV; amplitude, 25 mV; frequency, 15 Hz.

[0069] Wherein: the nucleic acid probe sequences of CD31 and CD45 used in step (c) are:

[0070] 5′-TTTTTTTTTAAGTG-3′ and 5′-TCTCTATCATTATTT-3′.

[0071] The sequences of H1 and H2 used in step (e) are:

[0072] 5'-TCTCTATCATTAATCTTCATAATGATAGAGACACTT-3',

[0073] 5'-ATTCTCATCTCTATCATTATGAAGATTAATG-3'

[0074] The sequence of the crRNA used in step (f) is:

[0075] 5'-UAAUUUCUACUAAGUGUAGAUAAGUGUGAAGAU-3';

[0076] The signal chain sequence with methylene blue (MB) labeling at the 3' end is:

[0077] 5'-ATGCCGATCCCCAAAAGTGTACACTTAATCGAAGCTTT-MB-3'.

[0078] Example 1: Verification of the electrochemical signal amplification effect for hematopoietic stem cell detection

[0079] The steps are as follows:

[0080] (a) Prepare capture probes by mixing 8-10 μL of CD34 antibody and 80-100 μL of activated carboxyl-functionalized magnetic beads. Incubate at 25°C for 2-3 hours, then magnetically separate the beads and discard the solution. Add 45-100 μL of 10-15 mM PBS (pH 7.5), mix thoroughly again, and then magnetically separate the beads. Resuspend the CD34 antibody-functionalized beads in 1-2 mL of 10 mM PBS (pH 7.5) for later use.

[0081] (b) Antibody-functionalized magnetic beads capture hematopoietic stem cells. Take 80-100 μL of antibody-functionalized magnetic beads, add 150-200 μL of hematopoietic stem cells, and incubate at 4°C overnight. The next day, remove the beads and vortex to remove unbound cells.

[0082] (c) Antibody-nucleic acid probe synthesis. First, 10-15 μL of CD31 and CD45, respectively, was mixed with 10-15 μL of 10-15 mM SMCC and 80-100 μL of PBS and reacted at room temperature for 2-37 hours. The activated antibodies were then ultrafiltered at 10,000-12,000 rpm for 10-15 minutes using a 30K ultrafiltration tube. The activated DNA strands were then reacted with the ultrafiltered antibodies at room temperature for 2-3 hours to produce antibody-nucleic acid probes S1 and S2.

[0083] (d) Click chemistry reaction: 80-100 μL of the hematopoietic stem cells captured in step (b) were taken and added to the prepared antibody-nucleic acid composite probes S1 and S2, respectively. The mixture was reacted at 37°C for 1-1.5 hours to allow S1 and S2 to fully bind to the hematopoietic stem cells. The mixture was then magnetically separated to remove unbound S1 and S2 in the supernatant.

[0084] (e) Catalytic hairpin assembly reaction: First, H1 (8-10 μL, 20-30 μM) and H2 chains (8-10 μL, 20-30 μM) were separately annealed at 95°C for 5 minutes and then naturally cooled to room temperature to form a stable hairpin loop structure. Then, 8-10 μL and 20-30 μM of H1 and H2, respectively, were added to the system in (d) and reacted at 37°C for 2 hours. The supernatant was then magnetically aspirated.

[0085] (f) CRISPR / cas reaction: DEPC water (42-45 μL), 10× Buffer (10-15 μL), crRNA (20-25 mM, 2-5 μL), cas12a protein (20-30 mM, 4-6 μL) and long-chain SS connected with MB signal (100-150 mM, 2-5 μL) were added to the system in (e) and reacted at 37°C for 1-1.5 hours.

[0086] (g) Modification of graphite electrode: The specific steps are as follows: PDDA solution (3.5-5 mg / ml) is applied to the polished graphite electrode and incubated for 20-30 minutes; after gently rinsing with ultrapure water, the gold nanoparticle solution is applied to the surface of the graphite electrode and incubated for 1-1.5 hours; finally, CB[7] (1.1-2.0 mg / ml) is applied to the surface of the graphite electrode and incubated for 1-1.5 hours.

[0087] (h) 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 a CB[7] / AuNP / PDDA functionalized graphite electrode. The solution was thoroughly deoxygenated by high-purity nitrogen gas, and nitrogen flow was used throughout the electrochemical detection process to maintain the solution anaerobic. The parameters of square wave voltammetry (SWV) were as follows: potential scan range, Potential step, 4 mV; amplitude, 25 mV; frequency, 15 Hz.

[0088] like Figure 2 As shown in the figure, curves a and b are the detection results of the full system and the system without Cas protein, respectively. It can be seen that after the signal amplification reaction assisted by Cas12a protein, the electrode surface can be significantly improved, while in the system without Cas protein, only a very low electrochemical signal can be obtained.

[0089] Example 2 Specific detection of hematopoietic stem cells

[0090] The steps are as follows:

[0091] (a) Prepare capture probes by mixing 8-10 μL of CD34 antibody and 80-100 μL of activated carboxyl-functionalized magnetic beads. Incubate at 25°C for 2-3 hours, then magnetically separate the beads and discard the solution. Add 45-100 μL of 10-15 mM PBS (pH 7.5), mix thoroughly again, and then magnetically separate the beads. Resuspend the CD34 antibody-functionalized beads in 1-2 mL of 10 mM PBS (pH 7.5) for later use.

[0092] (b) Antibody-functionalized magnetic beads capture hematopoietic stem cells. Take 80-100 μL of antibody-functionalized magnetic beads, add 150-200 μL of hematopoietic stem cells, and incubate at 4°C overnight. The next day, remove the beads and vortex to remove unbound cells.

[0093] (c) Antibody-nucleic acid probe synthesis. First, 10-15 μL of CD31 and CD45 were mixed with 10-15 μL of 10-15 mM SMCC and 80-100 μL of PBS, respectively, and reacted at room temperature for 2-3 hours. The activated antibodies were then ultrafiltered at 10,000-12,000 rpm for 10-15 minutes using a 30K ultrafiltration tube. The activated DNA strands were then reacted with the ultrafiltered antibodies at room temperature for 2-3 hours to produce antibody-nucleic acid probes S1 and S2.

[0094] (d) Click chemistry reaction: 80-100 μL of the hematopoietic stem cells captured in step (b) were taken and added to the prepared antibody-nucleic acid composite probes S1 and S2, respectively. The mixture was reacted at 37°C for 1-1.5 hours to allow S1 and S2 to fully bind to the hematopoietic stem cells. The mixture was then magnetically separated to remove unbound S1 and S2 in the supernatant.

[0095] (e) Catalytic hairpin assembly reaction: First, H1 (8-10 μL, 20-30 μM) and H2 chains (8-10 μL, 20-30 μM) were separately annealed at 95°C for 5 minutes and then naturally cooled to room temperature to form a stable hairpin loop structure. Then, 8-10 μL and 20-30 μM of H1 and H2, respectively, were added to the system in (d) and reacted at 37°C for 2 hours. The supernatant was then magnetically aspirated.

[0096] (f) CRISPR / cas reaction: DEPC water (42-45 μL), 10× Buffer (10-15 μL), crRNA (20-25 mM, 2-5 μL), cas12a protein (20-30 mM, 4-6 μL) and long-chain SS connected with MB signal (100-150 mM, 2-5 μL) were added to the system in (e) and reacted at 37°C for 1-1.5 hours.

[0097] (g) Modification of graphite electrode: The specific steps are as follows: PDDA solution (3.5-5 mg / ml) is applied to the polished graphite electrode and incubated for 20-30 minutes; after gently rinsing with ultrapure water, the gold nanoparticle solution is applied to the surface of the graphite electrode and incubated for 1-1.5 hours; finally, CB[7] (1.1-2.0 mg / ml) is applied to the surface of the graphite electrode and incubated for 1-1.5 hours.

[0098] (h) 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 a CB[7] / AuNP / PDDA functionalized graphite electrode. The solution was thoroughly deoxygenated by high-purity nitrogen gas, and nitrogen flow was used throughout the electrochemical detection process to maintain the solution anaerobic. The parameters of square wave voltammetry (SWV) were as follows: potential scan range, Potential step, 4 mV; amplitude, 25 mV; frequency, 15 Hz.

[0099] like Figure 3 As shown, curves a and b are respectively: hematopoietic stem cell system and normal cell MCF-10A. It can be seen that when target cells exist, the electrochemical signal on the electrode surface can be significantly improved.

[0100] Example 3 Quantitative Detection of Hematopoietic Stem Cells

[0101] The steps are as follows:

[0102] (a) Prepare capture probes by mixing 8-10 μL of CD34 antibody and 80-100 μL of activated carboxyl-functionalized magnetic beads. Incubate at 25°C for 2-3 hours, then magnetically separate the beads and discard the solution. Add 45-100 μL of 10-15 mM PBS (pH 7.5), mix thoroughly again, and then magnetically separate the beads. Resuspend the CD34 antibody-functionalized beads in 1-2 mL of 10 mM PBS (pH 7.5) for later use.

[0103] (b) Antibody-functionalized magnetic beads capture hematopoietic stem cells. Take 80-100 μL of antibody-functionalized magnetic beads, add 150-200 μL of hematopoietic stem cells, and incubate at 4°C overnight. The next day, remove the beads and vortex to remove unbound cells.

[0104] (c) Antibody-nucleic acid probe synthesis. First, 10-15 μL of CD31 and CD45 were mixed with 10-15 μL of 10-15 mM SMCC and 80-100 μL of PBS, respectively, and reacted at room temperature for 2-3 hours. The activated antibodies were then ultrafiltered at 10,000-12,000 rpm for 10-15 minutes using a 30K ultrafiltration tube. The activated DNA strands were then reacted with the ultrafiltered antibodies at room temperature for 2-3 hours to produce antibody-nucleic acid probes S1 and S2.

[0105] (d) Click chemistry reaction: 80-100 μL of the hematopoietic stem cells captured in step (b) were taken and added to the prepared antibody-nucleic acid composite probes S1 and S2, respectively. The mixture was reacted at 37°C for 1-1.5 hours to allow S1 and S2 to fully bind to the hematopoietic stem cells. The mixture was then magnetically separated to remove unbound S1 and S2 in the supernatant.

[0106] (e) Catalytic hairpin assembly reaction: First, H1 (8-10 μL, 20-30 μM) and H2 chains (8-10 μL, 20-30 μM) were separately annealed at 95°C for 5 minutes and then naturally cooled to room temperature to form a stable hairpin loop structure. Then, 8-10 μL and 20-30 μM of H1 and H2, respectively, were added to the system in (d) and reacted at 37°C for 2 hours. The supernatant was then magnetically aspirated.

[0107] (f) CRISPR / cas reaction: DEPC water (42-45 μL), 10× Buffer (10-15 μL), crRNA (20-25 mM, 2-5 μL), cas12a protein (20-30 mM, 4-6 μL) and long-chain SS connected with MB signal (100-150 mM, 2-5 μL) were added to the system in (e) and reacted at 37°C for 1-1.5 hours.

[0108] (g) Modification of graphite electrode: The specific steps are as follows: PDDA solution (3.5-5 mg / ml) is applied to the polished graphite electrode and incubated for 20-30 minutes; after gently rinsing with ultrapure water, the gold nanoparticle solution is applied to the surface of the graphite electrode and incubated for 1-1.5 hours; finally, CB[7] (1.1-2.0 mg / ml) is applied to the surface of the graphite electrode and incubated for 1-1.5 hours.

[0109] (h) 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 a CB[7] / AuNP / PDDA functionalized graphite electrode. The solution was thoroughly deoxygenated by high-purity nitrogen gas, and nitrogen flow was used throughout the electrochemical detection process to maintain the solution anaerobic. The parameters of square wave voltammetry (SWV) were as follows: potential scan range, Potential step, 4 mV; amplitude, 25 mV; frequency, 15 Hz.

[0110] Subsequently, electrochemical analysis was performed on a series of different concentrations of hematopoietic stem cells, including 1×10 4 , 1×10 5 , 1×10 6 and 1×10 7 The electrochemical quantitative results of different numbers of hematopoietic stem cells are as follows. Figure 4 As shown in Figure 2, the peak current increases with the increase in the number of hematopoietic stem cells. When the number of cells reaches 1×10 7 When the current value reaches saturation, it is consistent with the expected result. The increase in the number of hematopoietic stem cells increases the number of activation chains marked on their surface, enhances the trans-cleavage activity of the Cas12a protein, and further enhances the response value of the electrochemical signal.

[0111] Project name:Compositions for electrochemical detection of hematopoieticstem cells and methids thereof

[0112] Status: Generated

[0113] Creation Date:2023-05-11

[0114] Basic Information

[0115] Current Application

[0116] Application number:before

[0117] Intellectual Property Office: CN

[0118] Application Number:

[0119] Application date:

[0120] Applicant file name: 021009.23106XL

[0121] Invention Name

[0122] language Inventor's name zh Composition and method for electrochemical detection of hematopoietic stem cells

[0123] Applicant and Inventor:

[0124] Inventor Name: Zhang Yi

[0125] Language:zh

[0126] Latin name: Zhang Yi

[0127] Residential address:

[0128] Correspondence address:

[0129] Name of applicant: China Stem Cell Group Shanghai Biotechnology Co., Ltd.

[0130] Language:zh

[0131] Latin name: China stem cell group Shanghai Biotechnology Co., Ltd

[0132] Residential address:

[0133] Correspondence address:

[0134] sequence

[0135] Sequence 1: "CD31 antibody-bound nucleic acid probe"

[0136]

[0137] feature

[0138]

[0139] residue

[0140] tttttttttta agtg 14 sequence 2: "CD45 antibody-bound nucleic acid probe"

[0141]

[0142] feature

[0143]

[0144] residue

[0145] tctctatcat tattt 15 sequence 3: "crRNA"

[0146]

[0147] feature

[0148]

[0149] residue

[0150] taatttctac taagtgtaga taagtgtgaa gat 33 sequence 4: "signal nucleic acid chain"

[0151]

[0152] feature

[0153]

[0154] residue

[0155] atgccgatcc ccaaaagtgt acacttaatc gaagcttt 38 Sequence 5: "H1 chain"

[0156]

[0157] feature

[0158]

[0159] residue

[0160] tctctatcat taatcttcat aatgatagag acactt 36 Sequence 6: "H2 chain"

[0161]

[0162] feature

[0163]

[0164] residue

[0165] atcttcatct ctatcattat gaagattaat g 31

Claims

1. A method for electrochemical detection of hematopoietic stem cells, characterized in that include: The test solution is mixed with CD34 antibody-functionalized magnetic beads and incubated overnight. After that, the magnetic beads bound to hematopoietic stem cells are extracted. Then, a nucleic acid probe bound to a CD31 antibody and a nucleic acid probe bound to a CD45 antibody are added, and after reaction, magnetic separation is performed; Then, the catalytic hairpin assembly reaction H1 chain of the hairpin loop structure and the catalytic hairpin assembly reaction H2 chain of the hairpin loop structure were added, and after the reaction, the supernatant was magnetically aspirated; After that, crRNA and signal nucleic acid chain are added and CRISPR / cas reaction is carried out; Finally, the generated free methylene blue molecules were incubated with a graphite electrode functionalized with cucurbit[7]uril, gold nanoparticles, and polydiallyldimethylammonium chloride. They were captured by the hydrophobic cavity of cucurbit[7]uril to form a stable host-guest complex, generating an electrochemical signal. The electrochemical signal of methylene blue enriched on the surface of the functionalized graphite electrode was measured to detect hematopoietic stem cells. The nucleic acid probe sequence bound by the CD31 antibody is: 5′-TTTTTTTTTAAGTG-3′; The nucleic acid probe sequence bound by the CD45 antibody is: 5′-TCTCTATCATTATTT-3′; The crRNA sequence is: 5'-UAAUUUCUACUAAGUGUAGAUAAGUGUGAAGAU-3'; The sequence of the signal nucleic acid chain is: 5'-ATGCCGATCCCCAAAAGTGTACACTTAATCGAAGCTTT-MB-3'; The sequence of the H1 chain of the catalytic hairpin assembly reaction is: 5'-TCTCTATCATTAATCTTCATAATGATAGAGACACTT-3'; The sequence of the H2 chain catalyzing the hairpin assembly reaction is: 5'-ATTCTCATCTCTATCATTATGAAGATTAATG-3'.

2. The method for electrochemical detection of hematopoietic stem cells according to claim 1, characterized in that The electrochemical detection analysis was performed by square wave voltammetry with a scanning range of 0 V to 0.6 V, an amplitude of 25 mV, a potential step of 4 mM, and a frequency of 15 Hz.

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