Method for detecting hematopoietic stem cells based on DNA self-replication circulation

Abstract

A method for detecting hematopoietic stem cells based on DNA self-replication circulation comprises five nucleic acid chains of T, H1, H2, H3 and H4, the 5'end of the T chain is covalently connected with a CD45 antibody to form a probe, and H1, H2, H3 and H4 are hairpin nucleic acid chains. CD34 and CD45 on the surfaces of the hematopoietic stem cells are selected as targets, a fluorescent detection method for accurate recognition and quantitative analysis of the hematopoietic stem cells is constructed by means of a tree-shaped DNA structure formed by DNA self-replication circulation formed by catalytic hairpin structure reaction, and sensitive detection of the hematopoietic stem cells can be achieved.

Description

Technical Field

[0001] The invention relates to a method for detection, in particular to a method for applying a DNA self-replication cycle to perform qualitative and quantitative detection on hematopoietic stem cells. Background Art

[0002] Hematopoietic stem cells refer to cells that have not yet matured. They are the origin of all hematopoietic cells and immune cells. They can not only differentiate into red blood cells, white blood cells and platelets, but also differentiate into cells of various tissues and organs across systems. They have the potential for self-renewal, multidirectional differentiation and homing (i.e., directional migration to hematopoietic tissues and organs), so they are also called multifunctional stem cells. At present, hematopoietic stem cell transplantation can treat 75 fatal diseases such as malignant blood diseases, some malignant tumors, and some genetic diseases. Including acute leukemia, chronic leukemia, hematopoietic stem cell diseases, myeloproliferative diseases, macrophage diseases, inherited metabolic diseases, etc.

[0003] Due to the insulating properties of the proteins on the cell surface, direct detection will greatly weaken the detection signal of the biosensor, thereby reducing the detection sensitivity of the biosensor. Therefore, in order to achieve the purpose of accurate detection, it is necessary to develop a biorecognition device that can reduce the obstruction of cells and cell surfaces. Among them, the self-replication cycle reaction is an autocatalytic system initiated by T. The tree-like DNA structure of the product formed can not only eliminate the influence of the cell's own steric hindrance on the interface reaction, but also the method has a relatively strong versatility and can be used to detect a variety of cancer markers.

[0004] At present, the main methods for detecting and collecting hematopoietic stem cells include colony culture method, flow cytometry detection, etc. However, these methods are not only susceptible to interference, but also have high experimental technical requirements and costs, and also have the disadvantage of cumbersome data processing. We proposed a tree-like DNA structure mediated by hematopoietic stem cell surface markers CD34 and CD45, which is used for the detection of hematopoietic stem cells through catalytic hairpin assembly and cascade amplification reactions. Compared with single-stranded DNA, the tree-like DNA structure can not only eliminate the influence of the cell's own steric hindrance on the interface reaction, but also can achieve accurate identification and quantitative detection of hematopoietic stem cells without complex sample processing and analysis of precision instruments.

[0005] Fluorescence spectrometer is the main method used to study the interaction between small molecules and nucleic acids, and is also one of the commonly used instruments for studying proteins. The interaction between drugs and proteins may cause changes in the drug's autofluorescence spectrum, protein's autofluorescence spectrum, and synchronous fluorescence spectrum, which can provide information on the binding of drugs and proteins. Fluorescence spectrometers have the advantages of fast analysis speed, non-destructive analysis, high analysis accuracy, simple sample preparation, repeated measurement of the same sample, and reproducibility. They are widely used in chemistry, biochemistry, and environmental fields. Summary of the invention

[0006] One object of the present invention is to provide a method for detecting hematopoietic stem cells, which is based on the method of DNA self-replication cycle to perform efficient qualitative detection of hematopoietic stem cells.

[0007] Another object of the present invention is to provide a method for detecting hematopoietic stem cells, which can be used for efficient quantitative detection of hematopoietic stem cells based on a composition of a DNA self-replication cycle.

[0008] A method for detecting hematopoietic stem cells, comprising five nucleic acid chains, T, H1, H2, H3 and H4, each nucleic acid chain sequence is as follows:

[0009] T: 5'-GACACTCGCTTCATCTTCATCTCCGACACTC-3';

[0010] H1: 5'-GCTTCATCTTCGAGTGTCGGAGATGAAGATGAAGCCATCGTGCTTCAT CTTCATCTCCG-3';

[0011] H2: 5'-GCTTCATCTTCATCTCCGATCTCGTTTTGCGGAGATGAAGATGAAGCA CGATG-3';

[0012] H3: 5'-CAAAACGAGATCGGAGATGAAGATGAAGCTTGCCTGCTTCATCTTCATCTCCGATCTCCGACACTC-3';

[0013] H4: 5'-GCTTCATCTTCATCTCCGACACTCCGGAGATGAAGATGAAGCAGGCA A-3'.

[0014] The 5' end of the T strand of the DNA composition of the present invention is modified with a thiol group (-SH).

[0015] The 5' end of the H4 chain of the DNA composition of the present invention is modified with a first fluorescent group (FAM).

[0016] The 3' end of the H4 chain of the DNA composition of the present invention is modified by a second fluorescent group (TAMRA).

[0017] The 5' end of the T chain of the DNA composition of the present invention is covalently linked to the CD45 antibody to form a probe.

[0018] In the DNA composition of the present invention, H1, H2, H3 and H4 are hairpin nucleic acid chains.

[0019] Hairpin nucleic acids H1, H2, H3 and FAM-H4-TAMRA modified with fluorescent and quenching groups at both ends are added to the reaction system. The CD45-T probe can open the H1 hairpin, the opened H1 hairpin can open the H2 hairpin, the opened H2 hairpin can open the H3 hairpin, and the opened H3 can open the FAM-H4-TAMRA hairpin. Therefore, CD45-T can make the four hairpin nucleic acids H1, H2, H3, and FAM-H4-TAMRA form a loop unit through base complementary pairing. Because the opened FAM-H4-TAMRA hairpin can open the H1 hairpin and play the role of CD45-T probe, 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 play the role of CD45-T and continue to open the hairpin nucleic acid. Therefore, the hairpin nucleic acid gradually extends in the shape of a tree based on T.

[0020] The composition of the present invention uses CD34 and CD45 on the surface of hematopoietic stem cells as targets, and constructs a fluorescent detection method for accurate identification and quantitative analysis of hematopoietic stem cells by means of a tree-like DNA structure formed by a DNA self-replication cycle formed by a catalytic hairpin structure reaction, thereby enabling sensitive detection of hematopoietic stem cells.

[0021] The method for using the DNA composition of the present invention to detect hematopoietic stem cells is as follows:

[0022] Dilute the hematopoietic stem cells after centrifugation, take 100-150 μL of CD34 antibody functionalized magnetic beads, add 200 μL of hematopoietic stem cells, place at 4°C overnight, take out the vortex and magnetically absorb the next day to remove unbound cells;

[0023] Then, the hematopoietic stem cells were incubated with 50-60 μL of CD45 antibody-functionalized CD45-T for about 30 minutes, and then washed with PBS (e.g., 10 mM, pH 7.5) after magnetic absorption;

[0024] Then, add 50-60 μL of a mixed solution of H1, H2, H3 and FAM-H4-TAMRA (all at 1 μM concentration, diluted with PBS), incubate for 60-90 minutes, separate by magnetic absorption, wash with PBS (10 mM, pH 7.5), and resuspend (e.g., 100 μL PBS);

[0025] Finally, fluorescence spectrometer was used for detection.

[0026] Compared with the prior art, the technical solution of the present invention has the following beneficial effects:

[0027] The CD34 protein on the surface of hematopoietic stem cells can specifically bind to CD34 antibody-functionalized magnetic beads and thus efficiently capture hematopoietic stem cells on the magnetic bead interface. It has the advantages of good selectivity and high capture efficiency, and can simply and efficiently enrich hematopoietic stem cells even in a complex serum environment.

[0028] 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.

[0029] Compared with single-stranded DNA products, the tree-like DNA structure not only enhances the signal amplification efficiency, but also significantly improves the sensitivity and accuracy of detection through combination with fluorescent signal output, eliminates the influence of the cell's own steric hindrance on the interface reaction, and does not require complex sample processing and precision instrument analysis to achieve accurate identification and quantitative detection of hematopoietic stem cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 This is a schematic diagram of the principle of detecting hematopoietic stem cells according to the present invention;

[0031] Figure 2 The figure is the result of agarose gel electrophoresis;

[0032] Figure 3 It is the fluorescence detection result diagram of each detection system;

[0033] Figure 4 This is the fluorescence detection result of hematopoietic stem cells and normal breast cells MCF-10A at the same concentration;

[0034] Figure 5 The graph shows the fluorescence intensity response results of various hematopoietic stem cell concentrations. DETAILED DESCRIPTION

[0035] 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 only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention is 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 can be modified or replaced by equivalents without departing from the spirit and scope of the technical solution of the present invention, which should be included in the scope of the claims of the present invention.

[0036] Figure 1 The schematic diagram of the present invention for detecting hematopoietic stem cells. The specific method includes:

[0037] (1) Preparation and capture of functional magnetic beads: The activated carboxylated magnetic beads can react with the exposed amino groups on the CD34 antibody to form a stable amide bond, which can then be used for the specific capture of hematopoietic stem cells.

[0038] (2) The CD45-T probe, which is antibody-conjugated to CD45, can specifically bind to the CD45 protein on the surface of hematopoietic stem cells. When hematopoietic stem cells are present in the reaction system, the CD45-T probe can be further coupled to the hematopoietic stem cells enriched on the functional magnetic beads. Subsequently, when H1, H2, H3 and FAM-H4-TAMRA with hairpin structures are added to the reaction system, the CD45-T probe can stimulate a self-replication cycle, eventually forming a tree-like DNA structure.

[0039] (3) The specific process of the self-replication cycle reaction is as follows: hairpin nucleic acids H1, H2, H3 and FAM-H4-TAMRA modified with fluorescent and quenching groups at both ends are added to the above reaction solution, and the CD45-T probe coupled to the hematopoietic stem cell 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 FAM-H4-TAMRA, thereby restoring the FAM fluorescence. Therefore, the CD45-T probe can restore the FAM fluorescence of H1, H2, H3, FAM-H4-TAMRA. A four hairpin nucleic acids form a loop unit through base complementary pairing. The opened FAM-H4-TAMRA can continue to open the hairpin structure of H1 and act as a CD45-T probe, 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 CD45-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 CD45-T probe, and finally forms a tree-like DNA structure.

[0040] (4) When hematopoietic stem cells are present in the reaction system, they can be captured by functionalized magnetic beads and coupled to the CD45-T probe, which then triggers a self-replication cycle to form a tree-like DNA structure. After the H4 hairpin structure is opened, the FAM fluorescence is restored. Therefore, hematopoietic stem cells can be sensitively detected by measuring the fluorescence intensity of the final reaction system.

[0041] (5) When there are no hematopoietic stem cells in the detection system, the added CD45 antibody functionalized nucleic acid probe CD45-T is free in the reaction system and is removed after magnetic cleaning. It cannot activate the self-replication cycle, the H4 hairpin structure will not be opened, and the FAM fluorescence is quenched by the quenching group. Therefore, no fluorescence signal can be detected in the final reaction system.

[0042] Example 1

[0043] The steps for verifying the DNA self-replication cycle are as follows:

[0044] (a) Preparation and verification of dendritic DNA. Since the number of bases in dendritic DNA is relatively large, agarose gel electrophoresis is used to verify the formation of dendritic DNA in the verification stage of the experiment. In the preparation stage of the experiment, the four DNA single strands need to be annealed. The specific operation is to place the DNA strands in a 95℃ metal bath for 5 to 10 minutes and then slowly cool them to room temperature. Then, the mixed solution of T and H1, H2, H3 and H4 is placed in a 37℃ metal bath for reaction for 120 to 130 minutes, and then 5 to 6 μL of the reaction solution is mixed with the DNA ladder and 5× Loading buffer, and then electrophoresed for 30 to 40 minutes under 1×TAE buffer and 80 to 120V voltage. The agarose is imaged using the Doc XR+ gel imaging system.

[0045] The sequences of the DNA used in step (a) are:

[0046] T: 5'-SH-GACACTCGCTTCATCTTCATCTCCGACACTC-3';

[0047] H1: 5'-GCTTCATCTTCGAGTGTCGGAGATGAAGATGAAGCCATCGTGCTTCAT CTTCATCTCCG-3';

[0048] H2: 5'-GCTTCATCTTCATCTCCGATCTCGTTTTGCGGAGATGAAGATGAAGCA CGATG-3';

[0049] H3: 5'-CAAAACGAGATCGGAGATGAAGATGAAGCTTGCCTGCTTCATCTTCATCTCCGATCTCCGACACTC-3';

[0050] FAM-H4-TAMRA: 5'-FAM-GCTTCATCTTCATCTCCGACACTCCGGAGATGAAGATGAAGCAGGCAA-TAMRA-3'.

[0051] The results of agarose gel electrophoresis verification of DNA self-replication cycle are as follows Figure 2As shown, a tree-like DNA structure can only be formed when T, H1, H2, H3 and H4 exist at the same time (lane 6). The mixed sample of H1, H2, H3 and H4 cannot form a tree-like DNA due to the absence of T (lane 5), thereby verifying the occurrence of the DNA self-replication cycle and the formation of the tree-like DNA structure product.

[0052] Example 2

[0053] Fluorescence qualitative analysis of hematopoietic stem cells, the steps are as follows:

[0054] (a) Activate carboxyl functionalized magnetic beads. Take 50-60 μL of commercial magnetic beads, add 1-1.2 mL PBS (10 mM, pH 7.5), mix and wash thoroughly, then perform magnetic separation and discard the solution. Repeat the washing three times. After magnetic absorption and discarding the supernatant, resuspend in 450-500 μL (0.22 M) EDC / NHS mixed solution for activation, react at room temperature for 30-40 minutes, wash three times and resuspend in 1 mL PBS for use. Take the above activated magnetic beads and incubate with 10-15 μL CD34 antibody at room temperature for 2-2.5 hours to obtain antibody functionalized magnetic beads.

[0055] (b) Preparation and verification of dendritic DNA. Since the number of bases in dendritic DNA is relatively large, agarose gel electrophoresis is used to verify the formation of dendritic DNA in the verification stage of the experiment. In the preparation stage of the experiment, the four DNA single strands need to be annealed. The specific operation is to place the DNA strands in a 95°C metal bath for 5 to 10 minutes and then slowly cool them to room temperature. Then, the mixed solution of T and H1, H2, H3 and H4 is placed in a 37°C metal bath for reaction for 120 to 130 minutes, and then 5 to 6 μL of the reaction solution is mixed with the DNA ladder and 5× Loading buffer, and then electrophoresed for 30 to 40 minutes under 1×TAE buffer and 80 to 120V voltage. The agarose is imaged using the Doc XR+ gel imaging system.

[0056] (c) Preparation of CD45-T probe functionalized with CD45 antibody, specific process: CD45 antibody (8-10 μL) and sulfo-SMCC (10-15 μL) were incubated in PBS (80-100 μL) at 30°C for 60-90 minutes. At the same time, T was reacted with TCEP (10-15 μL, 200 μM) in PBS (80-100 μL) at 30°C for 60-90 minutes. Then, the above solution was placed in a centrifuge tube at room temperature for 60-120 minutes, after which the mixture was transferred to a 30kD ultrafiltration centrifuge tube and centrifuged at 14,000 rpm for 5-10 minutes. After repeating the centrifugation three times, the upper filtrate was collected as a CD45-T probe.

[0057] (d) The specific process of the self-replication cycle reaction is as follows: hairpin nucleic acids H1, H2, H3 and FAM-H4-TAMRA modified with fluorescent and quenching groups at both ends are added to the reaction system, CD45-T probe can open H1 hairpin, the opened H1 hairpin can open H2 hairpin, the opened H2 hairpin can open H3 hairpin, and the opened H3 can open FAM-H4-TAMRA hairpin, so CD45-T can make H1, H2, H3, FAM-H4-TAMRA four A hairpin nucleic acid forms a loop unit through base complementary pairing. Because the opened FAM-H4-TAMRA hairpin can open the H1 hairpin and act as a CD45-T probe, 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 play the role of CD45-T and continue to open the hairpin nucleic acid. Therefore, the hairpin nucleic acid gradually extends in the shape of a tree based on T.

[0058] (e) Dilute the hematopoietic stem cells after centrifugation to the required concentration, take 100-150 μL antibody-functionalized magnetic beads, add 200 μL hematopoietic stem cells, place at 4°C overnight, take out the vortex magnetic aspiration the next day, and remove unbound cells. Then, incubate the hematopoietic stem cells with 50-60 μL CD45 antibody-functionalized CD45-T for about 30 minutes, wash three times with PBS (10mM, pH 7.5) after magnetic aspiration, and then add 50-60 μL of a mixed solution of H1, H2, H3 and FAM-H4-TAMRA (all at 1 μM, diluted with PBS), incubate for 60-90 minutes, separate by magnetic aspiration, wash three times with PBS (10mM, pH 7.5), and resuspend in 100 μL PBS.

[0059] (f) Fluorescence spectrometer detection: The solution in (e) was detected by fluorescence spectrometer.

[0060] in:

[0061] The sequences of the DNA used in step (b) are:

[0062] T: 5'-SH-GACACTCGCTTCATCTTCATCTCCGACACTC-3';

[0063] H1: 5'-GCTTCATCTTCGAGTGTCGGAGATGAAGATGAAGCCATCGTGCTTCAT CTTCATCTCCG-3';

[0064] H2: 5'-GCTTCATCTTCATCTCCGATCTCGTTTTGCGGAGATGAAGATGAAGCA CGATG-3';

[0065] H3: 5'-CAAAACGAGATCGGAGATGAAGATGAAGCTTGCCTGCTTCATCTTCATCTCCGATCTCCGACACTC-3';

[0066] FAM-H4-TAMRA: 5'-FAM-GCTTCATCTTCATCTCCGACACTCCGGAGATGAAGATGAAGCAGGCAA-TAMRA-3'.

[0067] The fluorescence detection results are as follows Figure 3 As shown, when hematopoietic stem cells are present, the CD45-T probe can be coupled to the hematopoietic stem cells and stimulate the self-replicating DNA circulation system, the FAM fluorescence is restored, and a strong fluorescence signal can be monitored (curve a), while the fluorescence signal of the blank control group without hematopoietic stem cells is greatly reduced (curve d). At the same time, if the excitation chain CD45-T (curve b) or H1 (curve c) is not added, a fluorescence signal almost consistent with the blank group will be obtained. The above results prove that the method provided in this embodiment successfully detects hematopoietic stem cells qualitatively.

[0068] Example 3

[0069] The specific detection and analysis of hematopoietic stem cells is as follows:

[0070] (a) Activate carboxyl functionalized magnetic beads. Take 50-60 μL of commercial magnetic beads, add 1-1.2 mL PBS (10 mM, pH 7.5), mix and wash thoroughly, then perform magnetic separation and discard the solution. Repeat the washing three times. After magnetic absorption and discarding the supernatant, resuspend in 450-500 μL (0.22 M) EDC / NHS mixed solution for activation, react at room temperature for 30-40 minutes, wash three times and resuspend in 1 mL PBS for use. Take the above activated magnetic beads and incubate with 10-15 μL CD34 antibody at room temperature for 2-2.5 hours to obtain antibody functionalized magnetic beads.

[0071] (b) Preparation and verification of dendritic DNA. Since the number of bases in dendritic DNA is relatively large, agarose gel electrophoresis is used to verify the formation of dendritic DNA in the verification stage of the experiment. In the preparation stage of the experiment, the four DNA single strands need to be annealed. The specific operation is to place the DNA strands in a 95°C metal bath for 5 to 10 minutes and then slowly cool them to room temperature. Then, the mixed solution of T and H1, H2, H3 and H4 is placed in a 37°C metal bath for reaction for 120 to 130 minutes, and then 5 to 6 μL of the reaction solution is mixed with the DNA ladder and 5× Loading buffer, and then electrophoresed for 30 to 40 minutes under 1×TAE buffer and 80 to 120V voltage. The agarose is imaged using the Doc XR+ gel imaging system.

[0072] (c) Preparation of CD45-T probe functionalized with CD45 antibody, specific process: CD45 antibody (8-10 μL) and sulfo-SMCC (10-15 μL) were incubated in PBS (80-100 μL) at 30°C for 60-90 minutes. At the same time, T was reacted with TCEP (10-15 μL, 200 μM) in PBS (80-100 μL) at 30°C for 60-90 minutes. Then, the above solution was placed in a centrifuge tube at room temperature for 60-120 minutes, after which the mixture was transferred to a 30kD ultrafiltration centrifuge tube and centrifuged at 14,000 rpm for 5-10 minutes. After repeating the centrifugation three times, the upper filtrate was collected as a CD45-T probe.

[0073] (d) The specific process of the self-replication cycle reaction is as follows: hairpin nucleic acids H1, H2, H3 and FAM-H4-TAMRA modified with fluorescent and quenching groups at both ends are added to the reaction system, CD45-T probe can open H1 hairpin, the opened H1 hairpin can open H2 hairpin, the opened H2 hairpin can open H3 hairpin, and the opened H3 can open FAM-H4-TAMRA hairpin, so CD45-T can make H1, H2, H3, FAM-H4-TAMRA four A hairpin nucleic acid forms a loop unit through base complementary pairing. Because the opened FAM-H4-TAMRA hairpin can open the H1 hairpin and act as a CD45-T probe, 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 play the role of CD45-T and continue to open the hairpin nucleic acid. Therefore, the hairpin nucleic acid gradually extends in the shape of a tree based on T.

[0074] (e) Dilute the hematopoietic stem cells after centrifugation to the required concentration, take 100-150 μL antibody-functionalized magnetic beads, add 200 μL hematopoietic stem cells, place at 4°C overnight, take out the vortex magnetic aspiration the next day, and remove unbound cells. Then, incubate the hematopoietic stem cells with 50-60 μL CD45 antibody-functionalized CD45-T for about 30 minutes, wash three times with PBS (10mM, pH 7.5) after magnetic aspiration, and then add 50-60 μL of a mixed solution of H1, H2, H3 and FAM-H4-TAMRA (all at 1 μM, diluted with PBS), incubate for 60-90 minutes, separate by magnetic aspiration, wash three times with PBS (10mM, pH 7.5), and resuspend in 100 μL PBS.

[0075] (f) Fluorescence spectrometer detection: The solution in (e) was detected by fluorescence spectrometer.

[0076] in:

[0077] The sequences of the DNA used in step (b) are:

[0078] T: 5'-SH-GACACTCGCTTCATCTTCATCTCCGACACTC-3';

[0079] H1: 5'-GCTTCATCTTCGAGTGTCGGAGATGAAGATGAAGCCATCGTGCTTCAT CTTCATCTCCG-3';

[0080] H2: 5'-GCTTCATCTTCATCTCCGATCTCGTTTTGCGGAGATGAAGATGAAGCA CGATG-3';

[0081] H3: 5'-CAAAACGAGATCGGAGATGAAGATGAAGCTTGCCTGCTTCATCTTCATCTCCGATCTCCGACACTC-3';

[0082] FAM-H4-TAMRA: 5'-FAM-GCTTCATCTTCATCTCCGACACTCCGGAGATGAAGATGAAGCAGGCAA-TAMRA-3'.

[0083] The method of this example was used to analyze the fluorescence signals obtained from hematopoietic stem cells and normal breast cells MCF-10A at the same concentration. The results are as follows: Figure 4 As shown, the fluorescence signal obtained by analyzing hematopoietic stem cells is much higher than that of MCF-10A cells, which proves that the detection method of this embodiment has good specificity.

[0084] Example 4

[0085] Fluorescence quantitative detection of hematopoietic stem cells, the steps are as follows:

[0086] (a) Activate carboxyl functionalized magnetic beads. Take 50-60 μL of commercial magnetic beads, add 1-1.2 mL PBS (10 mM, pH 7.5), mix and wash thoroughly, then perform magnetic separation and discard the solution. Repeat the washing three times. After magnetic absorption and discarding the supernatant, resuspend in 450-500 μL (0.22 M) EDC / NHS mixed solution for activation, react at room temperature for 30-40 minutes, wash three times and resuspend in 1 mL PBS for use. Take the above activated magnetic beads and incubate with 10-15 μL CD34 antibody at room temperature for 2-2.5 hours to obtain antibody functionalized magnetic beads.

[0087] (b) Preparation and verification of dendritic DNA. Since the number of bases in dendritic DNA is relatively large, agarose gel electrophoresis is used to verify the formation of dendritic DNA in the verification stage of the experiment. In the preparation stage of the experiment, the four DNA single strands need to be annealed. The specific operation is to place the DNA strands in a 95°C metal bath for 5 to 10 minutes and then slowly cool them to room temperature. Then, the mixed solution of T and H1, H2, H3 and H4 is placed in a 37°C metal bath for reaction for 120 to 130 minutes, and then 5 to 6 μL of the reaction solution is mixed with the DNA ladder and 5× Loading buffer, and then electrophoresed for 30 to 40 minutes under 1×TAE buffer and 80 to 120V voltage. The agarose is imaged using the Doc XR+ gel imaging system.

[0088] (c) Preparation of CD45-T probe functionalized with CD45 antibody, specific process: CD45 antibody (8-10 μL) and sulfo-SMCC (10-15 μL) were incubated in PBS (80-100 μL) at 30°C for 60-90 minutes. At the same time, T was reacted with TCEP (10-15 μL, 200 μM) in PBS (80-100 μL) at 30°C for 60-90 minutes. Then, the above solution was placed in a centrifuge tube at room temperature for 60-120 minutes, after which the mixture was transferred to a 30kD ultrafiltration centrifuge tube and centrifuged at 14,000 rpm for 5-10 minutes. After repeating the centrifugation three times, the upper filtrate was collected as a CD45-T probe.

[0089] (d) The specific process of the self-replication cycle reaction is as follows: hairpin nucleic acids H1, H2, H3 and FAM-H4-TAMRA modified with fluorescent and quenching groups at both ends are added to the reaction system, CD45-T probe can open H1 hairpin, the opened H1 hairpin can open H2 hairpin, the opened H2 hairpin can open H3 hairpin, and the opened H3 can open FAM-H4-TAMRA hairpin, so CD45-T can make H1, H2, H3, FAM-H4-TAMRA four A hairpin nucleic acid forms a loop unit through base complementary pairing. Because the opened FAM-H4-TAMRA hairpin can open the H1 hairpin and act as a CD45-T probe, 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 play the role of CD45-T and continue to open the hairpin nucleic acid. Therefore, the hairpin nucleic acid gradually extends in the shape of a tree based on T.

[0090] (e) Dilute the hematopoietic stem cells after centrifugation to the required concentration, take 100-150 μL antibody-functionalized magnetic beads, add 200 μL hematopoietic stem cells, place at 4°C overnight, take out the vortex magnetic aspiration the next day, and remove unbound cells. Then, incubate the hematopoietic stem cells with 50-60 μL CD45 antibody-functionalized CD45-T for about 30 minutes, wash three times with PBS (10mM, pH 7.5) after magnetic aspiration, and then add 50-60 μL of a mixed solution of H1, H2, H3 and FAM-H4-TAMRA (all at 1 μM, diluted with PBS), incubate for 60-90 minutes, separate by magnetic aspiration, wash three times with PBS (10mM, pH 7.5), and resuspend in 100 μL PBS.

[0091] (f) Fluorescence spectrometer detection: The solution in (e) was detected by fluorescence spectrometer.

[0092] in:

[0093] The sequences of the DNA used in step (b) are:

[0094] T: 5'-SH-GACACTCGCTTCATCTTCATCTCCGACACTC-3';

[0095] H1: 5'-GCTTCATCTTCGAGTGTCGGAGATGAAGATGAAGCCATCGTGCTTCAT CTTCATCTCCG-3';

[0096] H2: 5'-GCTTCATCTTCATCTCCGATCTCGTTTTGCGGAGATGAAGATGAAGCA CGATG-3';

[0097] H3: 5'-CAAAACGAGATCGGAGATGAAGATGAAGCTTGCCTGCTTCATCTTCATCTCCGATCTCCGACACTC-3';

[0098] FAM-H4-TAMRA: 5'-FAM-GCTTCATCTTCATCTCCGACACTCCGGAGATGAAGATGAAGCAGGCAA-TAMRA-3'.

[0099] The test results of each concentration are as follows Figure 5 As shown, the measured fluorescence signal increases with the increase in the concentration of hematopoietic stem cells added, which is due to the coupling of more CD45-T probes, thereby stimulating more DNA self-replication cycles, thereby enhancing the fluorescence signal.

Claims

1. A method for detecting hematopoietic stem cells based on DNA self-replication cycle, characterized in that It includes five nucleic acid chains, T, H1, H2, H3 and H4, and the sequences of each nucleic acid chain are as follows: T: 5'-GACACTCGCTTCATCTTCATCTCCGACACTC-3'; H1: 5'-GCTTCATCTTCGAGTGTCGGAGATGAAGATGAAGCCATCGTGCTTCAT CTTCATCTCCG-3'; H2: 5'-GCTTCATCTTCATCTCCGATCTCGTTTTGCGGAGATGAAGATGAAGCA CGATG-3'; H3: 5'-CAAAACGAGATCGGAGATGAAGATGAAGCTTGCCTGCTTCATCTTCATCTCCGATCTCCGACACTC-3'; H4: 5'-GCTTCATCTTCATCTCCGACACTCCGGAGATGAAGATGAAGCAGGCA A-3'.

2. The method for detecting hematopoietic stem cells based on DNA self-replication cycle according to claim 1, characterized in that The 5' end of the T nucleic acid chain is modified by a thiol group.

3. The method for detecting hematopoietic stem cells based on DNA self-replication cycle according to claim 1, characterized in that The 5' end of the T nucleic acid chain is covalently linked to the CD45 antibody to form a probe.

4. The method for detecting hematopoietic stem cells based on DNA self-replication cycle according to claim 1, characterized in that H1, H2, H3 and H4 are hairpin nucleic acid chains.

5. The method for detecting hematopoietic stem cells based on DNA self-replication cycle according to claim 1, characterized in that The 5' end of the H4 strand is modified with the first fluorescent group FAM.

6. The method for detecting hematopoietic stem cells based on DNA self-replication cycle according to claim 1, characterized in that The 3' end of the H4 strand was modified with a second fluorescent group, TAMRA.

7. The method for detecting hematopoietic stem cells based on DNA self-replication cycle according to claim 1, characterized in that Also includes: Dilute the hematopoietic stem cells after centrifugation, take 100-150 μL of CD34 antibody functionalized magnetic beads, add 200 μL of hematopoietic stem cells, place at 4°C overnight, take out the vortex and magnetically absorb the next day to remove unbound cells; Then, the hematopoietic stem cells were incubated with CD45-T functionalized with CD45 antibody for 30 minutes, and then washed after magnetic absorption; Then, a mixed solution of H1, H2, H3 and H4 nucleic acid chains was added, separated after incubation, washed and resuspended; Finally, fluorescence spectrometer was used for detection.

8. The method for detecting hematopoietic stem cells based on DNA self-replication cycle according to claim 7, characterized in that The hematopoietic stem cells are incubated with 50-60 μL of CD45 antibody functionalized CD45-T for 30 minutes.

9. The method for detecting hematopoietic stem cells based on DNA self-replication cycle according to claim 7, characterized in that The volume of the mixed solution of H1, H2, H3 and H4 nucleic acid chains is 50-60 μL, and incubated for 60-90 minutes.

10. The method for detecting hematopoietic stem cells based on DNA self-replication cycle according to claim 7, characterized in that The mixed solution of H1, H2, H3 and H4 nucleic acid chains contains 1 μM H1 nucleic acid chain.

11. The method for detecting hematopoietic stem cells based on DNA self-replication cycle according to claim 7, characterized in that The mixed solution of H1, H2, H3 and H4 nucleic acid chains contains 1 μM H2 nucleic acid chain.

12. The method for detecting hematopoietic stem cells based on DNA self-replication cycle according to claim 7, characterized in that The mixed solution of H1, H2, H3 and H4 nucleic acid chains contains 1 μM H3 nucleic acid chain.

13. The method for detecting hematopoietic stem cells based on DNA self-replication cycle according to claim 7, characterized in that The mixed solution of H1, H2, H3 and H4 nucleic acid chains contains 1 μM H4 nucleic acid chain.

14. The method for detecting hematopoietic stem cells based on DNA self-replication cycle according to claim 7, characterized in that The 5' end of the H4 nucleic acid chain is modified by FAM, and the 3' end is modified by TAMRA.

15. The method for detecting hematopoietic stem cells based on DNA self-replication cycle according to claim 7, characterized in that Wash with PBS solution, concentration: 10 mM, pH 7.5.

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