Composition and method for detecting mesenchymal stem cells using dual markers

Abstract

A composition and method for detecting mesenchymal stem cells using dual markers include magnetic beads bound to a CD73 antibody, a CD44 antibody-modified DNA probe with SH- (C6 Spacer) modification at the 5' end, a signal strand modified with NH2- (C6 Spacer) at the 5' end, a primer modified with Biotin- (C6 Spacer) at the 5' end, and streptavidin-modified magnetic beads. Compared with traditional detection and analysis methods, coating the surface of magnetic beads with specific antibodies, streptavidin, and other functional molecules facilitates the enrichment of target molecules in the presence of complex components such as biochemical samples, effectively improving the efficiency and speed of mesenchymal stem cell and signal product enrichment. Simultaneously, it helps to transfer nucleic acid assembly and detection reactions to a simpler solution environment, reducing background signal interference and thus improving the sensitivity and specificity of the analysis and detection.

Description

Technical Field

[0001] This invention relates to a detection composition for cell-derived substances, and more particularly to a composition and method thereof for qualitative and quantitative detection of biological substances, such as stem cells, using an electrochemical method. Background Technology

[0002] Mesenchymal stem cells (MSCs) are pluripotent stem cells, possessing all the common characteristics of stem cells, namely self-renewal and multipotent differentiation capabilities. In recent years, MSCs have attracted considerable attention due to their immense potential in cell therapy. Indeed, they secrete a variety of immunomodulatory factors of interest for the treatment of immune-related and inflammatory diseases. MSCs can be extracted from multiple tissues in the human body. However, the heterogeneity of MSCs from different sources, particularly the differences in surface markers, places high demands on the sensitivity and specificity of detecting specific subpopulations of MSCs. Summary of the Invention

[0003] One object of the present invention is to provide a composition for detecting mesenchymal stem cells with dual markers, which is applied to the electrochemical detection of cell capture and release.

[0004] Another objective of this invention is to provide a composition for detecting mesenchymal stem cells using dual markers, which detects mesenchymal stem cells by an electrochemical method, effectively reducing false positive results.

[0005] Another objective of this invention is to provide a method for detecting mesenchymal stem cells using dual markers, which combines primer exchange reaction with electrochemical technology to achieve efficient, sensitive and specific detection of mesenchymal stem cells.

[0006] Another objective of this invention is to provide a method for detecting mesenchymal stem cells using dual markers, thereby achieving quantitative detection of mesenchymal stem cells.

[0007] In the technical solution of this invention, based on the expression level characteristics of specific biomarkers such as CD73, CD44, CD105 and CD90 on the surface of mesenchymal stem cells, a method for detecting mesenchymal stem cells that identifies two surface biomarkers is established, enabling more detailed and sensitive analysis of mesenchymal cells.

[0008] Compared with traditional detection and analysis methods, coating the surface of magnetic beads with specific antibodies, streptavidin and other functional molecules is beneficial for enriching target molecules in the presence of complex components such as biochemical samples. This effectively improves the efficiency and speed of mesenchymal stem cell and signal product enrichment. At the same time, it helps to transfer nucleic acid assembly reactions and detection reactions to a simpler solution environment, reduce background signal interference, and thus improve the sensitivity and specificity of analysis and detection.

[0009] This invention constructs magnetic beads bound to a CD73 antibody. By using CD73 protein as a specific biomarker for mesenchymal stem cells, it is enriched on the surface of the immunomagnetic beads. A DNA probe chain modified with a CD44 antibody recognizes and binds to a second molecular marker on the surface of mesenchymal stem cells, triggering a nucleic acid assembly reaction centered on primer exchange. This proposes an electrochemical analysis method for mesenchymal stem cells. Specific advantages include:

[0010] (1) This method involves the design and use of two types of magnetic beads. Among them, CD73 antibody-functionalized immunomagnetic beads are used to capture and enrich mesenchymal stem cells, which can avoid non-specific adsorption in complex environments and have high selectivity; streptavidin magnetic beads enrich biotin-labeled double-stranded DNA products, which helps to reduce background signals, avoid false positive signals, and improve the accuracy and sensitivity of detection.

[0011] (2) When CD44 antibody-modified DNA probes are used together with CD73 antibody-functionalized magnetic beads, they can identify two biomarkers on the surface of mesenchymal stem cells, such as CD73 and CD44, through antigen-antibody recognition, thereby improving the accuracy and specificity of cell cluster analysis. At the same time, the DNA probes fixed on the cell surface can be used to trigger nucleic acid assembly reactions, generate signal amplification, and improve the sensitivity of detection.

[0012] (3) The technical solution of the present invention can be applied in clinical practice. By adjusting the surface markers to identify antibodies, more diversified and more accurate mesenchymal stem cell detection can be achieved.

[0013] A composition for detecting mesenchymal stem cells using dual biomarkers, comprising:

[0014] Magnetic beads incorporating CD73 antibody;

[0015] The CD44 antibody-modified DNA probe contains the following nucleic acid sequence, with the 5' end modified with SH-(C6 Spacer), where C6 Spacer is a 6-carbon alkyl intercalary arm;

[0016] 5'-SH-(C6 Spacer)-CATCTATCCCTACGGGTTTTCCCGTAGGGATAGATGTACTCGGTATAA-3';

[0017] The signal chain, labeled with cadmium sulfide quantum dots, contains the following selected nucleic acid sequences, with the 5' end modified with NH2-(C6Spacer):

[0018] 5'-NH2-(C6 Spacer)-GTAGGGATAGATGTACTCGGTA-3';

[0019] The primers contain the following nucleic acid sequences, with the 5' end modified using Biotin-(C6 Spacer):

[0020] 5'-Biotin-(C6 Spacer)-TACCGAGTA-3'; and

[0021] Streptophilic magnetic beads.

[0022] The composition of the present invention comprises a CD44 antibody-modified DNA probe with a fluorescein Cy3 label at the 3' end.

[0023] The composition of the present invention further includes a functionalized graphite electrode, specifically a mercury film-modified graphite electrode.

[0024] A method for detecting mesenchymal stem cells using dual biomarkers includes:

[0025] After mixing magnetic beads with CD73 antibody and mesenchymal stem cells, the mixture was incubated at room temperature for 2 hours, magnetically separated, and washed twice with PBS. Then, it was incubated with a DNA probe modified with CD44 antibody for 30-60 minutes, and then in a primer exchange reaction buffer containing a quantum dot-modified signal strand, and incubated at 37°C for 90-120 minutes to promote complementary hybridization with the quantum dot-labeled signal strand, producing a DNA product double-labeled with biotin and cadmium sulfide quantum dots.

[0026] The final double-stranded product obtained from the reaction was enriched on streptavidin-modified magnetic beads and treated with nitric acid (e.g., 0.5–2 M) to release cadmium ions.

[0027] Electrochemical signals of cadmium atoms deposited on the electrode surface were collected using differential pulse voltammetry (DPV).

[0028] The acid-hydrolyzed reaction solution was mixed with 3.8–3.9 mL of 0.5–1 M sodium acetate solution to serve as the electrolyte. Cadmium ions were deposited onto a graphite electrode using anodic stripping voltammetry (ASV), and the electrochemical signal of the deposited cadmium atoms on the electrode surface was collected using differential pulse voltammetry (DPV). All measurements were performed on a CHI-660C electrochemical workstation. A three-electrode system was used for electrochemical measurements: a platinum wire as the auxiliary electrode, a saturated calomel electrode as the reference electrode, and a mercury-modified graphite electrode as the working electrode.

[0029] The primer exchange reaction is carried out in a reaction solution (e.g., 50–100 μL) which includes: 10 μM primers, 0.8 units / µL Bst DNA polymerase, 100 μM dCTP, dTTP and dATP, 1×Bst reaction buffer and a buffer containing magnesium ions (20 mM Tris-HCl, 10 mM (NH4)2SO4, 10 mM KCl, 12 mM MgSO4, 0.1% Triton X-100).

[0030] It has been verified that the method of the present invention works at a mesenchymal stem cell concentration range of 10. 3 cells / mL to 10 7 The concentration of cells / mL showed a linear correlation with the obtained electrochemical signal, enabling quantitative electrochemical detection within this concentration range. The peak current (I) was linearly related to the logarithm of the number of mesenchymal stem cells, with the linear equation being I0. Cd (microample) = 0.254 × lg cell concentration (cells / mL) - 0.535 (R) 2 =0.99), with a detection limit of 826 cells / mL, which is significantly better than most existing mesenchymal stem cell detection methods. Attached Figure Description

[0031] Figure 1 Fluorescent images of CD73 antibody-functionalized magnetic beads capturing mesenchymal stem cells and binding to Cy3-labeled CD44 antibody-modified DNA strands;

[0032] Figure 2 Electrochemical response diagram for detecting mesenchymal stem cells using anodic stripping voltammetry;

[0033] Figure 3 The graph shows the results of electrochemical quantitative analysis of different mesenchymal stem cell concentrations.

[0034] Figure 4 Linear fitting plot of electrochemical signal response results for mesenchymal stem cells at different concentrations;

[0035] Figure 5 A schematic diagram illustrating the mechanism for implementing mesenchymal stem cell detection in the invention. Detailed Implementation

[0036] 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.

[0037] Figure 5The mechanism for implementing mesenchymal stem cell detection according to the present invention includes:

[0038] (1) Mesenchymal stem cells specifically express the biomarker CD73 protein on their surface. Therefore, we first captured and enriched mesenchymal stem cells by preparing CD73 antibody-functionalized immunomagnetic beads. CD73 antibody-functionalized immunomagnetic beads can avoid non-specific adsorption in complex environments and transfer the reaction to a simpler solution environment. At the same time, using this as an interface for the capture and enrichment of mesenchymal stem cells has high selectivity and enhances the cell fixation efficiency on the surface of the magnetic beads.

[0039] (2) The CD44 antibody-modified DNA probe can bind to CD44 protein, another molecular marker on the surface of mesenchymal stem cells, through the interaction between antigen and antibody. At the same time, the DNA part can serve as a template to participate in the primer exchange reaction. The amplification product is complementary to the quantum dot-labeled signal chain to generate a DNA product double-labeled with biotin and cadmium sulfide. The double-stranded product obtained is then enriched using streptavidin magnetic beads to reduce background signal interference and obtain quantitative information of mesenchymal stem cells.

[0040] (3) When mesenchymal stem cells are present in the system, they can be recognized and captured by CD73 protein-functionalized immunomagnetic beads; subsequently, the captured mesenchymal stem cells can be further recognized and bound to the DNA probe modified by CD44 antibody, thereby triggering primer exchange reaction and generating biotin-modified nucleic acid products.

[0041] (4) Primer exchange reaction in solution can generate biotin-labeled DNA products, which hybridize with quantum dot-labeled signal chains to form biotin- and quantum dot-labeled DNA products; streptavidin magnetic beads are used to enrich the dual-labeled products; the enriched cadmium sulfide quantum dots are dissolved, and the dissolution signal of the quantum dots after acid dissolution is measured by anodic stripping voltammetry to obtain a quantitative signal related to the number of mesenchymal stem cells.

[0042] Based on the above mechanism, the method used in the following embodiments of the present invention mainly includes the following steps:

[0043] (a) Preparation of CD73 antibody-functionalized immunomagnetic beads: 100–150 μL of 10 mg / mL carboxylated magnetic beads were washed three times with PBS and then resuspended in 500–750 μL of 0.22–0.25 M EDC / NHS mixed solution, and incubated at room temperature for 30 minutes to activate the carboxyl groups. After washing with PBS, 10–15 μL of CD73 antibody was added and reacted with the magnetic beads at room temperature for 2 hours. The beads were then magnetically washed again to remove any CD73 antibody that had not undergone the amino-carboxyl group reaction and bound to the carboxylated magnetic beads. Finally, the functionalized immunomagnetic beads were resuspended in 500–600 μL of PBS for further use.

[0044] (b) Preparation of CD44 antibody-modified DNA probe: 10–12 μL of CD44 antibody and 10–15 μL of 10–12 mM amine-thiol crosslinking agent sulfo-SMCC were added to 80–90 μL of PBS buffer and incubated at 30°C for 1–1.5 hours. Simultaneously, 10–12 μL of 20–25 μM DNA hairpin probe H and 10–15 μL of 200–220 μM TCEP were added to 80–100 μL of PBS buffer and reacted at 30°C for 1–1.5 hours. Then, the activated antibody and DNA probe H were mixed in a centrifuge tube and incubated at room temperature for 2–2.5 hours. The mixture was then transferred to a 30 kDa ultrafiltration centrifuge tube and centrifuged at 14,000 rpm for 10–15 minutes. This process was repeated three times, and the supernatant was collected as the prepared CD44 antibody-modified DNA probe.

[0045] (c) Preparation of quantum dot-labeled signal chains: The quantum dot-labeled signal chains are prepared via a carboxyl-amino reaction. In short, carboxylated cadmium sulfide quantum dots are dispersed in a mixed solution of EDC and NHS (50–100 μL, 0.2–0.4 M) at room temperature for 30 minutes to activate the carboxyl groups. Then, the activated quantum dots are incubated with amino-modified nucleic acid chains (100–150 μL, 5–10 μM) at 37 °C for 2 hours. Finally, the obtained quantum dot-labeled signal chains are transferred to 30 kD ultrafiltration centrifuge tubes and centrifuged at 14,000 rpm for 10 minutes. This process is repeated three times, and the supernatant is resuspended in PBS for further use.

[0046] (d) Electrochemical detection of mesenchymal stem cells: First, CD73 antibody-functionalized magnetic beads (100-150 μL) were mixed with mesenchymal stem cells and reacted at room temperature for 2 hours. The mixture was then magnetically separated and washed twice with PBS, followed by incubation at 25°C with CD44 antibody-modified DNA probes for 30-60 minutes. Then, the above solution was transferred to a primer exchange reaction buffer containing a quantum dot-modified signal strand. The primer exchange reaction was carried out in 50–100 μL of reaction solution (including 10 μM primers, 0.8 units / µL Bst DNA polymerase, 100 μM dCTP, dTTP and dATP, 1×Bst reaction buffer and a buffer containing magnesium ions (20 mM Tris-HCl, 10 mM (NH4)2SO4, 10 mM KCl, 12 mM MgSO4, 0.1% Triton X-100) and incubated at 37 °C for 90–120 minutes to promote complementary hybridization with the quantum dot-labeled signal strand.

[0047] (e) Specific process of primer exchange: The short biotin-modified primer strand spontaneously binds to the 3' end of the CD44 antibody-modified DNA probe and extends it using it as a template under the action of Bst DNA polymerase. Due to the lack of dGTP, the extension reaction stops at the C-rich sequence. Subsequently, the competitive binding between the replication domain of the biotin-modified extension product and the template domain within the DNA probe promotes the release of the biotin-modified extension product, while the free primer can continue to bind to the DNA probe and initiate a new round of extension reaction, producing a large amount of amplification products. Then, the biotin-modified extension product binds complementary to the quantum dot-modified signal strand, producing a biotin- and cadmium sulfide quantum dot-labeled DNA product. The final double-stranded product is enriched on streptavidin-modified magnetic beads and treated with nitric acid (200–500 μL, 0.5–2 M) to release cadmium ions.

[0048] (f) The acid-hydrolyzed reaction solution was mixed with 3.8–3.9 mL of 0.5–1 M sodium acetate solution to serve as the electrolyte. Cadmium ions were deposited onto a graphite electrode using anodic stripping voltammetry (ASV), and the electrochemical signal of the deposited cadmium atoms on the electrode surface was collected using differential pulse voltammetry (DPV). All measurements were performed on a CHI-660C electrochemical workstation. A three-electrode system was used for electrochemical measurements: a platinum wire as the auxiliary electrode, a saturated calomel electrode as the reference electrode, and a mercury-modified graphite electrode as the working electrode.

[0049] Wherein: the DNA strand sequence used in step (b) is: 5'-SH-(C6 Spacer)-CATCTATCCCTACGGGTTTTCCCGTAGGGATAGATGTACTCGGTATAA-3'.

[0050] The sequence of the signal chain used in step (c) is: 5'-NH2-(C6 Spacer)-GTAGGGATAGATGTACTCGGTA-3'.

[0051] The biotin-modified primer sequence used in step (d) is: 5'-Biotin-(C6 Spacer)-TACCGAGTA-3'.

[0052] The specific parameters of the anodic stripping voltammetry electrochemical deposition process used in step (f) are: electrodeposition at a potential of -1.2V for 480s; the specific parameters of the differential pulse voltammetry (DPV) used are: potential scan range of -1.0V to -0.5V, frequency of 15Hz, and amplitude of 50mV.

[0053] Example 1

[0054] The steps for capturing mesenchymal stem cells with immunomagnetic beads are as follows:

[0055] (a) Add 100-150 μL of prepared CD73 antibody immunomagnetic beads and mesenchymal stem cells, mix and incubate at room temperature for 2 hours, then magnetically separate and wash twice with PBS.

[0056] (b) Incubate the above solution with 10-15 μL of Cy3-labeled CD44 antibody-modified DNA probe at 25°C for 30-60 minutes.

[0057] (c) Observe the fluorescence on the surface of the immunomagnetic beads using a fluorescence microscope.

[0058] The relevant oligonucleotide sequences are as follows:

[0059] Cy3-labeled DNA probe: 5'-SH-(C6 Spacer)-CATCTATCCCTACGGGTTTTCCCGTAGGGATAGATGTACTCGGTATAA-Cy3-3'.

[0060] Figure 1 This is a fluorescence image of mesenchymal stem cells captured by CD73 antibody-functionalized magnetic beads and then modified with Cy3-labeled CD44 antibody. (Example:) Figure 1 As shown, strong fluorescence can be observed around the magnetic beads when mesenchymal stem cells are present, proving that CD73 antibody-functionalized magnetic beads can successfully capture mesenchymal stem cells and further bind to CD44 antibody to modify DNA chains.

[0061] Example 2

[0062] The qualitative analysis of mesenchymal stem cells follows these steps:

[0063] (a) Add 100-150 μL of the prepared CD73 antibody immunomagnetic beads and a solution containing or not containing mesenchymal stem cells, mix, react at room temperature for 2 hours, then magnetically separate and wash twice with PBS.

[0064] (b) Incubate the above solution with 100-150 μL of CD44 antibody-modified DNA probe (anti-CD44-H) at 25°C for 30-60 minutes.

[0065] (c) Transfer the above solution to a primer exchange reaction buffer containing a quantum dot-modified signal strand. The primer exchange reaction is performed in 50–100 μL of reaction solution (containing 10 μM primers, 0.8 units / µL Bst DNA polymerase, 100 μM dCTP, dTTP and dATP, 1×Bst reaction buffer and a magnesium-containing buffer (20 mM Tris-HCl, 10 mM (NH4)2SO4, 10 mM KCl, 12 mM MgSO4, 0.1% Triton X-100) and incubated at 37 °C for 90–120 min to promote complementary hybridization with the quantum dot-labeled signal strand.

[0066] (d) The final reaction yielded a DNA product double-labeled with biotin and cadmium sulfide quantum dots, which was enriched onto streptavidin-functionalized magnetic beads under the following conditions: incubation at 25°C for 1.5–2 hours.

[0067] (e) Magnetic beads enriched with quantum dots were treated with nitric acid (200–500 μL, 0.5–2 M) to release cadmium ions. The acid-hydrolyzed reaction solution was mixed with 3.8–3.9 mL of 0.5–1 M sodium acetate solution as the electrolyte to deposit cadmium ions onto the surface of a graphite electrode pre-coated with a mercury film. The corresponding electrochemical signals were then collected using differential pulse voltammetry. Electrochemical measurements were performed using a CHI660c electrochemical workstation. The three-electrode system consisted of a mercury film-functionalized electrode as the working electrode, a saturated calomel electrode as the reference electrode, and a platinum wire as the counter electrode.

[0068] The relevant oligonucleotide DNA strand sequences are as follows:

[0069] The DNA strand sequence modified by CD44 antibody is: 5'-CATCTATCCCTACGGGTTTTCCCGTAGGGATAGATGTACTCGGTATAA-3'.

[0070] The nucleic acid sequence contained in the signal strand is: 5'-GTAGGGATAGATGTACTCGGTA-3'.

[0071] The biotin-modified primer sequence is: 5'-Biotin-(C6 Spacer)-TACCGAGTA-3'.

[0072] The feasibility of using this method for mesenchymal stem cell analysis was investigated using electrochemical methods. Figure 2This study describes the electrochemical response of mesenchymal stem cells (MSCs) when detected using anodic stripping voltammetry. CD73 antibody-functionalized magnetic beads capture and enrich MSCs, subsequently binding to CD44 antibody-modified DNA strands. Following this, DNA probes on the MSC surface trigger primer exchange reactions in solution, yielding products that hybridize with cadmium sulfide quantum dot-modified DNA probes. The biotin-labeled DNA hybridization products are enriched using streptavidin and detected by electrochemical signal analysis. Figure 2 As shown, in the absence of mesenchymal stem cells, almost no electrochemical signal was detected in the solution (curve a), and the extremely low background signal further demonstrates that magnetic bead separation in this method can reduce the background signal. When normal cells L02 are present, this method can detect an electrochemical signal almost equivalent to the background signal (curve b), proving that this method can effectively avoid false positive results. When mesenchymal stem cells are present, a significant electrochemical signal response was detected in the solution (curve b). Therefore, the feasibility of this method for the identification and analysis of mesenchymal stem cells has been successfully verified.

[0073] Example 3

[0074] The quantitative analysis of mesenchymal stem cells follows these steps:

[0075] (a) CD73 antibody-functionalized immunomagnetic beads were mixed with different concentrations (10) 3 ~10 7 Incubate the mesenchymal stem cell solution (cells / mL) for 1.5–2 h, then magnetically separate and wash with PBS 2–3 times to remove any uncaptured mesenchymal stem cells. After that, incubate the above solution with CD44 antibody-modified DNA strands at 25 °C for 30–60 minutes.

[0076] (b) Incubate the above solution with 100-150 μL of CD44 antibody-modified DNA probe (anti-CD44-H) at 25°C for 30-60 minutes.

[0077] (c) Transfer the above solution to a primer exchange reaction buffer containing the quantum dot-modified signal strand. The primer exchange reaction is performed in 50–100 μL of reaction solution (containing 10 μM primers, 0.8 units / µL Bst DNA polymerase, 100 μM dCTP, dTTP and dATP, 1×Bst reaction buffer and a magnesium-containing buffer (20 mM Tris-HCl, 10 mM (NH4)2SO4, 10 mM KCl, 12 mM MgSO4, 0.1% Triton X-100) and incubated at 37 °C for 90–120 min to promote complementary hybridization with the quantum dot-labeled signal strand.

[0078] (d) The final reaction yielded a DNA product double-labeled with biotin and cadmium sulfide quantum dots, which was enriched onto streptavidin-functionalized magnetic beads under the following conditions: incubation at 25°C for 1.5–2 hours.

[0079] (e) Magnetic beads enriched with quantum dots were treated with nitric acid (200–500 μL, 0.5–2 M) to release cadmium ions. The acid-hydrolyzed reaction solution was mixed with 3.8–3.9 mL of 0.5–1 M sodium acetate solution as the electrolyte to deposit cadmium ions onto the surface of a graphite electrode pre-coated with a mercury film. The corresponding electrochemical signals were then collected using differential pulse voltammetry. Electrochemical measurements were performed using a CHI660c electrochemical workstation. The three-electrode system consisted of a mercury film-functionalized electrode as the working electrode, a saturated calomel electrode as the reference electrode, and a platinum wire as the counter electrode.

[0080] The relevant oligonucleotide DNA strand sequences are as follows:

[0081] The DNA strand sequence modified by CD44 antibody is: 5'-CATCTATCCCTACGGGTTTTCCCGTAGGGATAGATGTACTCGGTATAA-3'.

[0082] The signal chain sequence is: 5'-GTAGGGATA GATGTACTCGGTA-3'.

[0083] The biotin-modified primer sequence is: 5'-Biotin-(C6 Spacer)-TACCGAGTA-3'.

[0084] Electrochemical analysis was performed on a series of mesenchymal stem cells at different concentrations. Figure 3 This displays the electrochemical quantitative analysis results obtained under different mesenchymal stem cell concentrations, such as... Figure 3 As shown, the obtained current value increases with the increase of the number of mesenchymal stem cells. Figure 4 As shown, in 10 3 Up to 10 7 Within the range of cells / mL, the peak current (I) showed a linear relationship with the logarithm of the number of mesenchymal stem cells. The linear equation was I. Cd (microample) = 0.254 × lg cell concentration (cells / mL) - 0.535 (R) 2 =0.99), the detection limit is 826 cells / mL. Compared with existing mesenchymal stem cell detection methods (common detection limits are approximately 10), this is significantly better. 3 ~10 4 (per cell), the method in this embodiment has a lower detection limit and is more sensitive.

[0085] This paper compares the proposed method with existing methods for detecting and analyzing mesenchymal stem cells. Currently used methods include morphological identification, flow cytometry, and multi-lineage differentiation potential assessment. These methods are relatively cumbersome and complex, and the procedures can significantly impact cell viability, even leading to complete cell inactivation. In contrast, the key features of this invention are as follows:

[0086] This invention utilizes magnetic beads to enrich mesenchymal stem cells and signal marker products, which can avoid non-specific adsorption in complex environments, reduce background signals, effectively reduce false positive results, and simultaneously identify multiple surface markers, thereby improving the specificity and accuracy of mesenchymal stem cell analysis and contributing to the typing analysis of mesenchymal stem cells.

[0087] This invention combines primer exchange reaction with electrochemical technology, which has the advantages of high reaction efficiency, simple operation and high sensitivity, and is beneficial for the efficient, sensitive and specific detection of mesenchymal stem cells.

Claims

1. A method for detecting mesenchymal stem cells with double markers, characterized in that The method comprises the following steps: The magnetic beads combined with the CD73 antibody and the mesenchymal stem cells are mixed and incubated at room temperature for 2 hours, and then subjected to magnetic separation and washed twice with PBS, followed by incubation with the CD44 antibody modified DNA probe for 30-60 minutes, and then subjected to an exchange reaction with the primer containing the signal chain in the quantum dot modified signal chain primer exchange reaction buffer and incubated at 37°C for 90-120 minutes to produce the biotin and cadmium sulfide quantum dot double-labeled DNA product; The double-stranded product obtained in the final reaction is enriched on the streptavidin magnetic beads, and then treated with nitric acid to release cadmium ions; The cadmium ions are deposited on the electrode by anodic stripping voltammetry, and then the electrochemical signal of the cadmium atoms deposited on the surface of the electrode is collected by differential pulse voltammetry; The CD44 antibody modified DNA probe contains the following nucleic acid sequence, and the 5' end is modified with SH-(C6 Spacer): 5'-SH-(C6 Spacer)-CATCTATCCCTACGGGTTTTCCCGTAGGGATAGATGTACTCGGTATAA-3'; The signal chain is labeled with cadmium sulfide quantum dots, and the nucleic acid sequence contained therein is as follows, and the 5' end is modified with NH2-(C6 Spacer): 5'-NH2-(C6 Spacer)-GTAGGGATAGATGTACTCGGTA-3'; The primer contains the following nucleic acid sequence, and the 5' end is modified with Biotin-(C6 Spacer): 5'-Biotin-(C6 Spacer)-TACCGAGTA-3'.

2. The method of claim 1, wherein the mesenchymal stem cells are detected by double markers. A platinum wire is used as an auxiliary electrode, a saturated calomel electrode is used as a reference electrode, and a mercury film modified graphite electrode is used as a working electrode.

3. The method of claim 1, wherein the mesenchymal stem cells are detected by double markers. The specific parameters of the electrochemical deposition process using anodic stripping voltammetry are as follows: the electrodeposition is performed at a potential of -1.2V for 480s, and the electrochemical scanning range for detecting the silver ion signal by differential pulse voltammetry is from -1.0V to -0.5V, the frequency is 15Hz, and the amplitude is 50mV.

4. The method of claim 1, wherein the mesenchymal stem cells are detected by double markers. The cell concentration range from 10 3 cells / mL to 10 7 cells / mL is linearly correlated with the resulting electrochemical signal.

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