CN116242894A - Compositions and methods for capturing and releasing cells containing CD44 antigen - Google Patents
Compositions and methods for capturing and releasing cells containing CD44 antigen Download PDFInfo
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Abstract
A composition for capturing and releasing cells containing CD44 antigen and a method thereof, comprising cholesterol-aptamer, silver nano-modified aptamer, bleomycin-ferrous ion complex and the like, have higher biocompatibility and stability, and are favorable for forming a biological interface for capturing and releasing mesenchymal stem cells efficiently. The composition provided by the invention is applied to capturing and releasing electrochemical detection cells, has mild reaction conditions, has the advantages of simplicity, rapidness and sensitivity, and is convenient to be combined with other automatic reaction interfaces for commercialized development. By combining with the electrochemical biochip sensor array, more convenient mesenchymal stem cell capturing, detecting and releasing can be realized.
Description
Technical Field
The present invention relates to a composition for detecting substances of cellular origin, in particular to a method for electrochemical application, for example from: compositions and methods for performing qualitative and quantitative assays of biological substances of stem cells.
Background
Mesenchymal stem cells are multipotent stem cells that share all the common properties of stem cells, namely self-renewal and multipotent differentiation. In recent years, mesenchymal Stem Cells (MSCs) have been attracting attention due to their great potential in cell therapy. In fact, they secrete a variety of immune modulatory factors of interest for the treatment of immune-related and inflammatory diseases. MSCs can be extracted from multiple tissues of the human body. However, there are several factors that may limit their use in clinical applications, such as isolation requiring invasive procedures, limited numbers, and heterogeneity depending on the source or donor tissue. Because mesenchymal stem cell therapy still faces many challenges in the clinical application process, it is important to establish a high-efficiency and sensitive mesenchymal stem cell capturing, releasing and quantitative analysis method so as to develop deeper biological research.
Among the many non-biological contaminant materials that are highly resistant to protein adsorption, lipid-based materials are considered to be the closest biological systems, which create a cell membrane-mimicking environment for cell-cell and cell-biological material interactions. The lipid-based material can effectively avoid the adsorption of various non-interested substances in the solution due to the nonpolar and inert chemical properties of the lipid-based material, so that the lipid-based material can be strongly antifouling. In recent years, electrochemical technology is endlessly combined with an electrochemical biological sensing technology layer prepared by combining a self-assembled monomolecular film, a lipid bilayer film, a Langmuir-Blodgett layer and a supported lipid bilayer film, and a technology for separating cells by utilizing excellent characteristics of the lipid bilayer is also reported, so that a theoretical basis is provided for the development of new strategies for capturing and releasing cells.
Disclosure of Invention
It is an object of the present invention to provide a composition for capturing and releasing cells containing CD44 antigen for use in electrochemical detection of the capturing and release of cells.
It is another object of the present invention to provide a method for capturing and releasing cells containing CD44 antigen, which allows efficient monitoring of cell release by electrochemical means.
It is a further object of the present invention to provide a method for capturing and releasing cells containing CD44 antigen, obtaining differentiated electrochemical signal detection cells.
It is a further object of the present invention to provide a method for capturing and releasing cells containing CD44 antigen, which allows quantitative detection of cells.
The CD44 antigen is a group of membrane integrins with wide distribution, molecular weight of (85-160) multiplied by 10kD and high sugar content. CD44 has molecular weight of 85-250 kD, mediates the interaction between cells and extracellular matrix, and is glycoprotein composed of three parts of extracellular, transmembrane and cytoplasmic, and sugar chain is chondroitin sulfate and heparan sulfate.
The invention constructs a lipid bilayer membrane modified electrode, realizes the fixation of a nucleic acid aptamer by utilizing high affinity between a lipid bilayer and cholesterol molecules, and further combines the nucleic acid cleavage activity of a bleomycin-ferrous ion complex (bleomycin-ferrous ion complex) to realize the capture, release and detection of cells containing a CD44 antigen. The method has particularly great significance on mesenchymal stem cells.
A composition for capturing and releasing cells containing CD44 antigen for use in electrochemically detecting the capturing and releasing of cells, comprising:
a cholesterol-aptamer comprising the nucleotide sequence selected from the group consisting of:
5'-Cholesteryl-AAAGCGCGTAAGTGAAATGAGATTCATCACGCGCATAGTCCCAAGGCCTGCAAGGGAACCAAGGACACAGCGACTATGCGATGATGTCTTC-3'；
the silver nano modified aptamer comprises the following nucleotide sequences:
5'-CCCCCCCCCCCCGAGATTCATCACGCGCATAGTCCCAAGGCCTGCAAGGGAACCAAGGACACAGCGACTATGCGATGATGTCTTC-3'; and
bleomycin-ferrous ion complex.
(1) Compared with a gold electrode, the lipid bilayer membrane modified electrode can avoid nonspecific adsorption in a complex environment, and has higher biocompatibility and stability for fixing nucleic acid probe molecules.
(2) Bleomycin-ferrous complex formed by combining bleomycin and ferrous ions can specifically cleave 5'-GT and 5' -GC sequences under the action of oxygen, and the captured mesenchymal stem cells and cell surface-bound signaling probes are released efficiently.
(3) More convenient capture, detection and release of cells containing CD44 antigen (e.g., mesenchymal stem cells) can be achieved by combining with an electrochemical biochip sensor array.
The composition of the invention further comprises a functionalized gold electrode, specifically: a functionalized gold electrode of a lipid bilayer.
An embodiment of a functionalized gold electrode for preparing a lipid bilayer is provided, wherein the gold electrode is firstly arranged at a temperature of between 0.5 and 0.6M H 2 SO 4 In the solution, cyclic voltammetry scanning is carried out within the voltage range of 0-1.6V, the scanning turns are 25-30, and then the electrode is dried by nitrogen.
Next, an ethanol solution containing 2 to 2.5mM DPPTE was added to the surface of the gold working electrode, and incubated at room temperature for 16 to 20 hours, forming a first lipid layer by Au-S interaction.
Then, the electrode was rinsed with ethanol and treated with 20-25 mg/mL DPPC solution for 5-7 minutes, and then the electrode was placed at-20℃for 30-40 minutes, and then transferred to room temperature and then placed for 30-40 minutes to form a lipid bilayer membrane, thereby obtaining a lipid bilayer functionalized gold electrode.
A method of capturing and releasing cells comprising CD44 antigen comprising:
placing the functionalized gold electrode into a solution containing 1-2 mu M cholesterol-aptamer and 10-15 mM PBS, placing the solution into a 4 ℃ to react for 2-3 hours in a dark place, and then flushing the surface of the electrode by using 5-10 mLPBS;
the cell solution containing CD44 and the functionalized gold electrode are incubated for 1.5 to 2 hours, PBS is used for cleaning for 2 to 3 times to remove cells which are not captured, and then 1 to 1.5 mu M silver nano-modified aptamer is added to the electrode for capturing cells and reacts for 1 to 1.5 hours at room temperature.
Finally, 45-50 mu M bleomycin-ferrous complex is added for reaction for 60-65 min, and the reaction liquid is collected. The obtained reaction solution was centrifuged at 500 to 1000rpm, and the lower precipitate was collected as released cells.
The three-electrode system comprises a working electrode which is a lipid bilayer modified electrode, a saturated calomel electrode which is used as a reference electrode and a platinum wire which is used as a counter electrode.
Adding 0.5-1M nitric acid solution into the supernatant solution obtained by centrifugation, reacting for 2 hours at room temperature, and enriching silver ions on a graphite electrode by using a stripping voltammetry method. The electrochemical signal of the silver nano-particles was then detected with a 0.5-1M sodium acetate electrolyte in the range of-0.4V to 0.4V by differential pulse voltammetry. Electrochemical measurements were performed with the CHI660c electrochemical workstation. The three-electrode system comprises a working electrode which is a graphite electrode, a saturated calomel electrode which is used as a reference electrode and a platinum wire which is used as a counter electrode.
Proved by verification, the method of the invention has the concentration range of the exosomes of 10 2 Individual cells/mL to 10 6 The individual cells/mL are linearly related to the obtained electrochemical signal, and quantitative electrochemical detection can be realized in the concentration range. The linear equation is I (microampere) = 0.8192 ×lg cell concentration (individual/ml) +0.4149 (R) 2 =0.99), significantly better than most existing mesenchymal stem cell detection methods.
The technical scheme of the invention has the beneficial effects that:
the existing mesenchymal stem cell separation methods include an adherence method, a flow cytometry separation method, a density gradient centrifugation method, an immunomagnetic bead method and the like, and the processes of the methods are complicated. Compared with the method, the lipid bilayer membrane modified electrode can avoid nonspecific adsorption in a complex environment, has higher biocompatibility and stability, and is favorable for forming a biological interface for efficiently capturing and releasing mesenchymal stem cells.
Compared with the traditional separation and release method, the release method based on the bleomycin-ferrous complex cleavage activity not only can successfully release the cells captured by the interface to the ease, but also can release the signal molecules on the cell surface to the solution, and can collect and effectively quantitatively detect and analyze the mesenchymal stem cells.
The method has the advantages of convenient operation, mild reaction conditions, simplicity, rapidness and sensitivity, and is convenient to be combined with other automatic reaction interfaces for commercialized development. By combining with the electrochemical biochip sensor array, more convenient mesenchymal stem cell capturing, detecting and releasing can be realized.
Drawings
FIG. 1 is a graph of EIS results for electrode surfaces in different states;
FIG. 2 is an impedance diagram of mesenchymal stem cells immobilized on the surface of an electrode;
FIG. 3 is a graph showing electrochemical signals of mesenchymal stem cells after cleavage and release by bleomycin-ferrous complex;
FIG. 4 is a graph showing the results of electrochemical quantitative analyses of different mesenchymal stem cell concentrations;
FIG. 5 is a graph of a linear fit of electrochemical signal response of mesenchymal stem cells at different concentrations;
fig. 6 is a diagram of a mesenchymal stem cell capturing and releasing route implemented by the invention.
Detailed Description
The technical scheme of the present invention is described in detail below with reference to the accompanying drawings. The embodiments of the present invention are only for illustrating the technical scheme of the present invention and not for limiting the same, and 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 modifications and equivalents may be made thereto without departing from the spirit and scope of the technical scheme of the present invention, which is intended to be covered by the scope of the claims of the present invention.
FIG. 6 shows the mechanism of mesenchymal stem cell capture and release according to the present invention, including:
(1) The phospholipid bilayer membrane has good dirt resistance and biocompatibility, so that the stability and biocompatibility of an electrode interface can be improved by modifying the phospholipid bilayer membrane on the electrode, meanwhile, the phospholipid bilayer membrane is used as the interface for cell capturing, has good dirt resistance, can enhance the efficiency of capturing and releasing cells and ensure the activity of the captured and released cells, and therefore, the lipid bilayer membrane is modified on the surface of the working electrode for capturing mesenchymal stem cells by a phospholipid self-assembly mode.
(2) Cholesterol-modified CD44 aptamer probes (cholesterol-aptamer chains) can be inserted into a lipid bilayer membrane through the hydrophobic interaction of cholesterol groups with the lipid membrane, while DNA moieties act as aptamer sequences for the mesenchymal stem cell surface marker CD44, allowing specific capture of mesenchymal stem cells.
(3) When mesenchymal stem cells exist in the system, the mesenchymal stem cells can be identified and captured by cholesterol-aptamer chains on a gold electrode modified by a lipid bilayer membrane; the captured mesenchymal stem cells can then be further identified and bound to CD44 aptamer functionalized silver nanoparticles (aptamer chain-silver nanoparticles), which are labeled in situ by the identification and binding of CD44 aptamer to mesenchymal stem cells.
(4) Since the CD44 aptamer contains the bleomycin-ferrous complex cleavage sequence, after the bleomycin-ferrous complex is added, the CD44 aptamer can selectively cleave the 5'-GC-3' and 5'-GT-3' in the aptamer sequence, so that the captured mesenchymal stem cells are released from the electrode surface, and the signaling probes are also separated from the cell surface, so that the mesenchymal stem cells and the nanosilver signaling probes are released into the solution.
(5) Collecting a reaction solution, and obtaining a precipitate which is mesenchymal stem cells released by an interface after centrifugal treatment, wherein the precipitate can be used for subsequent biological research; the supernatant obtained by centrifugation contains the released silver nanoparticles, and a quantitative signal related to the number of mesenchymal stem cells can be obtained through an electrochemical technology.
According to the above mechanism, the method adopted in the following embodiments of the present invention mainly comprises the following steps:
(a) The preparation of the lipid bilayer gold electrode comprises the following specific processes: after polishing the gold electrode, an ethanol solution containing 2-2.5 mM DPPTE (1, 2-dipalmitoyl-sn-glycerophosphatidylthioethanol) is added to the surface of the gold working electrode, and incubated for 16-20 hours at room temperature, and a first lipid layer is formed through gold-thiol interaction. Thereafter, the electrode was rinsed with ethanol and treated with 20 to 25mg/mL DPPC (dipalmitoyl phosphatidylcholine) solution for 5 to 10 minutes, and then the electrode was placed at-20℃for 30 to 40 minutes, and then transferred to room temperature for another 30 to 40 minutes to form a lipid bilayer membrane.
(b) The preparation of the aptamer functionalized lipid bilayer gold electrode comprises the following specific processes: the electrode modified in the above steps was inverted in a solution containing 1-2. Mu.M cholesterol-aptamer chain and 10-15 mM PBS (pH about 7.4), and after 2-3 hours of reaction at 4℃in the absence of light, the electrode surface was rinsed with 5-10 mLPBS.
(c) The preparation of the aptamer chain-silver nanoparticle comprises the following specific processes: 3-5 mu L of 100-110 mu M CD44 aptamer chain is added into 1200-1500 mu L of silver nanoparticle solution, and the two are incubated for 1-2 hours at room temperature, so that DNA is adsorbed on the silver nanoparticle; adding 10-15 mu L of 500-510 mM trisodium citrate buffer (pH=3) into the solution, and incubating for 30-60 minutes at room temperature until the final concentration reaches 5-10 mM; adding 150-200 mu L of 200-210 mM PB buffer solution (pH=7.4) to make the pH of the silver nanoparticle solution neutral, and incubating for 10-15 minutes at room temperature; centrifuging at 14000rpm and 4 ℃ for 20-30 minutes to remove unbound aptamer; washing 3 times with 1-2 ml of 10-15 mM PB buffer (ph=7.4); after washing, the CD44 aptamer functionalized silver nanoparticle complex was dispersed in 1-2 ml 10mm PB buffer (ph=7.4) and stored at 4 ℃ for later use. Wherein, 200-210 mM PB buffer solution (pH=7.4), solution A: 3.5-3.6 g of disodium hydrogen phosphate dihydrate is dissolved in 100-110 mL of deionized water, and solution B: 3.1 to 3.2g of sodium hydrogen phosphate dihydrate is dissolved in 100 to 101mL of deionized water. Before use, the solution A and the solution B are mixed immediately, and the prepared PB buffer solution is stored at 4 ℃ for use.
(d) The mesenchymal stem cells are captured, released and detected, and the specific process is as follows: electrochemical workstation and three electrode systems are used to make electrochemical measurements. The three-electrode system includes: the working electrode is a graphite electrode or a gold electrode, the saturated calomel electrode is a reference electrode, and the platinum wire is a counter electrode. And (3) reversely buckling the mesenchymal stem cell solution on the aptamer functionalized lipid bilayer gold electrode for 1.5-2 h, washing with PBS for 2-3 times to remove the mesenchymal stem cells which are not captured, adding 1-1.5 mu M aptamer chain-silver nano particles on the electrode for capturing cells, reacting for 1-1.5 h at room temperature, and washing with PBS for 2-3 times. Subsequently, the electrode is immersed into 100 to 120 mu L of solution containing 45 to 50 mu M bleomycin-ferrous complex, and the reaction is carried out for 60 to 90 minutes, so that the simultaneous release of the mesenchymal stem cells and the signaling probes is realized. Then, collecting the reacted solution, adding 100-120 mu L of 0.5-1M nitric acid solution, and reacting for 1.5-2 hours at room temperature, so that the silver nano particles are acid-melted, and a large amount of silver ions are released. Finally, mixing the acidolysis reaction solution with 3.8-3.9 mL of 0.5-1M sodium acetate solution to be used as electrolyte, enriching silver ions on the surface of a graphite electrode through an electrochemical deposition process, and collecting electrochemical signals through a differential pulse voltammetry.
Wherein: the sequence of the cholesterol-aptamer chain used in step (b) is: 5 '-cholestyl-AAAGCGCGTAAGTGAAATGAGATTCATCACGCGCATAGTCCCAAGGCCTGCAAGGGAACCAAGGACACAGCGACTATGCGATGATGTCTTC-3'.
The sequence of the silver nano-modified aptamer chain used in step (c) is: 5'-CCCCCCCCCCCCGAGATTCATCACGCGCATAGTCCCAAGGCCTGCAAGGGAACCAAGGACACAGCGACTATGCGATGATGTCTTC-3'.
Specific parameters of the electrochemical deposition process used in step (d) are: -deposition at a potential of 1.2V for 8 minutes; specific parameters of the differential pulse voltammetry used are: the potential sweep ranged from-0.4V to 0.4V, amplitude 25mV, frequency 15Hz.
Example 1
The lipid bilayer gold electrode was prepared as follows:
(a) The gold electrode is firstly polished on abrasive paper, so that the surface of the gold electrode is smooth and the scratches are consistent. Then polishing with aluminum powder until the aluminum powder reflects light, repeatedly flushing with distilled water until impurities are removed, and then respectively ultrasonically treating the gold electrode in ethanol and distilled water for 2-3 minutes. The electrode surface was dried with nitrogen and then immersed in an freshly prepared aqua tiger fish solution (30% hydrogen peroxide: concentrated sulfuric acid=1:3, V/V) for 2-3 minutes, and then sonicated with ethanol and double distilled water for 2-3 minutes, respectively, to remove the residue.
(b) Subsequently, the gold electrode is placed in a range of 0.5M to 0.6. 0.6M H 2 SO 4 In the solution, cyclic voltammetry scanning is carried out within the voltage range of 0-1.6V, the scanning turns are 25-30, and then the electrode is dried by nitrogen.
(c) Subsequently, an ethanol solution containing 2 to 2.5mM DPPTE was added to the surface of the gold working electrode, and incubated at room temperature for 16 to 20 hours, forming a first lipid layer through Au-S interaction.
(d) After that, the electrode was rinsed with ethanol and treated with 20 to 25mg/mL DPPC solution for 5 to 7 minutes, and then placed at-20℃for 30 to 40 minutes, and then transferred to room temperature and placed for another 30 to 40 minutes to form a lipid bilayer membrane.
The modification of the lipid bilayer membrane on the surface of the electrode is critical to the implementation of the scheme, so that the assembly condition of the surface of the electrode is characterized by adopting an alternating current spectrum impedance method. As shown in fig. 1, the nyquist plot clearly shows EIS results for the electrode surface at different conditions. The impedance spectrum of the bare gold electrode is almost a straight line, which indicates that the electron transfer resistance at this time is extremely low (curve Au). When the lipid bilayer membrane was assembled on the electrode surface, a significant increase in the impedance spectrum (curve LB) was seen, indicating that the lipid bilayer membrane formed a significant steric barrier, resulting in an increase in the resistance to charge transfer, and thus indicating successful assembly of the lipid bilayer membrane on the electrode surface.
Example 2
Mesenchymal stem cells are captured and released as follows:
(a) And (3) reversely buckling the mesenchymal stem cell solution on the functionalized gold electrode for 1.5-2 h, washing for 2-3 times by using PBS (phosphate buffer solution) to remove the mesenchymal stem cells which are not captured, and then adding 1-1.5 mu M silver nano-modified aptamer chains on the electrode for capturing the cells for reacting for 1-1.5 h at room temperature.
(b) Then adding 45-50 mu M bleomycin-ferrous complex, reacting for 60-65 min, and collecting reaction liquid. The reaction solution obtained was centrifuged at 500-1000 rpm, and the lower pellet was used as released cells and collected for re-culture and further study. The three-electrode system comprises a working electrode which is a lipid bilayer modified electrode, a saturated calomel electrode which is used as a reference electrode and a platinum wire which is used as a counter electrode.
(c) Adding 0.5-1M nitric acid solution into the supernatant solution obtained by centrifugation, reacting for 2 hours at room temperature, and enriching silver ions on a graphite electrode by using a stripping voltammetry method. The electrochemical signal of the silver nano-particles was then detected with a 0.5-1M sodium acetate electrolyte in the range of-0.4V to 0.4V by differential pulse voltammetry. Electrochemical measurements were performed with the CHI660c electrochemical workstation. The three-electrode system comprises a working electrode which is a graphite electrode, a saturated calomel electrode which is used as a reference electrode and a platinum wire which is used as a counter electrode.
The relevant oligonucleotide DNA strand sequences are as follows:
the cholesterol-aptamer chain contains the following nucleotides, 5' of which contain a cholesterol group:
5'-Cholesteryl-AAAGCGCGTAAGTGAAATGAGATTCATCACGCGCATAGTCCCAAGGCCTGCAAGGGAACCAAGGACACAGCGACTATGCGATGATGTCTTC-3'。
the silver nano-modified aptamer chain contains the following nucleotides:
5'-CCCCCCCCCCCCGAGATTCATCACGCGCATAGTCCCAAGGCCTGCAAGGGAACCAAGGACACAGCGACTATGCGATGATGTCTTC-3'。
after successful modification of the lipid bilayer membrane on the electrode and immobilization of cholesterol modified aptamer chains, we studied their feasibility for mesenchymal stem cell capture and release using electrochemical methods. Fig. 2 is an impedance spectrum obtained by fixing and releasing mesenchymal stem cells on an electrode. As shown in fig. 2, in mesenchymal stem cells (10 6 And (3) the number of the semi-circles per milliliter) is fixed on the surface of the electrode, a very large semi-circle radius impedance chart can be obtained, and when the captured mesenchymal stem cells are released along with aptamer chain cutting, the semi-circle diameter of the obtained electrochemical impedance chart becomes smaller, and the electrochemical impedance chart is almost equivalent to the impedance obtained by the surface of the original lipid membrane modified electrode, so that the successful release of the cells captured on the surface of the electrode is proved.
The bleomycin-ferrous complex cleavage releases not only mesenchymal stem cells from the capture interface, but also silver nano signal molecules from the surface of mesenchymal stem cells, so that the supernatant solution thereof can be collected and silver nano particles in the supernatant solution can be characterized as quantitative basis. As shown in fig. 3, when the mesenchymal stem cells captured by the interface are cleaved and released by the bleomycin-ferrous complex, the signal molecules on the cell surface are released into the supernatant along with the cleavage reaction of the aptamer marker, and the obtained solution is detected, so that the electrochemical signal corresponding to the obtained solution can be obtained (curve a). However, when the silver nanoparticle-modified CD44 aptamer bound to the surface of mesenchymal stem cells, no corresponding electrochemical signal was detected in the co-cultured supernatant solution when no bleomycin-ferrous complex cleavage was added (curve b). From the above results, it is demonstrated that our established method is capable of effectively monitoring mesenchymal stem cell release by electrochemical means.
Example 3
Quantitative analysis of mesenchymal stem cells, the steps of which are as follows:
(a) Different concentrations (10-10) 6 And (3) reversely buckling the mesenchymal stem cell solution on the functionalized gold electrode for 1.5-2 h, washing with PBS for 2-3 times to remove the mesenchymal stem cells which are not captured, adding 1-1.5 mu M silver nanoparticle-aptamer complex on the electrode for capturing cells, and reacting for 1-1.5 h at room temperature.
(b) Then adding 45-50 mu M bleomycin-ferrous complex, and reacting for 60-65 min. Then 0.5-1M nitric acid solution is added to react for 2 hours at room temperature, and silver ions are enriched on the graphite electrode by using a stripping voltammetry method.
(c) The electrochemical signal of the silver nano-particles was then detected with a 0.5-1M sodium acetate electrolyte in the range of-0.4V to 0.4V by differential pulse voltammetry. Electrochemical measurements were performed with the CHI660c electrochemical workstation. The three-electrode system comprises a working electrode which is a graphite electrode, a saturated calomel electrode which is used as a reference electrode and a platinum wire which is used as a counter electrode.
A series of mesenchymal stem cells of different concentrations were subjected to electrochemical analysis. Fig. 4 shows a graph of electrochemical quantitative analysis results for different mesenchymal stem cell concentrations, as shown by the increase in electrochemical response current with increasing mesenchymal stem cell number, which is consistent with the expected results. The increase in the number of target cells increases the surface-bound silver nanoparticle-modified aptamer chains, while the increase in the number of signal molecules bound further increases the response value of the electrochemical signal. As shown in fig. 5, the peak current (I) increases with the increase in the number of mesenchymal stem cells. Peak current at 10 2 To 10 6 The number of mesenchymal stem cells in the range of individual/ml is in a linear relationship. The linear equation is I (microampere) = 0.8192 ×lg cell concentration (individual/ml) +0.4149 (R) 2 ＝0.99)。
Claims (10)
1. A composition for capturing and releasing cells containing CD44 antigen for use in electrochemically detecting the capturing and releasing of cells, comprising:
a cholesterol-aptamer comprising the nucleotide sequence selected from the group consisting of:
5'-Cholesteryl-AAAGCGCGTAAGTGAAATGAGATTCATCACGCGCATAGTCCCAAGGCCTGCAAGGGAACCAAGGACACAGCGACTATGCGATGATGTCTTC-3'；
the silver nano modified aptamer comprises the following nucleotide sequences:
5'-CCCCCCCCCCCCGAGATTCATCACGCGCATAGTCCCAAGGCCTGCAAGGGAACCAAGGACACAGCGACTATGCGATGATGTCTTC-3'; and
bleomycin-ferrous ion complex.
2. The composition for capturing and releasing cells containing CD44 antigen according to claim 1, wherein the cells are mesenchymal stem cells.
3. The composition for capturing and releasing cells containing CD44 antigen according to claim 1, further comprising a gold electrode functionalized with a lipid bilayer.
4. A composition for capturing and releasing cells containing CD44 antigen according to claim 3, characterized in that said lipid bilayer functionalized gold electrode is prepared by the following method:
the gold electrode is firstly arranged at 0.5M-0.6. 0.6M H 2 SO 4 In the solution, cyclic voltammetry scanning is carried out within the voltage range of 0-1.6V, the scanning turns are 25-30, and then the electrode is dried by nitrogen;
then adding an ethanol solution containing 2-2.5 mM DPPTE to the surface of the gold working electrode, and incubating for 16-20 hours at room temperature to form a first lipid layer through Au-S interaction;
then, the electrode was rinsed with ethanol and treated with 20-25 mg/mL DPPC solution for 5-7 minutes, and then the electrode was placed at-20℃for 30-40 minutes, and then transferred to room temperature and then placed for 30-40 minutes to form a lipid bilayer membrane, thereby obtaining a lipid bilayer functionalized gold electrode.
5. The composition for capturing and releasing cells containing CD44 antigen according to claim 1, further comprising a graphite electrode, a saturated calomel electrode and a platinum wire.
6. A method of capturing and releasing cells comprising CD44 antigen, comprising:
placing the functionalized gold electrode into a solution containing 1-2 mu M cholesterol-aptamer and 10-15 mM PBS, placing the solution into a 4 ℃ to react for 2-3 hours in a dark place, and then flushing the surface of the electrode by using 5-10 mLPBS;
incubating a cell solution containing CD44 with the functionalized gold electrode for 1.5-2 h, washing with PBS for 2-3 times to remove cells which are not captured, adding 1-1.5 mu M silver nano-modified aptamer to the electrode for capturing cells, and reacting for 1-1.5 h at room temperature;
finally, adding 45-50 mu M bleomycin-ferrous complex, reacting for 60-65 min, and collecting reaction liquid; centrifuging the obtained reaction liquid at 500-1000 rpm, collecting the lower layer precipitate as released cells;
the cholesterol-aptamer comprises the following nucleotide selection sequence, wherein 5' contains cholesterol groups:
5'-Cholesteryl-AAAGCGCGTAAGTGAAATGAGATTCATCACGCGCATAGTCCCAAGGCCTGCAAGGGAACCAAGGACACAGCGACTATGCGATGATGTCTTC-3'；
the silver nano-modified aptamer comprises the following nucleotide selection sequences:
5'-CCCCCCCCCCCCGAGATTCATCACGCGCATAGTCCCAAGGCCTGCAAGGGAACCAAGGACACAGCGACTATGCGATGATGTCTTC-3'。
7. the method of claim 6, wherein the method further comprises using platinum as an auxiliary electrode, a saturated calomel electrode as a reference electrode, and a platinum wire.
8. The method of claim 6, wherein the differential pulse voltammetry detection of silver ion signals has an electrochemical scan range of from-0.4V to 0.4V, a frequency of 15Hz, and an amplitude of 50mV.
9. The method of claim 6, wherein the cells containing CD44 antigen are quantitatively detected.
10. The method of capturing and releasing CD44 antigen containing cells according to claim 6, wherein said cell concentration range is 10 2 Individual cells/mL to 10 6 Individual cells/mL are linearly related to the electrochemical signal obtained.
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