Cell electric signal regulation and control membrane as well as preparation method and application thereof

Abstract

The invention discloses a cell electric signal regulation and control membrane and a preparation method and application thereof, and relates to the technical field of bioelectronics, and the cell electric signal regulation and control membrane comprises a DNA framework used for being specifically combined with a cell membrane lipid layer, a DNA functionalized carbon nanotube and a complementary hairpin DNA group used for connecting the DNA framework and the DNA functionalized carbon nanotube. On one hand, molecular-level precision self-assembly of the carbon nanotubes guided by a DNA framework is realized through complementary base pairing of DNA, so that the carbon nanotubes are directionally arranged, and the disorder of modification of the carbon nanotubes is overcome; on the other hand, by means of the structural rigidity and cell membrane anchoring capacity of the DNA framework, the stability of anchoring the carbon nano tube on the cell membrane is improved, and damage to the integrity of the cell membrane is avoided as much as possible. Under the comprehensive action, the energy transfer efficiency and biocompatibility of the cell electric signal regulation and control membrane are improved.

Description

Technical Field

[0001] The present application relates to the field of bioelectronics technology, and in particular to a cell electrical signal regulation membrane and a preparation method and application thereof. Background Art

[0002] Cellular electrical signaling is the core mediator driving life's activities. It regulates cell proliferation, differentiation, migration, and immune responses through membrane potential oscillations, ion channel dynamics, and calcium signaling cascades. For example, activation of voltage-gated calcium channels (VGCCs) can trigger neurotransmitter release and gene transcription, while mechanosensitive channels (such as Piezo1) regulate tissue repair by sensing changes in microenvironmental mechanics. However, traditional electrical stimulation techniques rely on macroscopic electrodes or chemical transmitters, and suffer from low spatial resolution, poor targeting, and susceptibility to damage cell activity, making precise regulation at the single-cell level difficult.

[0003] Currently, single-walled carbon nanotubes (SWCNTs) have been widely used due to their ultrahigh carrier mobility (>10 4 cm 2 Due to its high ionic strength (V / s) and ion channel-like transmembrane conductivity, SWCNTs are considered an ideal material for regulating cellular electrical signals. Studies have already been conducted to modify SWCNTs onto cell membrane surfaces through physical adsorption or chemical cross-linking.

[0004] However, since the disordered aggregation of single-walled carbon nanotubes can easily lead to the destruction of membrane integrity (such as membrane perforation or lipid layer disorder), existing modification methods lack controllability over the SWCNT assembly density and topological structure, and have low energy transfer efficiency (<30%), making it difficult to adapt to the conductivity requirements of different cell types, resulting in severe energy dissipation at the membrane interface and insufficient biocompatibility. Summary of the Invention

[0005] In view of the deficiencies in the above-mentioned related art, the present application provides a cell electrical signal regulation membrane, a preparation method thereof, and an application thereof. The present application constructs a DNA framework for specifically binding to the lipid layer of the cell membrane and functionalizes carbon nanotubes with DNA, and connects the DNA framework and the carbon nanotubes through a complementary hairpin DNA group. On the one hand, the DNA framework guides the carbon nanotubes to self-assemble with molecular precision through complementary base pairing of DNA, so as to arrange the carbon nanotubes in a directional manner, thereby overcoming the disorder of the modified carbon nanotubes. On the other hand, the structural rigidity and cell membrane anchoring ability of the DNA framework are utilized to improve the stability of the carbon nanotubes anchored to the cell membrane, thereby avoiding damage to the integrity of the cell membrane as much as possible. Under the combined effect, the energy transfer efficiency and biocompatibility of the cell electrical signal regulation membrane are improved.

[0006] In the first aspect, the present application provides a cell electrical signal regulation membrane using the following technical solutions: A cell electrical signal regulating membrane comprises a DNA framework for specifically binding to a cell membrane lipid layer, DNA-functionalized carbon nanotubes, and a complementary hairpin DNA group for connecting the DNA framework and the DNA-functionalized carbon nanotubes.

[0007] Preferably, the complementary hairpin DNA group includes a first hairpin DNA and a second hairpin DNA, the first hairpin DNA includes a nucleotide sequence for connecting to the DNA frame, a nucleotide sequence for connecting to the DNA-functionalized carbon nanotube and a nucleotide sequence for connecting to the second hairpin DNA, and the second hairpin DNA includes two different nucleotide sequences for connecting to the first hairpin DNA.

[0008] Preferably, the nucleotide sequence of the first hairpin DNA is SEQ ID NO: 1; and the nucleotide sequence of the second hairpin DNA is SEQ ID NO: 2.

[0009] Preferably, the DNA framework is prepared by the following steps: designing and synthesizing a first DNA, a second DNA, a third DNA and a fourth DNA, modifying cholesterol molecules on the first DNA, the second DNA and the third DNA to obtain a first cholesterol-modified DNA, a second cholesterol-modified DNA and a third cholesterol-modified DNA, and then incubating the first cholesterol-modified DNA, the second cholesterol-modified DNA, the third cholesterol-modified DNA and the fourth DNA with the target cells to form a DNA framework on the surface of the cell membrane lipid layer of the target cells.

[0010] Preferably, during co-incubation, the final concentrations of the first cholesterol-modified DNA, the second cholesterol-modified DNA, the third cholesterol-modified DNA and the fourth DNA are 0.9-1.1 μM.

[0011] Preferably, during co-incubation, the cell density of the target cells is 1.8-2.2×10 5 cells / mL.

[0012] Preferably, the nucleotide sequence of the first DNA is SEQ ID NO: 3; the nucleotide sequence of the second DNA is SEQ ID NO: 4; the nucleotide sequence of the third DNA is SEQ ID NO: 5; and the nucleotide sequence of the fourth DNA is SEQ ID NO: 6.

[0013] Preferably, the DNA-functionalized carbon nanotubes are prepared by modifying a fifth DNA on the carbon nanotubes.

[0014] Preferably, in the process of preparing the carbon nanotubes modified with the fifth DNA, the weight ratio of the carbon nanotubes to the fifth DNA is 1:8-12.

[0015] Preferably, the nucleotide sequence of the fifth DNA is SEQ ID NO:5.

[0016] In a second aspect, the present application provides a method for preparing a cell electrical signal regulation membrane using the following technical solution: A method for preparing a cell electrical signal regulation membrane comprises the following steps: constructing a DNA framework on the surface of a lipid layer of a cell membrane of a target cell, mixing a complementary hairpin DNA group with the target cell having the constructed DNA framework, forming a target cell covered with a three-dimensional dendritic network through a hybridization chain reaction, and mixing and incubating the target cell covered with the three-dimensional dendritic network with DNA-functionalized carbon nanotubes to obtain the cell electrical signal regulation membrane.

[0017] In a third aspect, the present application provides an application of a cell electrical signal regulation membrane in cell electrical signal regulation.

[0018] Preferably, the cell membrane electrical signal includes a calcium ion signal.

[0019] Preferably, the method comprises the following steps: constructing an indium tin oxide integrated chip, culturing target cells having the cell electrical signal regulation membrane formed thereon, and electrically stimulating the cell electrical signal regulation membrane in a static and / or oscillating mode through the integrated chip.

[0020] In summary, this application includes at least one of the following beneficial technical effects: 1. This application constructs a DNA framework for specific binding to the lipid layer of the cell membrane and functionalizes carbon nanotubes with DNA, and connects the DNA framework and carbon nanotubes via complementary hairpin DNA groups. Through complementary DNA base pairing, the DNA framework guides the carbon nanotubes to self-assemble with molecular precision, thereby directional arrangement of the carbon nanotubes, overcoming the disorder of carbon nanotube modification, and achieving improved energy transfer efficiency and biocompatibility of the cell electrical signal regulation membrane.

[0021] 2. This application utilizes the structural rigidity and cell membrane anchoring ability of the DNA framework to improve the stability of anchoring carbon nanotubes on the cell membrane, avoids damage to the integrity of the cell membrane as much as possible, and improves the biocompatibility of the cell electrical signal regulation membrane.

[0022] 3. This application adopts a base complementary pairing method, which is beneficial to improving the density of carbon nanotube modification and the controllability of the formed pore structure, thereby adapting to target cells with different conductivity requirements. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is a flow chart of the method for preparing the cell electrical signal regulation membrane of the present application; Figure 2This is a confocal microscope image of the target cell in which the DNA framework was constructed in Example 1 of the present application; Figure 3 This is a comparison of confocal microscope images of target cells constructed with a DNA framework and target cells constructed with single-stranded cholesterol DNA recovered after fluorescence bleaching in Example 1 of the present application; Figure 4 This is a fluorescence image of a cell formed with the cell electrical signal regulation membrane prepared in Example 1 of the present application; Figure 5 This is a comparison of Raman spectra of cells and carbon nanotubes formed with the cell electrical signal regulation membrane prepared in Example 1 of the present application and bare cells not formed with the cell electrical signal regulation membrane prepared in Example 1 of the present application; Figure 6 This is a cell impedance comparison diagram of cells formed with the cell electrical signal regulation membrane prepared in Example 1 of the present application and bare cells not formed with the cell electrical signal regulation membrane prepared in Example 1 of the present application; Figure 7 This is a comparison chart of the calcium ion fluorescence change curves of Application Example 1 and Comparative Example 1 of the present application; Figure 8 This is a comparison chart of the proliferation of cells after electrical stimulation in Application Example 1 of the present application and bare cells after electrical stimulation in Application Comparative Example 1; Figure 9 This is a comparison chart of gene expression in cells after electrical stimulation in Application Example 1 of the present application and in naked cells after electrical stimulation in Application Comparative Example 1. DETAILED DESCRIPTION

[0024] The present application is described in further detail below in conjunction with Examples and accompanying drawings. The following examples are merely intended to illustrate the present invention and are not to be construed as limiting the scope of the present invention. In the following examples, if specific conditions are not specified, the methods used are conventional methods well known in the art, and the consumable materials and reagents used are commercially available, unless otherwise specified. Unless otherwise indicated, the professional and scientific terms used herein have the same meaning as those familiar to those skilled in the art. In addition, any method or material similar or equivalent to the described content may also be applied to the present invention.

[0025] The raw materials used in the examples and comparative examples can all be obtained commercially.

[0026] Example 1 Example 1 of the present application provides a cell electrical signal regulation membrane, the preparation steps of which are as follows: Construction of DNA framework and membrane anchoring: design first DNA, second DNA, third DNA and fourth DNA, entrust Shengong Bioengineering (Shanghai) Co., Ltd. to synthesize and modify cholesterol molecules on the first DNA, second DNA and third DNA by thiol-maleimide reaction to obtain first cholesterol-modified DNA, second cholesterol-modified DNA and third cholesterol-modified DNA, and dissolve the first cholesterol-modified DNA, second cholesterol-modified DNA, third cholesterol-modified DNA and fourth DNA in Tris-EDTA buffer (10 mM Tris, 1 mM EDTA, pH = 8.0). The final concentration of the first cholesterol-modified DNA, second cholesterol-modified DNA, third cholesterol-modified DNA and fourth DNA is controlled to be 1 μM, and the buffer containing the first cholesterol-modified DNA, second cholesterol-modified DNA, third cholesterol-modified DNA and fourth DNA is incubated with HUVEC cells (cell density 2 × 10 5

[0027] Construction of three-dimensional dendritic network: design first hairpin DNA and second hairpin DNA of complementary hairpin DNA group, entrust Shengong Bioengineering (Shanghai) Co., Ltd. to synthesize, and add the first hairpin DNA and second hairpin DNA into the cell culture fluid containing the target cells with constructed DNA framework, control the final concentration of the first hairpin DNA and second hairpin DNA to be 2 μM, and react at 37°C for 2 h to obtain the cell culture fluid containing the target cells covered with three-dimensional dendritic network.

[0028] Directional assembly of carbon nanotubes: mix carbon nanotubes (single-walled carbon nanotubes, diameter 1-2 nm) with the fifth DNA at a weight ratio of 1:10 in buffer (1 × TBE and 100 mM NaCl, pH 8.3), ultrasonically treat to form a uniform dispersion liquid containing DNA functionalized carbon nanotubes, control the concentration of the DNA functionalized carbon nanotubes to be 50 μg / mL, and add excess dispersion liquid into the cell culture fluid containing the target cells covered with three-dimensional dendritic network, incubate at 37°C for 1 h, and shape the cell electrical signal regulation membrane on the surface of the target cells.

[0029] The nucleotide sequences of the first DNA, second DNA, third DNA, fourth DNA, fifth DNA, first hairpin DNA, second hairpin DNA, first cholesterol-modified DNA, second cholesterol-modified DNA and third cholesterol-modified DNA in this embodiment are shown in Table 1.

[0030] Table 1:

[0031] ​The "TATCACCAGGCAGTTGA" sequence in SEQ ID NO:3 is complementary to the "TCAACTGCCTGGTGATA" sequence in SEQ ID NO:4, the "ACGACACTACGTGGGAA" sequence in SEQ ID NO:4 is complementary to the "TTCCCACGTAGTGTCGT" sequence in SEQ ID NO:5, the "GTATTGGACCCTCGCAT" in SEQ ID NO:5 is complementary to the "ATGCGAGGGTCCAATAC" sequence in SEQ ID NO:3, the "GTGTAGCAAGCTGTAAT" sequence in SEQ ID NO:3 is complementary to the "ATTACAGCTTGCTACAC" sequence in SEQ ID NO:6, the "TACTATGGCGGCTCTTC" sequence in SEQ ID NO:4 is complementary to the "GAAGAGCCGCCATAGTA" sequence in SEQ ID NO:6, the "TTCAGACTTAGGAATGT" in SEQ ID NO:5 is complementary to the "ATGCGAGGGTCCAATAC" sequence in SEQ ID NO:3 NO:6 "ACATTCCTAAGTCTGAA" sequence base pairing. Through the above design, the first cholesterol modified DNA, the second cholesterol modified DNA, the third cholesterol modified DNA and the fourth DNA form a complementary base pairing as shown below. Figure 1 The triangular pyramid-shaped DNA framework shown in the figure and the cholesterol structure are located on the same plane, which improves the anchoring effect with the cell membrane surface.

[0032] The "AAAAAGAGAACCTGGG" sequence in SEQ ID NO:1 is base-complementarily paired with the "CCCAGGTTCTCTTTTT" sequence in SEQ ID NO:6, and the DNA framework triggers the unfolding of the first hairpin DNA and connects to the first hairpin DNA. The "GAACCTGGGCATCGTA" sequence in SEQ ID NO:2 is base-complementarily paired with the "TACGATGCCCAGGTTC" sequence in SEQ ID NO:1, the "CCCAGGTTCTCTTTTT" sequence in SEQ ID NO:2 is base-complementarily paired with the "AAAAAGAGAACCTGGG" sequence in SEQ ID NO:1, and the "AGAATAGCCTCGCATC" sequence in SEQ ID NO:7 is base-complementarily paired. By adopting the above scheme, after the first hairpin DNA is connected to the DNA framework, a portion of the second hairpin DNA is now connected to the first hairpin DNA. The first hairpin DNA still contains a sequence for complementary pairing with the fifth DNA, and the second hairpin DNA still contains a sequence for subsequent connection with the first hairpin DNA. In other words, by controlling the reaction time of the first and second hairpin DNAs, the amount of sequence for complementary pairing with the fifth DNA can be controlled, thereby regulating the binding density of the added DNA-functionalized carbon nanotubes. The "GAACCTGGGCATCGTA" sequence in the second hairpin DNA is consistent with the sequence in the DNA framework for base pairing with the first hairpin DNA. This streamlined structure of the first hairpin DNA is beneficial for balancing thermodynamic stability and kinetic responsiveness, reducing the probability of nonspecific binding and amplification problems that may be caused by excessively long sequences. At the same time, it improves the consistency of the unfolding direction of the second hairpin DNA when connected to the first hairpin DNA, thereby improving the orderliness of the carbon nanotube assembly.

[0033] Application Example 1 Application Example 1 of the present application provides an application of a cell electrical signal regulation membrane in cell electrical signal regulation, including the following steps: First, the indium tin oxide (ITO) conductive glass substrate (sheet resistance of 15-25 Ω / sq) was subjected to standard cleaning treatment: sequentially ultrasonic cleaning in a detergent solution, deionized water, acetone and isopropanol for 15 minutes, and then dried with high-purity nitrogen for standby. The preparation of the polydimethylsiloxane (PDMS) microfluidic chamber used a soft lithography process: the PDMS prepolymer and its curing agent were mixed at a ratio of 10:1 (w / w), and the bubbles were removed in a vacuum dryer for about 30 minutes. Then, the degassed PDMS mixture was poured into the pre-prepared silicon-based master mold, and heat-cured in an oven at 80°C for 2 hours. After complete curing, the PDMS layer was carefully peeled off from the master mold, and cut into a chamber structure with a length of 2 cm x 2 cm according to experimental requirements. To achieve effective bonding of the PDMS chamber and the ITO glass substrate, the contact surface of the cut PDMS chamber and the clean ITO glass sheet were simultaneously subjected to surface activation in a plasma cleaner (oxygen plasma, power 50 W, treatment time 60 seconds). After treatment, the activated surface of the PDMS chamber was conformally attached to the conductive surface of the ITO glass, and a slight pressure was applied to ensure tight contact, thereby forming an irreversible sealed bonding structure, and finally obtaining an integrated chip that can be used for subsequent cell electrical stimulation experiments.

[0034] The cells shaped with the cell electrical signal regulation membrane prepared in Example 1 were adherently cultured on the integrated chip, and subjected to electrical stimulation by a Shanghai Chenhua Company electrochemical workstation (CHI660E). The stimulation voltage was 0.8 V, the electric field intensity was 0.8 V / cm, the voltage rise time was 85 s, and the maximum value was maintained for 100 s, and the electrical stimulation was performed in a static mode.

[0035] Comparative Example 1 The difference between Comparative Example 1 and Example 1 is that Comparative Example 1 uses naked cells without a cell electrical signal regulation membrane on the surface for electrical stimulation.

[0036] Test detection (1) The target cells with DNA framework constructed in Example 1 were observed by confocal microscopy, and the images are shown in Figure 2 The target cells with DNA framework constructed in Example 1 and the target cells with cholesterol-modified DNA single strand were detected by confocal microscopy combined with fluorescence recovery after photobleaching (FRAP) technology, and the comparison of confocal microscopy images is shown in Figure 3

[0037] (2) The cells shaped with the cell electrical signal regulation membrane prepared in Example 1 were detected by fluorescence DNA labeling combined with confocal microscopy observation, and the results are as follows:​ Figure 4 shown.

[0038] (3) Detect the Raman spectra of cells and carbon nanotubes formed with the cell electrical signal regulation membrane prepared in Example 1 and bare cells not formed with the cell electrical signal regulation membrane prepared in Example 1 of the present application, and obtain the Raman spectrum comparison diagram as shown in FIG. Figure 5 shown.

[0039] (4) In order to monitor the physiological state of cells in real time and without labeling, the real-time cell analysis (RTCA) system was used to detect the cell impedance, and the time-dependent impedance spectrum reflecting the different HCR reaction times of cell SWCNT was obtained, such as Figure 6 shown.

[0040] (5) The cells with the cell electrical signal regulation membrane formed on their surfaces after electrical stimulation in Application Example 1 and the bare cells after electrical stimulation in Comparative Example 1 were co-incubated with the Fluo-4 calcium ion fluorescent probe (methyl acetate type) probe (final concentration 5 μM) at 37°C for 30 min, and the changes in the ΔF / F signal were recorded using a confocal microscope. Figure 7 shown.

[0041] (6) The cells with the cell electrical signal regulation membrane formed on their surfaces after the electrical stimulation of Application Example 1 and the bare cells after the electrical stimulation of Comparative Example 1 were co-incubated with 5-ethynyl-2'-deoxyuridine-Alexa Fluor 647 fluorescent marker (EdU-647) (final concentration 10 μM) at 37°C for 2 h to detect cell proliferation. The results are as follows: Figure 8 shown.

[0042] (7) A commercial RNA extraction kit (Thermo Fisher Scientific) was used to extract RNA from cells with cell electrical signal regulation membranes formed on their surfaces after electrical stimulation in Application Example 1 and from naked cells after electrical stimulation in Comparative Example 1. A typical three-step thermal cycling method was used on a real-time fluorescence quantitative PCR instrument. The program was: pre-denaturation at 95°C for 10 minutes; followed by 40-45 cycles of denaturation at 95°C for 15 seconds and annealing / extension at 60°C for 30-60 seconds; finally, melting curve analysis was performed to confirm the specificity of the product and to detect the activation of target genes of calmodulin-dependent protein kinase I (CAMK1) and nuclear factor of activated T cells type 1 (NFATC1). The results are as follows: Figure 9 shown.

[0043] Result Analysis The following is combined with Table 1 and Figure 1-9 This application is described in detail.

[0044] Reference Figure 2The fluorescence signal on the cell membrane surface confirmed that the DNA framework had been successfully constructed and positioned on the cell membrane, showing a relatively uniform ring distribution. Through the hydrophobic effect of cholesterol molecules, the cholesterol-modified DNA was able to anchor in the lipid bilayer of the cell membrane, providing a foundation for the subsequent assembly of functionalized carbon nanotubes.

[0045] Reference Figure 3 The results show fluorescence recovery of two groups of cells (top row: target cells constructed with single-stranded cholesterol DNA, green fluorescence; bottom row: target cells constructed with the DNA framework described in Example 1, red fluorescence) before photobleaching, after photobleaching (0 seconds), and 20 seconds after photobleaching. The bleached area is marked by a white dashed box. The fluorescence recovery rate and degree of recovery at the cell membrane surface in target cells constructed with the DNA framework described in Example 1 were slower and less complete than those in target cells constructed with single-stranded cholesterol-modified DNA. This analysis is based on the fact that fluorescence recovery after photobleaching (FRAP) is used to monitor the lateral diffusion of macromolecules. Single-stranded cholesterol DNA molecules are relatively small and can move relatively freely on the cell membrane, resulting in faster fluorescence recovery in the bleached area. However, in target cells constructed with the DNA framework described in Example 1, due to its larger size and more stable structure formed by multiple anchor points, the DNA framework exhibits significantly reduced mobility on the cell membrane, resulting in slower and incomplete fluorescence recovery in the bleached area, which facilitates the subsequent precise and stable assembly of other functional units.

[0046] Reference Figure 4 , where the cell nucleus appears as blue fluorescence (stained nucleus), while the cell membrane and surrounding areas exhibit yellow speckled or network-like fluorescence signals (carbon nanotube network signals). Fluorescently labeled DNA-functionalized carbon nanotubes successfully connect to the DNA framework on the cell surface via complementary hairpin DNA groups, forming a cell electrical signal regulation membrane covering the cell surface. The yellow fluorescence signal is distributed on the cell membrane surface, indicating that through complementary DNA base pairing, the DNA-functionalized carbon nanotubes are precisely guided and assembled onto the DNA framework on the cell membrane surface, successfully constructing the cell electrical signal regulation membrane.

[0047] Reference Figure 5 The results showed that the spectrum at 1367 cm -1 (D peak), 1591 cm -1 (G peak) and 2585 cm -1 Characteristic peaks such as (2D peak or G' peak) are typical Raman characteristics of carbon nanotubes. The Raman spectrum of cells formed with the cell electrical signal regulation membrane prepared in Example 1 clearly shows the characteristic peaks of carbon nanotubes (such as G peak and D peak). Their peak shapes and positions are similar to those of pure carbon nanotubes. However, these characteristic peaks are absent in the Raman spectrum of bare cells, confirming that the carbon nanotubes have been successfully modified onto the surface of the target cells.

[0048] Reference Figure 6 The results showed that compared with bare cells, cells molded with the cell electrical signal regulation membrane prepared in Example 1 showed a lower cell index / impedance. Cell impedance is an indicator of the degree to which the cell layer hinders the current, and is used for label-free real-time monitoring of cell status. Carbon nanotubes are highly conductive materials. When the cell surface is successfully modified with a regulatory membrane composed of DNA-functionalized carbon nanotubes, the conductivity of the cell-electrode interface will be enhanced, and the current will pass more easily, resulting in a decrease in the measured cell impedance. This result shows that the construction of the cell electrical signal regulation membrane effectively improves the coupling between cells and external electrical signals.

[0049] Reference Figure 7 The results showed that after applying electrical stimulation, the internal calcium ion concentration (Ca 2+ ) showed a more rapid response and a greater increase in fluorescence intensity. Calcium ions are important second messengers involved in various cellular electrical signaling activities, indicating that the presence of the cellular electrical signaling regulatory membrane significantly enhances the ability of electrical stimulation to regulate intracellular calcium signaling. This is attributed to the carbon nanotube network on the surface of the cellular electrical signaling regulatory membrane, which improves the efficiency of charge transfer to the cell membrane, more effectively activating voltage-gated calcium channels and other calcium ion channels on the cell membrane, leading to stronger calcium influx.

[0050] Reference Figure 8 The results showed that compared with bare cells, the proportion of red fluorescent cells labeled with EdU was higher in cells formed with the cell electrical signal regulation membrane of Example 1, showing more significant proliferation activity, indicating that the cell electrical signal regulation membrane constructed in Example 1 can significantly promote cell proliferation after mediating electrical stimulation. This may be because the cell electrical signal regulation membrane effectively transmits electrical stimulation signals to cells, thereby activating signal pathways related to cell cycle regulation and proliferation, thereby increasing the cell proliferation rate. This finding not only proves that the cell electrical signal regulation membrane constructed in Example 1 has good biocompatibility, but more importantly, reveals that it can effectively mediate electrical signals to produce a positive and significant regulatory effect on key physiological functions of cells (such as cell proliferation).

[0051] Reference Figure 9 The results showed that compared with bare cells, the relative expression levels of CAMK1 and NFATC1 genes were significantly increased after electrical stimulation of cells formed with the cell electrical signal regulation membrane of Example 1. CAMK1 and NFATC1 are key effector molecules downstream of the intracellular calcium signaling pathway. Their activation and expression level changes are signs of the activation of the calcium signaling pathway. Figure 8, cells molded with the cell electrical signal regulation membrane of Example 1 showed a stronger calcium ion response under electrical stimulation. This enhanced calcium signal further activated the downstream signaling pathway, leading to the upregulation of CAMK1 and NFATC1 gene expression. This shows that the cell electrical signal regulation membrane of Example 1 of the present application can effectively convert electrical stimulation into intracellular biochemical signals and regulate the expression of related genes, thereby achieving deep regulation of cell function.

[0052] This specific embodiment is merely an explanation of the present application and is not a limitation of the present application. After reading this specification, those skilled in the art may make non-creative modifications to the present embodiment as needed, but as long as they are within the scope of the claims of the present application, they are protected by the patent law.

Claims

1. A cell electrical signal regulation membrane, characterized in that: The invention comprises a DNA framework for specifically binding to a cell membrane lipid layer, a DNA-functionalized carbon nanotube and a complementary hairpin DNA group for connecting the DNA framework and the DNA-functionalized carbon nanotube.

2. The cell electrical signal regulating membrane according to claim 1, characterized in that: The complementary hairpin DNA group includes a first hairpin DNA and a second hairpin DNA, the first hairpin DNA includes a nucleotide sequence for connecting to the DNA framework, a nucleotide sequence for connecting to the DNA-functionalized carbon nanotube, and a nucleotide sequence for connecting to the second hairpin DNA, and the second hairpin DNA includes two different nucleotide sequences for connecting to the first hairpin DNA.

3. The cell electrical signal regulating membrane according to claim 2, characterized in that: The nucleotide sequence of the first hairpin DNA is SEQ ID NO: 1; the nucleotide sequence of the second hairpin DNA is SEQ ID NO:

2.

4. The cell electrical signal regulating membrane according to claim 1, characterized in that: The DNA framework is prepared by the following steps: designing and synthesizing a first DNA, a second DNA, a third DNA, and a fourth DNA, modifying cholesterol molecules on the first DNA, the second DNA, and the third DNA to obtain a first cholesterol-modified DNA, a second cholesterol-modified DNA, and a third cholesterol-modified DNA, and then co-incubating the first cholesterol-modified DNA, the second cholesterol-modified DNA, the third cholesterol-modified DNA, and the fourth DNA with target cells to form a DNA framework on the surface of the cell membrane lipid layer of the target cells.

5. The cell electrical signal regulating membrane according to claim 4, characterized in that: The nucleotide sequence of the first DNA is SEQ ID NO: 3; the nucleotide sequence of the second DNA is SEQ ID NO: 4; the nucleotide sequence of the third DNA is SEQ ID NO: 5; and the nucleotide sequence of the fourth DNA is SEQ ID NO:

6.

6. The cell electrical signal regulating membrane according to claim 1, characterized in that: The DNA functionalized carbon nanotube is prepared by modifying a fifth DNA on the carbon nanotube.

7. The cell electrical signal regulating membrane according to claim 6, characterized in that: The nucleotide sequence of the fifth DNA is SEQ ID NO:

7.

8. A method for preparing a cell electrical signal regulation membrane according to any one of claims 1 to 7, characterized in that: The following steps are involved: A DNA framework is constructed on the surface of the lipid layer of the cell membrane of the target cell, a complementary hairpin DNA group is mixed with the target cell with the constructed DNA framework, and a target cell covered with a three-dimensional dendritic network is formed through a hybridization chain reaction. The target cell covered with the three-dimensional dendritic network is mixed with DNA-functionalized carbon nanotubes and incubated to obtain the cell electrical signal regulation membrane.

9. Use of the cell electrical signal regulation membrane according to any one of claims 1 to 7 in cell electrical signal regulation.

10. The use according to claim 9, characterized in that: The following steps are involved: An indium tin oxide integrated chip is constructed, target cells formed with the cell electrical signal regulation membrane are placed on the integrated chip for culture, and the cell electrical signal regulation membrane is electrically stimulated in a static and / or oscillating mode through the integrated chip.

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