CN112014429A - Cell membrane vibration detection method based on ultramicro electroosmotic flow regulation - Google Patents
Cell membrane vibration detection method based on ultramicro electroosmotic flow regulation Download PDFInfo
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- 210000000170 cell membrane Anatomy 0.000 title claims abstract description 41
- 238000005370 electroosmosis Methods 0.000 title claims abstract description 20
- 230000033228 biological regulation Effects 0.000 title claims abstract description 15
- 238000001514 detection method Methods 0.000 title claims abstract description 9
- 239000002071 nanotube Substances 0.000 claims abstract description 42
- 210000004027 cell Anatomy 0.000 claims abstract description 37
- 239000010453 quartz Substances 0.000 claims abstract description 36
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- HKZLPVFGJNLROG-UHFFFAOYSA-M silver monochloride Chemical compound [Cl-].[Ag+] HKZLPVFGJNLROG-UHFFFAOYSA-M 0.000 claims description 6
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Abstract
The invention provides a cell membrane vibration detection method based on ultramicro electroosmotic flow regulation, which distinguishes different cells and different states of the same cell through cell membrane vibration frequency caused by electroosmotic flow regulation in a quartz nanotube, wherein the tip size of the quartz nanotube is 50nm-300 nm. The invention overcomes the defects of time consumption, difficult operation and the like of the traditional cell membrane monitoring, the quartz nanotube is directly used for monitoring from a specific position on the surface of a single living cell membrane, the voltage applied to the electrode end can accurately control the electroosmotic flow generated by the tip of the quartz nanotube, the electroosmotic flow can drive the cell membrane to vibrate, and the biophysics of the cell membrane at the level of a single cell can be more comprehensively understood. The method provides a simple and reliable method for simultaneously analyzing local and global membrane vibration of a single living cell, has small damage, and provides a tool for quantification of drugs, diseases or cell structure mutation.
Description
Technical Field
The invention relates to a cell imaging technology, in particular to a method for detecting cell membrane vibration based on an ultramicro electroosmotic flow regulation technology.
Background
The cell membrane, also called plasma membrane, is a very thin membrane surrounding the cell surface, and is mainly composed of phospholipids and membrane proteins. The basic role of the cell membrane is to maintain the relative stability of the intracellular microenvironment and to participate in material exchange, energy and information transfer with the external environment. In addition, they play an important role in the survival, growth, division and differentiation of cells. The cell membrane is not only the boundary of the cell, but also the expression platform of many proteins and glycans, and is used for regulating various cell essential functions such as cellular communication, metabolism and transportation. The cell membrane is composed of lipid bilayers that can vibrate and deform, which is critical for the regulation of cell shape. Membrane vibration plays an important role in cell behavior and is a key factor reflecting the physiological state of cells. However, previous studies of cell mechanical properties often required complex equipment or indirect studies through exogenous modification.
The traditional patch clamp measurement method comprises the following steps: when the open quartz electrode touches the cell membrane, the open end and the cell membrane jointly form a tiny sealing area, so that the sealing area is isolated from the surrounding environment, the ionic current of the area is monitored, and the traditional cell membrane has the defects of time consumption, difficult operation and the like.
Disclosure of Invention
The invention aims to provide a cell membrane vibration detection method based on ultramicro electroosmotic flow regulation and control, which does not need to use any external marker and only utilizes a quartz nanotube to accurately measure the vibration of a cell membrane.
The second purpose of the invention is to provide the application of the quartz nanotube in monitoring cell membranes.
In order to achieve the first object, the invention provides a cell membrane vibration detection method based on ultramicro electroosmotic flow regulation, which is characterized in that different cells and different states of the same cell are distinguished by cell membrane vibration frequency caused by electroosmotic flow regulation in a quartz nanotube, and the tip size of the quartz nanotube is 50nm-300 nm.
As a preferred scheme, the circuit in detection is formed by the following modes: soaking the silver wire in ferric chloride solution overnight, soaking one Ag/AgCl electrode in the quartz nanotube solution, and soaking the other Ag/AgCl electrode in cell culture solution.
As a preferred scheme, the quartz nanotube is drawn by a laser drawing instrument P-2000, and the instrument parameters are specifically set as follows: line 1: Heat 650, Fil 3, Vel 35, Del 145, Pul 75; line 2: Heat 920, Fil 2, Vel 15, Del 128, Pul 200.
As a preferable scheme, the tip size of the quartz nanotube is 100 nm.
In order to achieve the second object, the invention provides the application of the quartz nanotube in monitoring cell membranes, which is characterized in that different cells and different states of the same cell are distinguished by cell membrane vibration frequency caused by electroosmotic flow regulation in the quartz nanotube, and the tip size of the quartz nanotube is 50nm-300 nm.
The invention selects two cells: human breast cancer cells (MCF-7) and cervical cancer cells (HeLa) were cultured in adherent culture overnight. The nanotube is used for accurately measuring cell membranes of MCF-7 cells and HeLa cells, and voltage is regulated at two ends of an electrode to cause cell membrane vibration.
The invention has the advantages that the invention discloses a cell membrane vibration frequency detection method based on quartz nanopores, which does not need any external mark, overcomes the defects of time consumption, difficult operation and the like of the traditional cell membrane monitoring, directly monitors the specific position on the surface of a single living cell membrane through a quartz nanotube, can accurately control the electroosmotic flow generated by the tip of the quartz nanotube by the voltage applied at an electrode end, can drive the cell membrane to vibrate, and has more comprehensive understanding on the biophysics of the cell membrane at the level of a single cell. The method provides a simple and reliable method for simultaneously analyzing local and global membrane vibration of a single living cell, has small damage, and provides a tool for quantification of drugs, diseases or cell structure mutation.
Drawings
FIG. 1.a schematic of nanotube device, b vibration of cell membrane caused by electroosmotic flow at nanotube tip, c continuous current plot at 0mV of nanotube.
Fig. 2. from the current signal, the time domain of the current can be decomposed into two different frequencies: local vibrations under the effect of electroosmotic flow and the natural frequency of the cell membrane itself.
Detailed Description
Hereinafter, the technique of the present invention will be described in detail with reference to specific embodiments. It should be understood that the following detailed description is only for the purpose of assisting those skilled in the art in understanding the present invention, and is not intended to limit the present invention.
Example 1.
(1) The quartz nanotube is prepared by laser heating a quartz capillary tube by a P-2000 laser drawing instrument. The specific method comprises the following steps: the quartz capillary tube was sequentially washed in ethanol, acetone and pure water for about 30 minutes, and finally blown dry with nitrogen. And setting parameters of a laser drawing instrument program to obtain quartz nanopores with different sizes. First, the capillary tube was placed on a P-2000, placed symmetrically, and drawn after the carbon dioxide laser was stabilized. Different parameters in the procedure will affect the pore size of the quartz nanotubes and the length of the tip drawn. The parameters to be set are respectively the heating temperature (HEAT): 0-999, is used for heating and melting the quartz capillary used; FILAMENT (FILAMENT): 0-15, which is used for setting different scanning speeds of the laser; VELOCITY (VELOCITY): 0-255, adjusting the moving speed of the pull rod; DELAY (DELAY), which influences the length of the tip of the quartz nanotube, wherein when the DELAY is 128 values, the laser irradiation is finished, the pull rod starts to move, the DELAY is more than 128 values, a period of time is required to pass after the laser is finished, the DELAY is less than 128 values, and the pull rod starts to move before the laser is finished; PULL force (PULL): 0-255, which is used to adjust the pulling force on the quartz capillary, and the size of the pulling force also influences the diameter and length of the nanotube tip. The invention uses a two-step method to draw the nanotube, and the parameters are as follows:
Line 1:Heat 650,Fil 3,Vel 35,Del 145,Pul 75；
Line 2:Heat 920,Fil 2,Vel 15,Del 128,Pul 200。
(2) the cells are cultured by conventional methods. MCF-7 cell culture: RPMI 1640, fetal bovine serum (10%), streptomycin (100. mu.g mL)-1) And penicillin (100. mu.g mL)-1) And preparing a culture solution. HeLa cell culture: DMEM, fetal bovine serum (10%), streptomycin (100. mu.g mL)-1) And penicillin (100. mu.g mL)-1) And preparing a culture solution. The cells in both the flask and the dish were cultured at 37 ℃ in a 5% carbon dioxide atmosphere.
(3) A2 mM PBS solution was injected into the quartz nanotubes using a micro-syringe, and the remaining air in the nanotubes was removed by centrifugation to ensure that the solution reached the tip of the tube. One Ag/AgCl electrode is immersed in the solution inside the quartz nanotube, and the other Ag/AgCl electrode is immersed in the cell culture solution. The specific method of the experiment is as follows: temporarily not placing cells on the sample table, moving the tube tip of the nanotube to the middle of a visual field by moving in the X, Y and Z axis directions, ensuring that the XY axis does not move, only moving the Z axis to lift the nanotube, placing the cells for experiments on the sample table after the nanotube is lifted to a sufficient height, finding single MCF-7 cells (or HeLa cells) with good shapes, moving the cells to the visual field, keeping the sample table still at the moment, and descending the nanotube again until the nanotube descends into the culture solution but does not touch the cells. Then gradually focusing the tube and the cell until the two approach to the same plane, switching to a micro operation with smaller torque, starting recording by opening a current software, and when the current fluctuates momentarily, proving that the nanotube touches the cell membrane.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (5)
1.A cell membrane vibration detection method based on ultramicro electroosmotic flow regulation is characterized in that different cells and different states of the same cell are distinguished through cell membrane vibration frequency caused by electroosmotic flow regulation in a quartz nanotube, and the tip size of the quartz nanotube is 50nm-300 nm.
2. The method for detecting cell membrane vibration based on ultramicro electroosmotic flow regulation according to claim 1, wherein the circuit in detection is formed by: soaking the silver wire in ferric chloride solution overnight, soaking one Ag/AgCl electrode in the quartz nanotube solution, and soaking the other Ag/AgCl electrode in cell culture solution.
3. The method for detecting cell membrane vibration based on ultramicro electroosmotic flow regulation and control according to claim 1, wherein the quartz nanotube is drawn by a laser drawing instrument P-2000, and the instrument parameters are specifically set as follows: line 1: Heat 650, Fil 3, Vel 35, Del 145, Pul 75; line 2: Heat 920, Fil 2, Vel 15, Del 128, Pul 200.
4. The method for detecting the vibration of the cell membrane based on the regulation and control of the ultramicro electroosmotic flow according to claim 1, wherein the tip size of the quartz nanotube is 100 nm.
5. The application of the quartz nanotube in monitoring cell membranes is characterized in that different cells and different states of the same cell are distinguished by cell membrane vibration frequency caused by electroosmotic flow regulation in the quartz nanotube, and the tip size of the quartz nanotube is 50nm-300 nm.
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| CN115594857A (en) * | 2022-10-20 | 2023-01-13 | 华东理工大学（Cn） | MOFs nanoparticle interface dynamic growth method and MOFs nanoparticles |
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