CN112504946A - High-flux measuring device and method for tension of single cell membrane - Google Patents
High-flux measuring device and method for tension of single cell membrane Download PDFInfo
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Abstract
The invention discloses a high-flux measuring device and a method for the tension of a single cell membrane, wherein the device comprises: the system comprises a micro-fluidic chip module, a pressure control module, a multi-channel image acquisition module and a data analysis and processing module; the microfluidic chip module is used for cell separation and providing a multi-channel single cell measurement environment; the pressure control module is used for providing negative pressure, and the negative pressure drives the cell to move; the multi-channel image acquisition module is used for acquiring cell images; the data analysis and processing module is used for processing the image acquired by the multi-channel image acquisition module to obtain the deformation of the cell; and based on the cell deformation and the pressure of the negative pressure provided by the pressure control module, acquiring the tension of the cell membrane by adopting a single cell membrane tension equivalent mechanical model. The invention can effectively realize high-flux measurement of the tension of the cell membrane of the single cell.
Description
Technical Field
The invention relates to the technical field of cell membrane tension measurement, in particular to a high-flux measuring device and method for the tension of a single-cell membrane.
Background
Cells are the basic structure and functional unit of a living body, and are accompanied by proliferation, migration, invasion and differentiation of cells in the process of life activities of the living body. Human studies of cells have never been stopped since 1665 cells were discovered by robert. It can be said that the state of the cell directly or indirectly reflects the state of the living body.
However, previous cell research approaches have been directed primarily to population cells, resulting in an average result for the cell population, which often masks the differences that exist between cells. In many cases, the data obtained at single cell resolution more truly reflect cell state and cell-to-cell differences.
As an important part of single cell analysis, cell membrane tension can reflect the functional state of the cell. The vital activities of cells are often accompanied by changes in the cell membrane skeleton, the cell membrane structure, and these structural changes can be manifested as changes in cell membrane tension. For example, lung tumor cells can change the cell membrane tension during canceration due to the change of cytoskeleton and cell membrane proteins during canceration; for example, the cell membrane tension of liver cancer cells with different metastatic potentials shows difference; the epidermal tension of cells at different stages of mitosis also appears to be different.
The single cell has small volume and extremely large sample volume, and in the face of the large sample volume, the high-throughput detection of the tension of the cell membrane of the single cell has extremely important significance for practical application scenes. The conventional method for detecting the epidermal tension of the cell membrane mainly comprises a micropipette and an atomic force microscope. In an atomic force microscope, a probe at the tip of a cantilever exerts force on the surface of a cell, laser beam displacement is used for obtaining the deformation of the probe, and the deformation of the probe is combined with a model so as to obtain the cell membrane tension parameter of a single cell. In the micro-pipette method, negative pressure attracts cells into a capillary, the deformation process of the cells entering the capillary is analyzed through microscopic imaging and image analysis technology, and then a mechanical model is combined to cell membrane tension parameters. These methods successfully obtain the cell membrane tension parameters of single cells, but due to the complicated operation process, the measurement flux is low, and only results of a plurality of cells can be obtained, which cannot meet the actual application requirements. The microfluidic technology is a technology for operating and controlling a small amount of fluid on a microscale, and is very suitable for single cell detection due to the characteristic dimension of the microfluidic technology is close to the cell dimension. In recent years, single cell detection realizes the improvement of detection flux under the promotion of a microfluidic technology, but a high-flux single cell epidermal tension detection method does not appear yet.
Therefore, a high-throughput single-cell epidermal tension detection device and method are needed.
Disclosure of Invention
The invention aims to provide a device and a method for measuring the tension of a single cell membrane at high flux, which are used for solving the technical problems in the prior art and can effectively realize the high flux measurement of the tension of the single cell membrane.
In order to achieve the purpose, the invention provides the following scheme: the invention provides a high-flux measuring device for the tension of a single cell membrane, which comprises: the system comprises a micro-fluidic chip module, a pressure control module, a multi-channel image acquisition module and a data analysis and processing module; the microfluidic chip module is respectively connected with the pressure control module and the multi-channel image acquisition module, and the multi-channel image acquisition module is connected with the data analysis and processing module;
the microfluidic chip module is used for cell separation and providing a multi-channel single cell measurement environment;
the pressure control module is used for providing negative pressure, and the negative pressure drives the cell to move;
the multi-channel image acquisition module is used for acquiring cell images;
the data analysis and processing module is used for processing the image acquired by the multi-channel image acquisition module to obtain the deformation of the cell; and based on the cell deformation and the pressure of the negative pressure provided by the pressure control module, acquiring the tension of the cell membrane by adopting a single cell membrane tension equivalent mechanical model.
Preferably, the microfluidic chip module comprises a substrate, a carrier is arranged on the upper surface of the substrate, a plurality of compression channels are arranged in the carrier, a microfluidic chip inlet and a microfluidic chip outlet are respectively arranged at two ends of the carrier, and the compression channels are connected in parallel between the microfluidic chip inlet and the microfluidic chip outlet; the compression channel is used for allowing cells to flow along the direction of the compression channel.
Preferably, a cell inflow channel and a cell outflow channel are further arranged in the carrier body; the cell inflow channel is connected between the inlet of the microfluidic chip and the compression channel; the cell outflow channel is connected between the outlet of the microfluidic chip and the pressure control module.
Preferably, the cross section of the compression channel is smaller than that of the cell, and the size of the cross section of the compression channel is 5-20 μm.
Preferably, the cross section of the cell inflow channel and the cell outflow channel is larger than that of the cell; the cross sections of the cell inflow channel and the cell outflow channel are 30-1000 mu m in size.
Preferably, the multi-channel image acquisition module comprises a microscope, a camera and an image acquisition controller; the microscope is arranged in the area of the compression channel and used for collecting images of the cells in the compression channel; the camera is used for collecting the image amplified by the microscope; the image acquisition controller is respectively connected with the microscope and the camera and is used for controlling the operation of the microscope and the camera.
Preferably, the specific working method of the data analysis and processing module includes: extracting the front cell membrane equivalent radius and the rear cell membrane equivalent radius of cells in each compression channel by processing the images acquired by the multi-channel image acquisition module; and obtaining the cell membrane tension corresponding to the single cell by adopting a single cell membrane tension equivalent mechanical model based on the front end cell membrane equivalent radius and the rear end cell membrane equivalent radius of the cell and the pressure of the negative pressure provided by the pressure control module.
Preferably, the single-cell membrane tension equivalent mechanical model is as shown in formula 3:
wherein P represents the pressure of the negative pressure provided by the pressure control module, and Pf、PrPressure at the front and rear ends of the cell, TcFor cell membrane tension, Rf、RrThe equivalent radius of the front cell membrane and the equivalent radius of the rear cell membrane of the cell are respectively.
The invention also provides a high-throughput measuring method of the tension of the cell membrane of the single cell, which comprises the following steps:
s1, filling the micro-fluidic chip module with a solution;
s2, adding cells into the microfluidic chip module, and applying negative pressure to the compression channel to control the cells to move along the compression channel;
s3, collecting images of the cell moving through the compression channel;
s4, processing the collected images to obtain the front cell membrane equivalent radius and the rear cell membrane equivalent radius of the cells in each compression channel; and obtaining the cell membrane tension corresponding to the single cell by adopting a single cell membrane tension equivalent mechanical model based on the front end cell membrane equivalent radius and the rear end cell membrane equivalent radius of the cell and the pressure of the negative pressure applied to the compression channel.
Preferably, in step S1, the solution is isotonic with the cells, and the solution is one of a cell culture solution, a phosphate buffer solution and physiological saline.
The invention discloses the following technical effects:
according to the invention, the cells enter the plurality of compression channels which are connected in parallel through negative pressure, the cells deform when flowing through the plurality of compression channels which are connected in parallel, the cell deformation of the cells in the plurality of compression channels is collected, processed and analyzed, and then the single cell membrane tension parameter is obtained through calculation by combining with a single cell membrane tension equivalent mechanical model, so that the high-flux measurement of the single cell membrane tension is realized, the detection quantity of each sample can reach 1000 or more, the blank that the single cell membrane parameters cannot be obtained in large quantity is filled, and the development of the single cell mechanical characteristic field is facilitated; meanwhile, through high-flux measurement of the tension of the cell membrane of the single cell, the establishment of a large database of cell membrane tension parameters is facilitated, the research on the relation between the cell function state and the cell membrane tension is facilitated, and data support is provided for disease diagnosis, treatment, prevention and the like.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings needed to be used in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings without inventive exercise.
FIG. 1 is a schematic structural diagram of a high throughput measurement device for measuring the cell membrane tension of a single cell according to the present invention;
FIG. 2 is a schematic diagram of a single cell membrane tension equivalent mechanical model according to an embodiment of the present invention;
FIG. 3 is a flow chart of the method for high-throughput measurement of the cell membrane tension of a single cell according to the present invention.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
In order to make the aforementioned objects, features and advantages of the present invention comprehensible, embodiments accompanied with figures are described in further detail below.
Referring to fig. 1, the present embodiment provides a high throughput measurement apparatus for measuring the tension of a single cell membrane, including a microfluidic chip module, a pressure control module, a multi-channel image acquisition module, and a data analysis and processing module; the micro-fluidic chip module is respectively connected with the pressure control module and the multi-channel image acquisition module, and the multi-channel image acquisition module is connected with the data analysis and processing module.
The microfluidic chip module is used for cell separation and providing a multi-channel single cell measurement environment; the micro-fluidic chip module comprises a substrate, a carrier is arranged on the upper surface of the substrate, a plurality of compression channels are arranged in the carrier, a micro-fluidic chip inlet and a micro-fluidic chip outlet are respectively arranged at two ends of the carrier, and the compression channels are connected in parallel between the micro-fluidic chip inlet and the micro-fluidic chip outlet;
the substrate is made of one of glass, a silicon wafer, polymethyl methacrylate (PMMA) and Polydimethylsiloxane (PDMS), and other materials can be selected according to requirements;
the material of the bearing body is one of glass, SU-8 and silicon chip materials, and other materials can be selected according to the requirement;
the plurality of compression channels are used for being extruded by cells and moving along the direction of the compression channels; the cell detection flux can be effectively improved through a plurality of compression channels which are connected in parallel; the cross section of the compression channel is one of rectangular, circular and semicircular, and can be set to other shapes according to requirements; the cross section of the compression channel is smaller than that of the cell, and the size of the cross section of the compression channel is 5-20 micrometers; the number of the compression channels is any number, and is more than or equal to 1; the length of the compression channel is 2-10000 mu m, and the lengths of a plurality of compression channels are the same or different;
a cell inflow channel and a cell outflow channel are also arranged in the carrier body; the cell inflow channel is connected between the inlet of the microfluidic chip and the compression channel and used for enabling cells to smoothly enter the compression channel; the cell outflow channel is connected between the outlet of the microfluidic chip and the pressure control module; the cross section of the cell inflow channel and the cell outflow channel is larger than that of the cell; the cross sections of the cell inflow channel and the cell outflow channel are 30-1000 mu m in size.
The pressure control module is communicated with the outlet of the microfluidic chip and is used for providing negative pressure to drive the cell to move in the compression channel; the pressure control module comprises a pressure source, a pressure controller and a closed guide pipe; the pressure controller is connected with the pressure source; the pressure source is used for providing a negative pressure source; the pressure controller is used for outputting negative pressure and controlling the output negative pressure; the closed conduit is used for connecting the pressure controller and the outlet of the microfluidic chip, and applying negative pressure at the inlet of the microfluidic chip to provide power for cell movement; the pressure control module can also adopt the structure of a pump and a liquid communicating pipe as long as negative pressure drive can be provided.
The multi-channel image acquisition module is used for acquiring images of cells passing through the compression channel; the multi-channel image acquisition module comprises a microscope, a camera and an image acquisition controller; the microscope is arranged in the area of the compression channel and used for collecting images of the cells in the compression channel; the camera is used for collecting the image amplified by the microscope; the image acquisition controller is respectively connected with the microscope and the camera and is used for controlling the operation of the microscope and the camera; wherein, the microscope and the camera are conventional structures in the field, and are not described herein again.
The data analysis and processing module is used for processing the image acquired by the multi-channel image acquisition module to obtain the deformation of the cell in the compression channel, and the tension of the cell membrane is acquired by adopting a single-cell membrane tension equivalent mechanical model. The data analysis and processing module includes various forms of computing devices, such as general purpose computers, Application Specific Integrated Circuits (ASICs), Field Programmable Gate Arrays (FPGAs). The data analysis and processing module can work according to the method flow by loading programs and code segments stored in the storage device so as to complete image processing and cell cytoplasm viscosity calculation. The data analysis and processing module also includes an input device, such as a mouse, a keyboard, etc., for inputting user commands, data, and an output device, such as a display, for outputting processing results (e.g., prediction results, etc.). In this embodiment, the combination of the input device and the output device is implemented as a touch screen.
The specific principle of the embodiment that the cell membrane tension is obtained by the high-flux measuring device for the cell membrane tension of the single cell is as follows: the cells are driven by the pressure control module to continuously enter a plurality of compression channels which are connected in parallel, deformation is generated in the compression channels, and the multi-channel image acquisition module acquires images of the cells in the compression channels; transmitting the collected image data to the data analysis and processing module, and extracting the front cell membrane equivalent radius R of the cell in each compression channel through image processingfRear cell membrane equivalent radius Rr(ii) a Cell-based front cell membrane equivalent radius RfRear cell membrane equivalent radius RrAnd the pressure P of the negative pressure provided by the pressure control module adopts a single cell membrane tension equivalent mechanical model to obtain the cell membrane tension corresponding to the single cell.
In this example, the equivalent mechanical model of the cell membrane tension of a single cell is shown in FIG. 2.
For the front cell membrane there are:
for the posterior cell membrane there are:
wherein, Pf、PrPressure at front and back ends of the cell, respectively, and having Pr-PfP is the pressure of the negative pressure provided by the pressure control module, PinIs the pressure inside the cell, TcIs the cell membrane tension.
The formula (1) and the formula (2) are combined to obtain:
the front cell membrane equivalent radius R of each cellfRear cell membrane equivalent radius RrSubstituting the pressure P of the negative pressure provided by the pressure control module into a formula (3) to obtain the cell membrane tension T of each single cellc。
Referring to fig. 3, the present embodiment further provides a high throughput measurement method of tension of a single cell membrane, which specifically includes the following steps:
s1, filling the micro-fluidic chip module with a solution to realize that the added cells are in a suspension state;
specifically, the solution is isotonic to cells, and one of a cell culture solution, Phosphate Buffered Saline (PBS), and physiological saline is used.
S2, adding cells into the microfluidic chip module, and applying negative pressure to the compression channel to control the cells to move along the compression channel;
s3, collecting images of the cell moving through the compression channel;
s4, processing the collected image to obtain the equivalent radius R of the front cell membrane of the cell in each compression channelfRear cell membrane equivalent radius Rr(ii) a Cell-based front cell membrane equivalent radius RfRear cell membrane equivalent radius RrAnd the pressure P of the negative pressure applied to the compression channel adopts a single cell membrane tension equivalent mechanical model to obtain the cell membrane tension corresponding to the single cell.
The equivalent mechanical model of single cell membrane tension is as follows:
for the front cell membrane there are:
for the posterior cell membrane there are:
wherein, Pr、PfPressure at front and back ends of the cell, respectively, and having Pr-PfP is the pressure provided by the pressure control module, PinIs the pressure inside the cell, TcIs the cell membrane tension.
The formula (1) and the formula (2) are combined to obtain:
the front cell membrane equivalent radius R of each cellfRear cell membrane equivalent radius RrThe pressure P generated by the pressure control module is substituted into formula (3) to obtain the cell membrane tension T of each single cellc。
The above-described embodiments are merely illustrative of the preferred embodiments of the present invention, and do not limit the scope of the present invention, and various modifications and improvements of the technical solutions of the present invention can be made by those skilled in the art without departing from the spirit of the present invention, and the technical solutions of the present invention are within the scope of the present invention defined by the claims.
Claims (10)
1. A device for high throughput measurement of the cell membrane tension of a single cell, comprising: the system comprises a micro-fluidic chip module, a pressure control module, a multi-channel image acquisition module and a data analysis and processing module; the microfluidic chip module is respectively connected with the pressure control module and the multi-channel image acquisition module, and the multi-channel image acquisition module is connected with the data analysis and processing module;
the microfluidic chip module is used for cell separation and providing a multi-channel single cell measurement environment;
the pressure control module is used for providing negative pressure, and the negative pressure drives the cell to move;
the multi-channel image acquisition module is used for acquiring cell images;
the data analysis and processing module is used for processing the image acquired by the multi-channel image acquisition module to obtain the deformation of the cell; and based on the deformation of the cell and the pressure of the negative pressure provided by the pressure control module, acquiring the tension of the cell membrane by adopting a single cell membrane tension equivalent mechanical model.
2. The device for measuring the tension of the single cell membrane with high flux as claimed in claim 1, wherein the microfluidic chip module comprises a substrate, a carrier is disposed on the upper surface of the substrate, a plurality of compression channels are disposed in the carrier, a microfluidic chip inlet and a microfluidic chip outlet are disposed at two ends of the carrier, and the plurality of compression channels are connected in parallel between the microfluidic chip inlet and the microfluidic chip outlet; the compression channel is used for allowing cells to flow along the direction of the compression channel.
3. The device for high throughput measurement of the tension of the single cell membrane according to claim 2, wherein a cell inflow channel and a cell outflow channel are further provided in the carrier; the cell inflow channel is connected between the inlet of the microfluidic chip and the compression channel; the cell outflow channel is connected between the outlet of the microfluidic chip and the pressure control module.
4. The device for high throughput measurement of the tension of the single cell membrane according to claim 2, wherein the cross section of the compression channel is smaller than that of the cell, and the size of the cross section of the compression channel is 5-20 μm.
5. The device for high throughput measurement of the cell membrane tension of a single cell according to claim 3, wherein the cross section of the cell inflow channel and the cell outflow channel is larger than the cross section of the cell; the cross sections of the cell inflow channel and the cell outflow channel are 30-1000 mu m in size.
6. The device for high-throughput measurement of the tension of the cell membrane of a single cell according to claim 2, wherein the multi-channel image acquisition module comprises a microscope, a camera and an image acquisition controller; the microscope is arranged in the area of the compression channel and used for collecting images of the cells in the compression channel; the camera is used for collecting the image amplified by the microscope; the image acquisition controller is respectively connected with the microscope and the camera and is used for controlling the operation of the microscope and the camera.
7. The device for high throughput measurement of the cell membrane tension of a single cell according to claim 2, wherein the specific working method of the data analysis and processing module comprises: extracting the front cell membrane equivalent radius and the rear cell membrane equivalent radius of cells in each compression channel by processing the images acquired by the multi-channel image acquisition module; and obtaining the cell membrane tension corresponding to the single cell by adopting a single cell membrane tension equivalent mechanical model based on the front end cell membrane equivalent radius and the rear end cell membrane equivalent radius of the cell and the pressure of the negative pressure provided by the pressure control module.
8. The device for high throughput measurement of the tension of single-cell membrane according to claim 7, wherein the equivalent mechanical model of single-cell membrane tension is represented by formula 3:
wherein P represents the pressure of the negative pressure provided by the pressure control module, and Pf、PrPressure at the front and rear ends of the cell, TcFor cell membrane tension, Rf、RrThe equivalent radius of the front cell membrane and the equivalent radius of the rear cell membrane of the cell are respectively.
9. The method for high throughput measurement of the cell membrane tension of a single cell according to any one of claims 2 to 8, comprising the steps of:
s1, filling the micro-fluidic chip module with a solution;
s2, adding cells into the microfluidic chip module, and applying negative pressure to the compression channel to control the cells to move along the compression channel;
s3, collecting images when the cells move through the compression channel;
s4, processing the collected images to obtain the front cell membrane equivalent radius and the rear cell membrane equivalent radius of the cells in each compression channel; and obtaining the cell membrane tension corresponding to the single cell by adopting a single cell membrane tension equivalent mechanical model based on the front end cell membrane equivalent radius and the rear end cell membrane equivalent radius of the cell and the pressure of the negative pressure applied to the compression channel.
10. The method for high-throughput measurement of cell membrane tension of single cell according to claim 9, wherein in step S1, the solution is isotonic with the cell, and the solution is one of cell culture solution, phosphate buffer solution and physiological saline.
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| CN114112807A (en) * | 2021-11-05 | 2022-03-01 | 国家纳米科学中心 | Quantitative characterization method of cell membrane tension |
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