阅读说明：本技术 检测细胞膜电势的装置及其检测方法 (Device and method for detecting cell membrane potential ) 是由 陈健 张毅 谭惠文 梁红雁 陈德勇 王军波 于 2020-12-03 设计创作，主要内容包括：本发明公开了一种检测细胞膜电势的装置及其检测方法,其中,检测细胞膜电势的装置,包括：微流控芯片模块、电压测量模块、压力控制模块,其中,微流控芯片模块包括：绝缘承载体、绝缘衬底；绝缘承载体依次包括：细胞流入通道、主压缩通道、侧压缩通道、细胞流出通道；绝缘衬底上包含有金属电极,用于实现与电压测量模块的连接；电压测量模块与微流控芯片模块连接；压力控制模块与微流控芯片模块连接；其中,检测细胞膜电势的方法包括操作流程,以及基于等效模型进行数据处理的方法。本发明提供的装置和方法避免了繁琐的捕获细胞、形成高阻封接的过程,提高了细胞膜电势测量的通量；同时不需要染色等过程,实现了细胞膜电势的直接测量。(The invention discloses a device and a method for detecting cell membrane potential, wherein the device for detecting the cell membrane potential comprises the following steps: micro-fluidic chip module, voltage measurement module, pressure control module, wherein, micro-fluidic chip module includes: an insulating carrier, an insulating substrate; insulating supporting body includes in proper order: the cell inflow channel, the main compression channel, the side compression channel and the cell outflow channel; the insulating substrate comprises a metal electrode for realizing the connection with the voltage measuring module; the voltage measuring module is connected with the microfluidic chip module; the pressure control module is connected with the microfluidic chip module; the method for detecting the cell membrane potential comprises an operation flow and a method for processing data based on an equivalent model. The device and the method provided by the invention avoid the fussy processes of capturing cells and forming high-resistance sealing, and improve the flux of measuring the potential of the cell membrane; meanwhile, processes such as dyeing and the like are not needed, and direct measurement of the cell membrane potential is realized.)

1. A device for detecting cell membrane potential comprising: a micro-fluidic chip module, a voltage measuring module, and a pressure control module, wherein,

the microfluidic chip module includes: an insulating carrier, an insulating substrate; the insulating bearing body sequentially comprises: the cell inflow channel, the main compression channel, the side compression channel and the cell outflow channel; the insulating substrate comprises a metal electrode for realizing conductive connection with the voltage measuring module;

the voltage measuring module is connected with the microfluidic chip module;

the pressure control module is connected with the microfluidic chip module.

2. The device for detecting cell membrane potential according to claim 1, wherein the microfluidic chip module is formed by aligned bonding of the insulating carrier and the insulating substrate.

3. The device for detecting membrane potential of a cell according to claim 1, wherein,

the cross-sectional areas of the cell inflow channel and the cell outflow channel are each greater than or equal to 30 μm × 30 μm;

the cell inflow channel is used for enabling cells to flow rapidly so as to ensure detection flux;

the cell outflow channel is used for recovering the detected cells so as to prevent the detected cells from blocking the outlet of the main compression channel.

4. The apparatus for detecting cell membrane potential according to claim 1, wherein the main compression channel has a cross-sectional length and height of 9-11 μm for initially compressing the cell and preventing the cell membrane from being damaged.

5. The device for detecting membrane potential of a cell according to claim 1, wherein,

the side compression passages are arranged on two sides of the preset position of the main compression passage;

the voltage measurement module includes: the data acquisition card is connected with the electrodes in the microfluidic chip module through the shielding wires;

the pressure control module includes: the pressure calibrator is connected with the through hole in the microfluidic chip module through the air guide hose.

6. The apparatus for detecting cell membrane potential according to claim 5, wherein the lateral compression channel has a cross-sectional width of 2 to 4 μm, a height of 9 to 11 μm, and a length of 3 to 8 μm at one side near the predetermined position of the main compression channel; one side of the side compression channel, which is far away from the preset position of the main compression channel, is of a widening structure, the cross section width is 6-10 mu m, the height is 9-11 mu m, which is the same as that of the main compression channel, and the length is larger than or equal to 5 mu m.

7. The apparatus of claim 6, wherein the lateral compression channel is used for cell flowing to the predetermined position far away from the main compression channel, and the cell membrane is locally damaged to form a high-resistance seal.

8. A method of processing a microfluidic chip module, comprising:

forming a positive film of a main compression passage and a side compression passage on a first substrate to obtain a first substrate containing the positive film of the main compression passage and the side compression passage;

forming a positive membrane of a cell inflow channel and a cell outflow channel on the first substrate of the obtained positive membrane comprising the main compression channel and the side compression channel to obtain a first substrate comprising a positive membrane with a specific structure;

pouring the first substrate containing the male die with the specific structure by using a pouring mixed solution based on a molding process, and curing and demolding to obtain a bearing body containing the microfluidic channel;

manufacturing a metal electrode on a second substrate to obtain the second substrate containing the metal electrode;

and punching holes at corresponding positions of the carrier containing the microfluidic channels, and aligning and bonding the carrier with the second substrate containing the metal electrodes to form the microfluidic chip module.

9. A method of detection based on a device for detecting cell membrane potential comprising:

the micro-fluidic chip module is respectively connected with the voltage measuring module and the pressure control module;

filling a preset solution into the microfluidic chip module to prevent the pressure control module from generating bubbles when exerting pressure on cells;

injecting cell suspension into a cell inflow channel in the microfluidic chip module, and respectively applying negative pressure to the compression channels at two sides and the main compression channel by using the pressure control module to drive cells to flow into the main compression channel and the side compression channel;

when the cells flow to the widening structure of the side compression channel, the cells are locally damaged and flow into a cell outflow channel after detection;

and in the cell flowing process, continuously measuring the voltage between the electrodes respectively connected with the outlets of the single side compression channel and the main compression channel by using the voltage measuring module to obtain the cell membrane potential original voltage data of the cells.

10. The method according to claim 9, further comprising:

the equivalent electrical model of the cells after being damaged at the side compression channel widening structure;

and carrying out data processing on the measured data through the equivalent electrical model to obtain the cell membrane potential of the cell in the outlet direction of the main compression channel.

Technical Field

The invention relates to the field of cell membrane potential detection, in particular to a device and a method for detecting cell membrane potential.

Background

The cell membrane potential, which refers to the voltage difference between the inside and outside of the membrane of the biological cell, ranges between-3 and-90 mV, is the result of the ion concentration gradient (in particular potassium, sodium and chloride ions) existing inside and outside the cell membrane, the difference in the permselectivity of the cell membrane for these ions and the combined action of the sodium and potassium pumps. The change of cell membrane potential is closely related to the physiological and pathological processes of cells, for example, the aging of cells is accompanied by the depolarization of cell membrane potential, the cell membrane potential changes during the proliferation, migration and differentiation of cells, and the process of cell canceration is also accompanied by the depolarization of cell membrane potential. Therefore, the detection of cell membrane potential is of great significance.

Methods for detecting the cell membrane potential are mainly microelectrode-based methods and fluorescent dye-based methods. The working principle of the microelectrode-based method is that a micromanipulator is used for controlling the microelectrode to pierce through cells to form a good seal, a millivoltmeter is used for measuring the cell membrane potential between the microelectrode and a reference electrode positioned in bath liquid, and the cell membrane potential is recorded. The method has the advantages that the direct measurement of the potential of the single cell membrane can be realized; the defects are that the operation is complex, the formation of good sealing is difficult and the time is long.

The method based on the fluorescent dye has the working principle that the potential of cell membranes can cause the change of the optical characteristics of proteins, and certain fluorescent dyes have the light-driven outward diffusion effect, so that the molecules of the fluorescent dyes can form unequal distribution inside and outside the cell membranes due to the potential difference between the inside and the outside of the cell membranes. The cell membrane potential can be represented by using a fluorescence microscope to collect signals, utilizing the concentration difference of the molecular distribution of the fluorescent dye and combining a standard curve. The method has the advantages of high response speed, capability of simultaneously representing the cell membrane potential of a plurality of cells and no damage to the cells during measurement; the drawback is that the fit of the standard curve may be biased and that fluorescent dyes may have an effect on the permeability of the cell membrane.

Disclosure of Invention

In view of the above, aiming at the defects in the method, the invention provides a device and a method for detecting cell membrane potential, which avoid the processes of capturing cells and forming high-resistance sealing, and effectively reduce the operation difficulty; the value of the cell membrane potential can be calculated based on the equivalent electrical model, so that the direct measurement of the cell membrane potential is realized; cells can continuously pass through the detection area, and the detection flux is ensured. In order to achieve the above objects, in one aspect, the present invention provides an apparatus for detecting a membrane potential of a cell, comprising: micro-fluidic chip module, voltage measurement module, pressure control module, wherein, micro-fluidic chip module includes: an insulating carrier, an insulating substrate; insulating supporting body includes in proper order: the cell inflow channel, the main compression channel, the side compression channel and the cell outflow channel; the insulating substrate comprises a metal electrode for realizing the connection with the voltage measuring module; the voltage measuring module is connected with the microfluidic chip module; the pressure control module is connected with the microfluidic chip module.

According to an embodiment of the present invention, the microfluidic chip module is formed by aligned bonding of the insulating carrier and the insulating substrate.

According to an embodiment of the present invention, wherein the cross-sectional areas of the cell inflow channel and the cell outflow channel are each greater than or equal to 30 μm × 30 μm; the cell inflow channel is used for enabling cells to flow rapidly so as to ensure detection flux; the cell outflow channel is used for recovering the detected cells to prevent the detected cells from blocking the outlet of the main compression channel.

According to the embodiment of the invention, the length and the height of the cross section of the main compression channel are both 9-11 μm, so that the main compression channel is used for preliminarily compressing cells and ensuring that cell membranes are not damaged.

According to the embodiment of the present invention, wherein the side compression passages are provided at both sides of the preset position of the main compression passage; the voltage measurement module includes: the data acquisition card is connected with the microfluidic chip module through the shielding wire; a pressure control module comprising: the pressure calibrator is connected with the microfluidic chip module through the air guide hose.

According to the embodiment of the invention, the cross section of one side of the side compression passage close to the preset position of the main compression passage has the width of 2-4 μm, the height of the side compression passage is the same as that of the main compression passage and is 9-11 μm, and the length of the side compression passage is 3-8 μm; the side of the side compression channel, which is far away from the preset position of the main compression channel, is of an expanded structure, the cross section width is 6-10 mu m, the height is the same as that of the main compression channel and is 9-11 mu m, and the length is larger than or equal to 5 mu m.

According to the embodiment of the invention, when the side compression channel is used for flowing the cells to the side away from the preset position of the main compression channel, the cell membrane is locally damaged, and a high-resistance seal is formed.

In another aspect, the present invention provides a method for processing a microfluidic chip module, including: forming a positive film of a main compression passage and a side compression passage on a first substrate to obtain a first substrate containing the positive film of the main compression passage and the side compression passage; forming a positive membrane of a cell inflow channel and a cell outflow channel on a first substrate on which the positive membrane comprising a main compression channel and a side compression channel is obtained, to obtain a first substrate comprising a positive membrane of a specific structure; pouring the first substrate containing the male die with the specific structure by using a pouring mixed solution based on a molding process, and curing and demolding to obtain a bearing body containing the microfluidic channel; manufacturing a metal electrode on a second substrate to obtain the second substrate containing the metal electrode; and punching holes at corresponding positions of the carrier containing the microfluidic channels, and aligning and bonding the carrier with a second substrate containing metal electrodes to form the microfluidic chip module.

In another aspect, the present invention also provides a detection method based on the device for detecting cell membrane potential, comprising: the micro-fluidic chip module is respectively connected with the voltage measuring module and the pressure control module; filling a preset solution into the microfluidic chip module to prevent bubbles from being generated when the pressure control module applies pressure to the cells; injecting cell suspension into a cell inflow channel in the microfluidic chip module, and respectively applying negative pressure to the compression channels at the two sides and the main compression channel by using a pressure control module to drive cells to flow into the main compression channel and the side compression channel; when the cells flow to the widening structure of the side compression channel, the cells are locally damaged and flow into the cell outflow channel after detection; in the process of cell flowing, continuously measuring the voltage between electrodes respectively connected with the outlets of the single side compression channel and the main compression channel by using a voltage measuring module to obtain cell membrane potential original voltage data of cells;

according to an embodiment of the present invention, the detection method based on the device for detecting cell membrane potential further includes:

the equivalent electrical model of the damaged cells at the side compression channel widening structure; and carrying out data processing on the measured data through an equivalent electrical model to obtain the cell membrane potential of the detected cell in the outlet direction of the main compression channel.

According to the embodiment of the invention, the device for detecting the cell membrane potential, the detection method thereof and the processing method of the microfluidic chip module in the device are provided, so that the technical problems that the operation for detecting the cell membrane potential is complex, the formation of high-resistance sealing is difficult and the value of the cell membrane potential cannot be accurately obtained in the prior art are solved, and the direct and high-flux detection of the cell membrane potential is realized.

Drawings

FIG. 1 is a schematic view showing an apparatus for detecting a membrane potential of a cell according to an embodiment of the present invention;

fig. 2 schematically shows a schematic view of a microfluidic chip module structure in the device according to an embodiment of the present invention;

fig. 3 schematically shows a process flow diagram of a microfluidic chip module in the device according to an embodiment of the invention;

fig. 4 schematically shows a flow chart of a method of processing a microfluidic chip module in the device according to an embodiment of the present invention;

FIG. 5 schematically shows a flow chart of a detection method based on a device for detecting cell membrane potential according to an embodiment of the present invention;

FIG. 6 is a diagram (a) schematically showing an equivalent electrical model of a cell after breakage of a membrane at the distal end of a lateral compression passage according to an embodiment of the present invention; fig. b is an equivalent circuit of fig. a.

Detailed Description

In order that the objects, technical solutions and advantages of the present invention will become more apparent, the present invention will be further described in detail with reference to the accompanying drawings in conjunction with the following specific embodiments.

FIG. 1 is a schematic diagram of an apparatus for detecting membrane potential of a cell according to an embodiment of the present invention. As shown in FIG. 1, the apparatus for detecting a membrane potential of a cell comprises: the device comprises a micro-fluidic chip module 1, a voltage measuring module 2 and a pressure control module 3. The voltage measuring module 2 is electrically connected with the microfluidic chip module 1, and the pressure control module 3 is connected with the microfluidic chip module 1.

The microfluidic chip module 1 is a core module of the device for detecting cell membrane potential, and comprises: insulating carrier, insulating substrate.

Fig. 2 schematically shows a schematic diagram of a microfluidic chip module structure in the device according to an embodiment of the present invention.

As shown in fig. 2, the insulating carrier sequentially includes, according to a predetermined direction shown in fig. 2: a cell inflow passage 21, a main compression passage 22, a side compression passage 23, a cell outflow passage 24; the insulating substrate contains a metal electrode 25.

According to the embodiment of the invention, the microfluidic chip module 1 is formed by aligning and bonding the insulating carrier and the insulating substrate.

According to the embodiment of the invention, the metal electrodes 25 on the insulating substrate are respectively bonded with the corresponding positions of one of the two side compression channels 23 and the cell outflow channel 24 on the insulating carrier body in an alignment manner, so as to realize the connection between the one of the two side compression channels 23 and the outlet of the main compression channel 22 in the insulating carrier body and an external voltage measuring module.

According to the embodiment of the present invention, the contact area between the metal electrode 25 on the insulating substrate and the one side compression channel 23 and the cell outflow channel 24 is greater than or equal to 100 μm × 100 μm, so as to facilitate the aligned bonding of the insulating carrier and the insulating substrate.

According to the embodiment of the present invention, the cross-sectional area of the cell inflow channel 21 is larger than the mean cell diameter by 15 μm, and the cross-sectional area of the cell inflow channel 21 is larger than or equal to 30 μm × 30 μm for allowing the cells to flow rapidly in the channel, and can continuously pass through the main compression channel 22, ensuring the cell detection throughput when detecting the cell membrane potential.

According to an embodiment of the present invention, the channel length of the cell inflow channel 21 is greater than or equal to 5000 μm to ensure convenience in the punching of the microfluidic chip module.

According to the embodiment of the present invention, the main compression passage 22 is used for primarily compressing the cells after the cells enter the main compression passage, so that the cells are more easily sucked into the side compression passages 23 at both sides of the main compression passage, and meanwhile, the cells are prevented from being excessively compressed and damaged, which affects the effectiveness of the measurement.

According to the embodiment of the present invention, the cross-sectional width and height of the main compression passage 22 are both smaller than the average cell diameter by 15 μm, i.e., the cross-sectional width and height of the main compression passage 22 are both 9 μm to 11 μm, and the length is greater than or equal to the stretched length of the largest-sized cell and is 100 μm to 200 μm.

According to the embodiment of the present invention, the length of the main compression channel 22 should ensure that the cells are maintained relatively stable at the detection position, that is, the cells are all located in the main compression channel 22 and no part of the cells in the cell inflow channel 21 and the cell outflow channel 24 is located in the detection position.

According to the embodiment of the present invention, the side compression passages 23 are provided on both sides of the preset position of the main compression passage 22, and the position close to the preset position of the main compression passage is referred to as the front end position of the side compression passage 23, i.e., the front end side compression passage; the preset position away from the main compression passage is referred to as a rear end position of the side compression passage 23, i.e., a rear end side compression passage.

According to the embodiment of the present invention, the front end side compression passage has a cross-sectional width of 2 μm to 4 μm, a height in conformity with the main compression passage 22 of 9 μm to 11 μm, and a length of 3 μm to 8 μm; the cross section width of the rear end side compression channel is 6-10 mu m, the height of the rear end side compression channel is consistent with that of the main compression channel and is 9-11 mu m, and the length of the rear end side compression channel is greater than or equal to 5 mu m, namely, the rear end side compression channel is in an expanded structure.

According to the embodiment of the present invention, the large difference in the cross-sectional dimension between the front-end side compression passage and the rear-end side compression passage in the side compression passage 23 causes breakage due to sudden loss of the inner wall support of the passage when the cell passes therethrough, that is, partial breakage of the cell membrane when the side compression passage 23 is used for the cell to flow to a predetermined position away from the main compression passage.

According to the embodiment of the invention, the cross section size of the front end side compression passage in the side compression passages 23 is smaller than that of the rear end side compression passage, so that the formation of high-resistance sealing is facilitated.

According to the embodiment of the present invention, the cell outflow channel 24 has the structural characteristics similar to those of the cell inflow channel 21, and has a cross-sectional area greater than the mean cell diameter by 15 μm and greater than or equal to 30 μm by 30 μm. The cell outflow channel 24 is used to recover the cells that have been tested, to prevent the tested cells from clogging the outlet of the main compression channel 22.

The voltage measuring module 2 includes: data acquisition card and shielded wire.

According to the embodiment of the invention, the voltage measuring module 2 can accurately detect at least a voltage signal of-100 mV, the minimum detection voltage is less than 0.01mV, and the input impedance is 10¹⁰The sampling rate can reach 100kS/s and is more than omega.

According to the embodiment of the invention, the interface of the voltage measurement module 2 connected with the microfluidic chip module 1 is a metal clamp or other metal clamps connected with a shielding wire.

The pressure control module 3 includes: a pressure calibrator and an air guide hose.

According to the embodiment of the invention, the pressure calibrator needs to comprise at least two pressure output ports and can output any pressure between-50 kPa and 50kPa through manual control; the pressure calibrator is connected with the electrodes in the microfluidic chip module 1 through the air guide hose.

According to the embodiment of the invention, the technical problems that the operation for detecting the cell membrane potential is complex, the formation of high-resistance sealing is difficult and the numerical value of the cell membrane potential cannot be accurately obtained in the prior art are solved through the provided device for detecting the cell membrane potential, and the direct and high-flux detection of the cell membrane potential is realized.

In the embodiment of the present invention, the microfluidic chip module in the apparatus for detecting a cell membrane potential is used as a core module thereof, and a method for processing the microfluidic chip module processed according to the present invention will be described in detail below.

Fig. 3 and 4 schematically show a flow chart of a process of the microfluidic chip module in the device and a flow chart of a process method thereof according to an embodiment of the present invention.

Referring to fig. 3 and 4, the method of manufacturing the microfluidic chip module includes operations S401 to S405.

In operation S401, a positive film of the main compression path and the side compression path is formed on the first substrate, resulting in a first substrate including the positive film of the main compression path and the side compression path.

According to the embodiment of the invention, the first substrate is a silicon substrate, a layer of AZ 5214E photoresist is spin-coated on the silicon substrate, and the silicon substrate coated with the AZ 5214E photoresist is subjected to processes such as pre-baking, exposure, reverse baking, flood exposure, development and the like to form masks of a main compression channel and a side compression channel, as shown in a-d diagrams in FIG. 3.

According to the embodiment of the invention, the mask for forming the main compression passage and the side compression passage is etched back, and after the etching is finished, the remaining mask is removed, so as to form the positive films of the main compression passage and the side compression passage, as shown in a diagram e in fig. 3.

In operation S402, a positive membrane of a cell inflow channel and a cell outflow channel is formed on a first substrate on which a positive membrane including a main compression channel and a side compression channel is obtained, resulting in a first substrate including a positive membrane of a specific structure.

According to the embodiment of the invention, a layer of SU8-25 photoresist is continuously spin-coated on the silicon substrate on which the positive films of the main compression passage and the side compression passage are formed, and the silicon substrate coated with the SU8-25 photoresist is subjected to processes of pre-baking, exposure, post-baking, development, film hardening and the like to form the positive films of the cell inflow passage and the cell outflow passage, as shown in the f-h diagram in FIG. 3.

In operation S403, a first substrate including a male mold with a specific structure is cast by using a casting mixture based on a molding process, and the carrier including the microfluidic channel is obtained by curing and demolding.

According to an embodiment of the present invention, the specific structure positive membrane includes a positive membrane having a compression channel and a side compression channel and a positive membrane having a cell inflow channel and a cell outflow channel, i.e., a microfluidic channel positive membrane.

According to an embodiment of the present invention, the casting mixture is a certain volume of Polydimethylsiloxane (PDMS), Polymethylmethacrylate (PMMA), or the like.

According to the embodiment of the invention, the mixed liquid is poured on the prepared mould and then cured, and after a preset period of curing, demoulding is carried out to obtain the carrier containing the microfluidic channel, namely, the insulating carrier part in the microfluidic chip module, as shown in an i-j diagram in fig. 3.

In operation S404, a metal electrode is formed on the second substrate, and a second substrate including the metal electrode is obtained.

According to the embodiment of the invention, the second substrate is a glass substrate, a layer of AZ 1500 photoresist is spin-coated on the glass substrate, and the glass substrate coated with the AZ 1500 photoresist is subjected to the processes of pre-baking, exposure, development and the like to form a patterned photoresist, i.e., there is no photoresist at the electrode position and there is no photoresist at the electrode position, as shown in l in fig. 3.

According to the embodiment of the invention, metal Cr/Au is sputtered on the second substrate containing the patterned photoresist, as shown in an m diagram in FIG. 3; the sputtering process is completed and the second substrate containing the metal electrode, i.e. the insulating substrate portion in the microfluidic chip module, is obtained, as shown in the n diagram in fig. 3.

In operation S405, holes are punched at corresponding positions of the carrier including the microfluidic channels, and aligned and bonded with a second substrate including metal electrodes to form a microfluidic chip module.

According to the embodiment of the invention, the corresponding position of the obtained carrier (shown as j in fig. 3) containing the microfluidic channel is punched, and the carrier is aligned and bonded with the second substrate (shown as n in fig. 3) containing the metal electrode obtained in step S404, so as to complete the processing of the microfluidic chip module (shown as o in fig. 3).

According to the embodiment of the invention, by designing the core device for detecting the cell membrane potential, the direct and high-throughput detection of the cell membrane potential can be realized.

FIG. 5 is a schematic flow chart of a detection method based on the device for detecting cell membrane potential according to an embodiment of the present invention.

As shown in FIG. 5, the flow of the detection method based on the apparatus for detecting a cell membrane potential includes operations S501 to S505.

In operation S501, the microfluidic chip module is connected to the voltage measurement module and the pressure control module, respectively.

According to the embodiment of the invention, the measuring end of the voltage measuring module is respectively connected with the on-chip electrodes corresponding to the outlets of the compression channel and the main compression channel on one side in the micro-fluidic chip module through the shielding wire; two pressure output ends of the pressure control module are respectively connected with two side compression channels and a cell outflow channel of the microfluidic chip module through air guide hoses.

In operation S502, a predetermined solution is filled in the microfluidic chip module to prevent bubbles from being generated when the pressure control module applies pressure to the cells.

According to the embodiment of the invention, the preset solution is a cell culture medium or a phosphate buffer solution, and the cell culture medium or the phosphate buffer solution is filled in all the channels in the microfluidic chip so as to prevent the pressure control module from generating air bubbles when applying pressure to the two side compression channels and the cell outflow channel in the microfluidic chip module to influence the cells to pass through the microfluidic chip module.

In operation S503, a cell suspension is injected into a cell inflow channel in a microfluidic chip module, and negative pressures are respectively applied to the two side compression channels and the main compression channel by using a pressure control module to drive cells to flow into the main compression channel and the side compression channel.

In operation S504, when the cells flow to the widening structure of the side compression path, the cells are partially damaged, and the cells flow into the cell outflow path after detection.

According to the embodiment of the present invention, when the cells pass through the main compression passage, the cells are initially compressed and enter the main compression passage while passing through the side compression passage, and the cells pass through the side compression passage, the cells reach the widening structure of the rear side compression passage due to the larger size of the cross section of the rear side compression passage relative to the front side compression passage, and the cells suddenly lose the support of the inner wall of the side compression passage, thereby causing local damage. The detected cells flow out of the main compression channel through the cell outflow channel.

In operation S505, while the cell flows in the microfluidic chip module, the voltage between the electrodes respectively connected to the outlets of the single side compression channel and the main compression channel is continuously measured by the voltage measurement module, and cell membrane potential raw voltage data of the cell is obtained.

According to the embodiment of the invention, the obtained cell membrane potential raw voltage data is subjected to data processing. The cell membrane potential original voltage data can be converted into the cell membrane potential of a single cell by using an equivalent electrical model of the cell after the cell membrane is damaged at the side compression channel widening structure.

For example, fig. 6 (a) schematically shows an equivalent electrical model of a cell after breakage of a membrane at the distal end of a lateral compression passage according to an embodiment of the present invention, and fig. (b) is an equivalent circuit of fig (a).

Shown in (a) and (b) in FIG. 6, wherein V_mIs the potential, R, of the cell membrane in the direction of the outlet opening in the main compression channel_mResistance of cell membrane in outlet direction in main compression channel, C_mCapacitance, R, of cell membrane in outlet direction in main compression channel_cyFor part of the cytoplasmic resistance, V, located in the circuit_ljFor the liquid-electric potential R between the cell culture medium (extracellular liquid) and the intracellular liquid in the compression channel after the cell membrane at the far end of the lateral compression channel is damaged_ch1For the resistance, R, of the solution between the electrode and the cell in the lateral compression channel_ch2Is the resistance R of the solution between the electrode and the cell in the main compression channel_sealSealing resistance, R, introduced to account for incomplete adhesion between cells and compression channels_vAnd measuring the input impedance of the channel for a data acquisition card in the voltage measuring module.

According to an embodiment of the invention, V in the circuit_ljThe values can be calculated according to the components of the solution in the compression channel during the experiment, and are in E1-10mV relative to the cell membrane potential V_mIs one order of magnitude smaller; r_cyThe value is-1 MOmega magnitude; since the side compression passages are sufficiently small in cross-sectional area, R_sealThe numerical value can reach 10 MOmega magnitude; r_ch1+R_ch2Is in the order of-1 MOmega; r_vCan reach the magnitude of 1G omega.

According to the embodiment of the invention, the data of the cell membrane damage instant is collected by the data acquisition card in the voltage measurement module, and the voltage value is V_VDue to then R_sealThe voltage across can be approximately V_vAnd R is_seal＞＞R_cySo that the cell membrane potential V_mThe absolute values are:

V_m＝V_v+V_lj

according to an embodiment of the invention, the direction of the cell membrane potential is negative on the inside of the cell. Because the on-chip electrode contacts with the solution, the potential difference can be generated after the double electric layers are formed, the solutions connected with the two measuring electrodes at two ends are completely the same, and the two potential differences generated by the double electric layers in the circuit are in opposite directions, so that the two potential differences can be mutually counteracted without influencing the measurement of the cell membrane potential.

According to the embodiment of the invention, the device for detecting the cell membrane potential, the detection method thereof and the processing method of the microfluidic chip module in the device are provided, so that the technical problems that the operation for detecting the cell membrane potential is complex, the formation of good sealing is difficult and the accurate value of the cell membrane potential cannot be obtained in the prior art are solved, the cell membrane potential value can be obtained by calculating by using the voltage value obtained by recording by the voltage measurement module in the device, and the direct measurement is realized; in the detection process, cells continuously pass through the detection area, so that the high-flux detection of the cell membrane potential is ensured.

The above-mentioned embodiments are intended to illustrate the objects, technical solutions and advantages of the present invention in further detail, and it should be understood that the above-mentioned embodiments are only exemplary embodiments of the present invention and are not intended to limit the present invention, and any modifications, equivalents, improvements and the like made within the spirit and principle of the present invention should be included in the protection scope of the present invention.