CN110187088B - Cell microsphere array chip device for measuring potential signals and method thereof - Google Patents
Cell microsphere array chip device for measuring potential signals and method thereof Download PDFInfo
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- G01N33/48707—Physical analysis of biological material of liquid biological material by electrical means
- G01N33/48728—Investigating individual cells, e.g. by patch clamp, voltage clamp
Abstract
The invention discloses a cell microsphere array chip device for measuring potential signals and a method thereof, relates to the field of biomedicine, and mainly aims to solve the problem that the prior art lacks of means for detecting the electrical properties of cell microspheres. The chip device comprises a cell microsphere flowing channel, a cell microsphere capturing channel, a separated electrical property measuring electrode and an external detection system, and can realize the electrical signal detection of a plurality of cell microspheres in the channel only by introducing a cell microsphere suspension without other external devices; because the captured cell microspheres are fixed in position, the electrical detection precision of the cells can be effectively improved, the coupling between the electrodes and the cells can be improved, and the signal-to-noise ratio is improved. The chip device has the advantages of simple processing technology, convenient operation, high repeatability and flexibility.
Description
Technical Field
The invention relates to the field of biomedicine, in particular to a technology for monitoring extracellular electrical properties of cell microspheres.
Background
Cells are basic units of life activities, and measurement of physicochemical properties of cells plays an important role in understanding cell activities and metabolism. The cell measurement generally includes biomechanical measurement, electrical measurement, motion behavior, biochemical characteristic measurement and the like, and the adopted measurement methods mainly include: microscopic observation, electrochemical measurement, optical measurement, and the like. The microscopic observation and optical measurement often need to carry out chemical or fluorescent labeling on the cells, and the operation is complex, the function is single, and the original characteristics of the cells are changed to a certain extent. The measuring method aiming at the cell electrical property analysis does not need to be marked, does not damage cells, can continuously measure the cells in real time in a natural culture state, and has the advantages of high sensitivity, full-automatic measurement, no need of manual intervention and the like.
Common cell membrane potential measurement methods mainly comprise methods such as a microelectrode array (MEA), a voltage clamp, a patch clamp and the like, but the methods are mostly used for detecting the electrical properties of planar two-dimensional cells or single cells and cannot detect the electrical properties of cell microspheres.
Compared with two-dimensional planar cells, the cell microspheres can better simulate an in-vivo microenvironment and simulate the interaction between in-vivo cells, are beneficial to analyzing various physiological functions of the cells, and provide a more accurate visual angle for observing cell behaviors. The establishment of the in vitro three-dimensional culture model is beneficial to spanning the gap between two-dimensional cell culture and a living body, so that the electrical detection of the cell microspheres has important application value in the aspects of drug screening, construction of disease and development models and the like. But a set of technology capable of accurately and conveniently monitoring the electrical properties of the cell microspheres is lacked at present.
Disclosure of Invention
The purpose of the invention is as follows: the invention provides a chip device for monitoring extracellular electrical properties of cell microspheres in real time in a high-flux manner, aiming at overcoming the defects of the prior art.
In order to realize the purpose of the invention, the technical scheme adopted by the invention is as follows: a cell microsphere array chip device for measuring potential signals comprises a cell microsphere flow channel, a cell microsphere capturing channel, a separated electrical property measuring electrode and an external detection system; the cell microsphere capturing channel is communicated with the cell microsphere flow channel and is used as a branch of the cell microsphere flow channel; one end of the cell microsphere capturing channel, which is communicated with the cell microsphere flowing channel, is used as an inlet end, the other end of the cell microsphere capturing channel is used as an outlet end, and a cell microsphere capturing and fixing part is arranged between the inlet end and the outlet end; the cell microsphere capture and fixation part contains at most one cell microsphere; the separated electrical performance measuring electrode comprises an extracellular potential detecting electrode and a lead, wherein the extracellular potential detecting electrode is arranged in the cell microsphere capturing and fixing part; the extracellular potential detecting electrode is connected with an external detecting system through a lead.
The cell microspheres of the present invention include a cell mass consisting of a large number of cells and microspheres containing cells and composed of materials such as gel or extracellular matrix.
Preferably, the width and height of the inlet end of the cell microsphere capturing channel are slightly larger than the diameter of the cell microspheres, the width and/or height of the subsequent part of the channel are slightly smaller than the diameter of the cell microspheres, so that the cell microspheres can be fixed at the target position of the channel and prevented from being washed away by liquid flowing through the channel, and the length from the target position of the fixed microspheres of the channel to the inlet end of the channel is slightly larger than the diameter of the microspheres, so that only one microsphere enters the capturing channel at a time. Preferably, the height and width of the inlet end are 1.1-2 times of the diameter of the cell microsphere, the width of the outlet end of the capturing channel is 0.3-0.9 times of the diameter of the cell microsphere, and the length of the capturing channel of the cell microsphere is 1.1-3 times of the diameter of the cell microsphere; the width of the cross section of the cell microsphere capturing channel is gradually reduced along the inflow direction, and the width of the outlet end is smaller than the diameter of the cell microsphere.
Preferably, the separated electrical property measuring electrode is horizontally fixed at the middle position of the tail end of the cell microsphere capturing channel, and the measuring part of the separated electrical property measuring electrode extends into the cell microsphere capturing channel along the axis of the cell microsphere capturing channel; the length of the channel extending into the cell microsphere capture channel is ensured to ensure that the measuring part is embedded into the cell microsphere and fully contacted with the cell microsphere when the cell microsphere is fixed at the cell microsphere capture fixing part. The protrusion length is typically in the range of 300 μm to 5mm, with different protrusion lengths being selected for different microsphere sizes.
Specifically, the microspheres have a certain speed after entering the capture channel, the microspheres are automatically inserted onto the electrode under the pushing of the fluid, and the measurement part of the electrode is embedded into the microspheres and fully contacted with the microspheres. The liquid in the capture channel is at a flow rate that ensures that the microspheres can be inserted onto the electrodes.
Preferably, the width and height of the cell microsphere flow channel should be greater than the measured diameter of the cell microsphere; the width is preferably 1.5-3 times the diameter of the cell microsphere and the height is 1.5-5 times the diameter of the cell microsphere.
Preferably, the material of the cell microsphere flow channel and the cell microsphere capture channel is independently selected from Polydimethylsiloxane (PDMS), polymethyl methacrylate (PMMA), inorganic glass, and Polystyrene (PS).
Preferably, the number of the cell microsphere capture channels is one or more, and the shape of the cell microsphere flow channel comprises a straight line shape, a curved line shape, a loop shape or a multi-channel shape.
Preferably, the angle between the central line of the cell microsphere flowing channel and the central line of the cell microsphere capturing channel is 0-90 degrees, preferably 30-60 degrees.
The microfluidic channel of the present invention may be a single channel in a curved arrangement, in which case only one inflow and one outflow are required; or a plurality of channels arranged in parallel, each channel having an inflow orifice and an outflow orifice communicating therewith.
The suspension can be recovered from the tail end of the cell microsphere capturing channel, the suspension and the unfixed cell microspheres can be recovered from the tail end of the flow channel, and the suspension and the unfixed cell microspheres can reenter the flow channel after being recovered.
Preferably, the electrode material of the extracellular potential detection electrode is selected from materials with certain rigidity and easily controlled size preparation, and the selected electrode material includes but is not limited to metal materials such as platinum, lead, nickel-chromium alloy, platinum-iridium alloy and the like; polymeric materials, mainly rigid conductive polymers, such as glassy carbon electrodes; inorganic non-metals, such as silicon electrodes and the like. Preferably, the selected separated electrical performance measuring electrode is a silicon-based microelectrode array electrode and mainly comprises a Michigan electrode and a Utah electrode, wherein the electrode site arrangement modes of the selected Michigan electrode comprise five arrangement modes of Linear, Edge, Tetron, Polytrode and Multi-shank.
Preferably, the external detection system comprises a preamplifier, a filter, a data acquisition card and a computer, wherein the preamplifier is connected with the separated electrical property measurement electrode through a lead; the detected electric signals are amplified by a preamplifier, then noise removal processing is carried out by using a filter, and then the amplified and filtered electric signals are collected by a data acquisition card and then are transmitted and stored in a computer.
The invention also discloses a cell microsphere potential signal detection method of the chip device, which comprises the following steps:
(1) the cell microsphere suspension flows into the cell microsphere capturing channel from the initial end of the cell microsphere flow channel of the chip device at a certain speed, the cell microsphere suspension flows into the cell microsphere capturing channel, the cross-sectional width of the capturing channel is gradually reduced to be smaller than the diameter of the cell microspheres along the inflow direction, the cell microspheres are clamped in the cell microsphere capturing channel and are inserted into an extracellular potential detection electrode which is fixed in the cell microsphere capturing channel in advance, the size of the capturing channel only allows one cell microsphere to flow in and be fixed, and the rest cell microspheres continue to flow downstream;
(2) observing whether the cell microspheres are captured in the cell microsphere capture channel;
(3) if the cell microspheres captured in the cell microsphere capture channel are observed, carrying out the next step, otherwise, repeating the step (1);
(4) the cell microspheres are clamped in the capture channel and inserted into a measuring electrode which is fixed in the capture channel in advance, and the measuring electrode detects extracellular potential signals of cells in the cell microspheres and is connected to a preamplifier through a lead; the electric property of the cells in the cell microspheres can be analyzed by processing the potential signals through an external detection system.
The invention has the following advantages:
(1) the chip device can measure the electrical characteristics of the 3D cell microspheres, the signal to noise ratio is high, and the detection precision is high;
(2) the chip device does not need other additional devices, and only needs to be introduced into the cell microsphere suspension liquid in the designed shunting fluid channel, so that the electric signal detection of a plurality of cell microspheres in the channel can be realized;
(3) because the position of the captured cell microspheres is fixed in the chip device, the electrical detection precision of cells can be effectively improved, the coupling of electrodes and the cells can be improved, and the signal-to-noise ratio is improved.
(4) The chip device can simultaneously measure the functional expression of the cell microspheres on the bioelectricity performance in the multiple capture channels, and can be used for researching the bioelectricity performance of cells;
(5) the chip device has multiple channels, and can realize the simultaneous parallel detection of the external cell electric signals in the cell microspheres by the multiple-channel measuring electrodes without damage, with long time and high flux;
(6) the chip device has the advantages of simple processing technology, convenient operation, high repeatability and flexibility.
Drawings
FIG. 1 is a schematic plan view of a chip device according to the present invention;
FIG. 2 is a schematic cross-sectional view of a chip arrangement according to the present invention;
FIG. 3 is a schematic diagram showing a partial enlargement of a cell microsphere capture channel of the chip device according to the present invention;
FIG. 4 is a schematic diagram of a simple planar structure of cell microsphere flow channels with different shapes and structures of the chip device of the present invention: a: linear shape, B: curve type, C: and (4) a loop shape.
Reference numerals: the cell microsphere measuring device comprises a cell microsphere flow channel 1, a cell microsphere capturing channel 2, a separated electrical property measuring electrode 3, an external detection system 4, cell microspheres 5 and an included angle alpha formed by the central line of the cell microsphere flow channel and the central line of the microsphere channel.
Detailed Description
The following detailed description of embodiments of the present invention is provided in connection with the accompanying drawings and examples. The following examples are intended to illustrate the invention and are not intended to limit the scope of the invention.
Example one
Fig. 1 shows a chip device with a designed linear structure, which includes a cell microsphere flow channel 1, a cell microsphere capture channel 2, a separated potential signal measurement electrode 3 and an external detection system 4, and is mainly used for testing local field potential of cardiomyocytes in the cardiomyocyte microspheres, and the size of the cardiomyocyte microspheres is 200 μm.
The cell microsphere flow channel is a flow channel of the myocardial cell microsphere suspension, and the width and the height of the channel are both 250 micrometers. The suspension of the myocardial cell microspheres flows from left to right, and the suspension can be recovered at the tail end of the channel.
The microsphere capture channel comprises a microsphere inlet, a microsphere capture fixing part and a liquid outlet, the channel is directly connected with the cell microsphere flow channel and is a branch of the flow channel, and the number of the channels is 6. The channel size of the microsphere capture fixing part is 200 μm, and the center line of the flow direction of the capture channel and the center line of the flow direction of the flow channel form an angle of 30 degrees.
The separated electrical performance measuring electrode comprises an electrode array and a lead wire used for connecting an external detection system, the separated electrical performance measuring electrode is fixed at the middle position of the tail end of the cell microsphere capturing channel, and the length of the separated electrical performance measuring electrode extending into the cell microsphere capturing channel ensures that when the cell microsphere is fixed at the cell microsphere capturing fixed position, the measuring position of the separated electrical performance measuring electrode is embedded into the cell microsphere and fully contacted with the cell microsphere, as shown in figure 3. Specifically, the microsphere of the myocardial cell has a certain speed after entering the capture channel, the microsphere is automatically inserted onto the electrode under the pushing of the fluid, and the measuring part of the electrode is embedded into the microsphere and fully contacted with the microsphere, so that the extracellular potential signal of the myocardial cell can be obtained.
The detected electric signal is amplified by a preamplifier, then is denoised by a filter, and then is collected and transmitted by a data acquisition card and stored in a computer, so that the frequency and amplitude of the myocardial cell potential signal in the measured cell microsphere can be finally obtained and can be further analyzed and processed.
Example two
The preparation method of the chip device for analyzing the cell microsphere potential signals is mainly suitable for a microfluidic chip for testing the cell microsphere potential signals with the size of below 200 mu m.
In the manufacturing process of this embodiment: firstly, SU8-3025 is dripped on the surface of a silicon chipThe photoresist (negative resist) was homogenized at 500rpm for 10s and at 100rpm for 30 s. Then, pre-baking at 65 ℃ for 1 minute and at 95 ℃ for 5 minutes; ultraviolet exposure (150-250 mJ/cm)2) (ii) a After exposure, baking at 65 ℃ for 1 minute and at 95 ℃ for 6 minutes; cooling to room temperature, developing by a developing solution for about 9 minutes; and then post-baking for 3 hours at 180 ℃ to solidify the die. Then, washing with deionized water, and blow-drying with an air gun to obtain the pipeline pattern with the height of 70-90 μm. After that, according to the PDMS: PDMS was prepared at a ratio of 10:1, and the pattern patterned by photolithography was transferred and cured by heating at 85 ℃. And packaging the cured PDMS with a glass sheet after plasma treatment.
In this way, the microchannel chip is manufactured. It is understood that the specific values in the above steps can be adjusted within a reasonable range, and only the PDMS channels are required to be successfully fabricated, which is not limited to the above actual values.
EXAMPLE III
A preparation method of the chip device for analyzing the cell microsphere potential signal comprises the following steps: drawing the designed graph with CAD software, wherein the width of the graph is 800 microns, and guiding the graph into an infrared cutting machine. Corresponding channels were cut in a PMMA substrate with a thickness of 1 mm. Adhering the PMMA substrate containing the channel to a glass or PMMA substrate, and then sealing the upper surface by using PMMA or glass. And fixing and clamping the whole three-layer structure by using screws. And fixing a detection electrode (shown in figure 3) at the corresponding part of the capture channel (shown in figure 2) to obtain the required PMMA multi-channel chip.
Example four
A method for analyzing the microsphere potential signals of the cardiac muscle cells by using the linear chip device is disclosed, and for the array chip device with the cell microsphere fluid channels with different shapes and structures, as shown in FIG. 4: a: linear shape, B: curve type, C: the detection principle and the detection method of the chip device of the loop shape are the same, and the detection analysis method comprises the following steps:
(1) enabling the cardiomyocyte microsphere suspension to flow in from the starting end of a cell microsphere flow channel of the chip device at a certain speed, enabling the cell microsphere suspension to flow in a cell microsphere capturing channel, enabling the cell microspheres to be clamped in the cell microsphere capturing channel as the cross-sectional width of the capturing channel is gradually reduced to be smaller than the diameter of the cell microspheres along the inflow direction, and inserting the cell microspheres on an extracellular potential detection electrode which is fixed in the cell microsphere capturing channel in advance, wherein the size of the capturing channel only allows one cell microsphere to flow in and be fixed, and the rest cell microspheres continue to flow downstream;
(2) observing under an optical microscope to see whether the cell microsphere capturing channel captures the cardiomyocyte microspheres;
(3) if the microspheres are observed to capture the cardiomyocyte microspheres captured in the apical-free channel, namely the capture channel is blocked by the cardiomyocyte microspheres, carrying out the next step, otherwise, repeating the step (1);
(4) the microsphere captures the microspheres of the captured cardiomyocytes in the non-apical channel, and the measuring electrode can detect extracellular potential signals of the cardiomyocytes in the cell microspheres and is connected to an external detection system through a lead; the detection system is used for processing the signals, so that the frequency and the amplitude of the myocardial cell potential signals in the myocardial cell microspheres can be analyzed.
The above embodiments are only for illustrating the invention and are not to be construed as limiting the invention, and those skilled in the art can make various changes and modifications without departing from the spirit and scope of the invention, therefore, all equivalent technical solutions also belong to the scope of the invention, and the scope of the invention is defined by the claims.
Claims (8)
1. A cell microsphere array chip device for measuring potential signals is characterized by comprising a cell microsphere flow channel, a cell microsphere capturing channel, a separated electrical property measuring electrode and an external detection system; the cell microsphere capturing channel is communicated with the cell microsphere flow channel and is a branch of the cell microsphere flow channel; one end of the cell microsphere capturing channel, which is communicated with the cell microsphere flowing channel, is used as an inlet end, the other end of the cell microsphere capturing channel is used as an outlet end, and a cell microsphere capturing and fixing part is arranged between the inlet end and the outlet end; the cell microsphere capture and fixation part contains at most one cell microsphere; the separated electrical performance measuring electrode comprises an extracellular potential detecting electrode and a lead, wherein the extracellular potential detecting electrode is arranged in the cell microsphere capturing and fixing part; the extracellular potential detection electrode is connected with an external detection system through a lead;
the separated electrical performance measuring electrode is horizontally fixed in the middle of the tail end of the cell microsphere capturing channel, and the measuring part of the separated electrical performance measuring electrode extends into the cell microsphere capturing channel along the axis of the cell microsphere capturing channel; the length of the channel extending into the cell microsphere capture channel is required to ensure that when the cell microsphere is fixed at the cell microsphere capture fixing part, the measuring part is embedded into the cell microsphere and fully contacted with the cell microsphere;
the center line of the capturing channel forms an angle of 30 degrees with the center line of the capturing channel;
the cross section width of the cell microsphere capturing channel is gradually reduced to be smaller than the diameter of the cell microspheres along the inflow direction, so that the cell microspheres are clamped in the cell microsphere capturing channel and are inserted into the extracellular potential detection electrode which is fixed in the cell microsphere capturing channel in advance, the size of the capturing channel only allows one cell microsphere to flow in and be fixed, and the rest cell microspheres continue to flow downstream.
2. The cellular microsphere array chip device for measuring potential signals of claim 1, wherein the height and width of the inlet end of the cellular microsphere capturing channel are 1.1 to 2 times of the diameter of the cellular microsphere, the width of the outlet end of the capturing channel is 0.3 to 0.9 times of the diameter of the cellular microsphere, and the length of the cellular microsphere capturing channel is 1.1 to 3 times of the diameter of the cellular microsphere; the width of the cross section of the cell microsphere capturing channel is gradually reduced along the inflow direction, and the width of the outlet end is smaller than the diameter of the cell microsphere.
3. The cellular microsphere array chip device for measuring potential signals of claim 1, wherein the width of the cellular microsphere flow channel is 1.5 to 3 times the diameter of the cellular microsphere and the height is 1.5 to 5 times the diameter of the cellular microsphere.
4. A cell microsphere array chip device for potential signal measurement according to any one of claims 1 to 3, wherein the material of the cell microsphere flow channel and the cell microsphere capture channel is independently selected from polydimethylsiloxane, polymethyl methacrylate, inorganic glass or polystyrene.
5. The cellular microsphere array chip device for measuring potential signals of claim 1, wherein the number of the cellular microsphere capturing channels is one or more, and the shape of the cellular microsphere flow channels comprises a straight line shape, a curved line shape, a loop shape or a multi-channel shape.
6. A cellular microsphere array chip device for measuring electric potential signals according to claim 1, wherein the extracellular electric potential detecting electrodes are silicon-based microelectrode array electrodes.
7. The cellular microsphere array chip device for measuring potential signals of claim 1, wherein the external detection system comprises a preamplifier, a filter, a data acquisition card and a computer, the preamplifier is connected with the separated electrical property measuring electrode through a lead; the detected electric signals are amplified by a preamplifier, then noise removal processing is carried out by using a filter, and then the amplified and filtered electric signals are collected by a data acquisition card and then are transmitted and stored in a computer.
8. A method for detecting a cell microsphere potential signal of the chip device of claim 1, wherein:
(1) the cell microsphere suspension flows into the cell microsphere capturing channel from the initial end of the cell microsphere flow channel of the chip device at a certain speed, the cell microsphere suspension flows into the cell microsphere capturing channel, the cross-sectional width of the capturing channel is gradually reduced to be smaller than the diameter of the cell microspheres along the inflow direction, the cell microspheres are clamped in the cell microsphere capturing channel and are inserted into an extracellular potential detection electrode which is fixed in the cell microsphere capturing channel in advance, the size of the capturing channel only allows one cell microsphere to flow in and be fixed, and the rest cell microspheres continue to flow downstream;
(2) observing whether the cell microspheres are captured in the cell microsphere capture channel;
(3) if the cell microspheres captured in the cell microsphere capture channel are observed, carrying out the next step, otherwise, repeating the step (1);
(4) the cell microspheres are clamped in the capture channel and inserted into a measuring electrode which is fixed in the capture channel in advance, and the measuring electrode detects extracellular potential signals of cells in the cell microspheres and is connected to a preamplifier through a lead; the electric property of the cells in the cell microspheres can be analyzed by processing the potential signals through an external detection system.
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| CN1284166A (en) * | 1997-12-25 | 2001-02-14 | 松下电器产业株式会社 | Cell potential measuring electrode and measuring appts using same electrode |
| CN102209779A (en) * | 2008-09-16 | 2011-10-05 | 马克思-普朗克科学促进协会 | Electrode device, generator device and method for power generation by means of membrane-potential shunting |
| CN108467835A (en) * | 2018-03-16 | 2018-08-31 | 东南大学 | Micro-fluidic chip and preparation method and mechanics electrology characteristic detection method for cardiac muscle cell's three-dimensional function culture |
| CN109097264A (en) * | 2018-07-31 | 2018-12-28 | 大连理工大学 | A kind of micro-current controlled cell absorption chip for membrane elastic property |
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| US20020144905A1 (en) * | 1997-12-17 | 2002-10-10 | Christian Schmidt | Sample positioning and analysis system |
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| CN1284166A (en) * | 1997-12-25 | 2001-02-14 | 松下电器产业株式会社 | Cell potential measuring electrode and measuring appts using same electrode |
| CN102209779A (en) * | 2008-09-16 | 2011-10-05 | 马克思-普朗克科学促进协会 | Electrode device, generator device and method for power generation by means of membrane-potential shunting |
| CN108467835A (en) * | 2018-03-16 | 2018-08-31 | 东南大学 | Micro-fluidic chip and preparation method and mechanics electrology characteristic detection method for cardiac muscle cell's three-dimensional function culture |
| CN109097264A (en) * | 2018-07-31 | 2018-12-28 | 大连理工大学 | A kind of micro-current controlled cell absorption chip for membrane elastic property |
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