CN103954658B - Dynamic real-time measuring apparatus for cell membrane potential - Google Patents
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Abstract
The invention relates to a dynamic real-time measuring apparatus for cell membrane potential, which comprises a gallium nitride (GaN) high electron mobility transistor (HEMT)device as a biosensor, a micro flow chamber, a micro injection pump and a liquid storage bottle. The micro flow chamber, the micro injection pump and the liquid storage bottle are connected in order to form a sealed circulatory system. The micro flow chamber is used for liquid flowing and cell injecting, and the liquid in the micro flow chamber is transmitted to the liquid storage bottle through the micro injection pump. The HEMT device as the biosensor is coupled with the micro flow chamber, and is used for detecting the real time cell membrane potential of cells in the micro flow chamber under dynamic condition.
Description
Technical Field
The invention relates to a device for dynamically measuring cell membrane potential in real time.
Background
The flow of body fluids will produce shear stress on cells, which in turn will cause changes in cell potentials. Shear forces can affect many aspects of the biological properties of human endothelial cells and are associated with a variety of cardiovascular diseases. Therefore, the detection of the change of the cell membrane potential has important physiological and pathological research significance.
At present, methods for measuring cell membrane potential under shear force mainly include a fluorescent dye method, a microelectrode array method and a patch clamp method.
The fluorescent dye method is a method for measuring the potential of cell membranes by utilizing the property that the fluorescence intensity can be changed under the action of shearing force and the change trend of the cell membrane potential obtained by patch clamp is the same. However, the reaction time and the precision of the method are greatly different, and the fluorescence intensity can not be proved to replace the cell membrane potential to represent the electrophysiological phenomenon of the cell at present.
The microelectrode array method adopts an integrated circuit silicon process, and can represent action potential of single muscle cells or nerve cells with large volume through a microelectrode array. However, this method has the disadvantages of long preparation time for measurement and low measurement accuracy and sensitivity.
The patch clamp method is the most common method at present, and is to measure the voltage or current difference inside and outside the membrane of a single cell through a glass microelectrode and obtain the potential of the cell membrane through certain amplification and conversion. However, this technique has the following congenital deficiencies: biological tissues are formed by closely arranging a large number of cells, and the patch clamp can only measure single cells and cannot represent the response condition of the actual multi-cell membrane potential to the shearing force; during measurement, cell membranes can be damaged, the characteristics of cells are changed, and the cells die in a short time, so that the research on the action potential of the cell membranes and the phenomenon of the ion channel in the early stage is limited; when the test is carried out in a fluid environment, cells are easy to fall off from the patch clamp due to the existence of shearing force; the patch clamp technique can record only one cell (or a pair of cells) at a time, and the data quantity which can be obtained every day is only a few to dozens of cells, which is time-consuming and labor-consuming.
In conclusion, the fluorescent dye method, the microelectrode array method and the patch clamp method can not solve the problem of measuring the multi-cell membrane potential of the living body, so as to accurately represent the influence of the shearing force on the cell membrane potential.
Therefore, there is a need for a device and method with the advantages of rapid detection, high sensitivity, real-time measurement, accurate result, high biocompatibility, and multi-cell in vivo measurement for measuring the cell membrane potential.
Disclosure of Invention
Accordingly, the present invention has been made keeping in mind the above problems occurring in the prior art, and an object of the present invention is to provide an apparatus for accurately measuring in real time a shear force-affected membrane potential of a living body in a fluid environment, which uses a gallium nitride (GaN) High Electron Mobility Transistor (HEMT) device technology, and is connected to a shear force micro flow chamber, a micro injection pump and a liquid storage bottle, which are made of a polymer material having a high elasticity, a small surface tension and a high transmittance, so as to dynamically simulate a multi-cell environment and monitor in real time the membrane potential of the living body in the fluid environment.
The invention is characterized in that a micro-injection pump is used for providing a dynamic fluid environment so as to realize real-time monitoring of living multi-cell membrane potential in the fluid environment.
The basic structure of the device of the invention is as follows: the HEMT device used as the biosensor is connected with the micro-flow chamber, the micro-injection pump and the liquid storage bottle, and the living multi-cell membrane potential in the fluid environment is monitored in real time through the HEMT device on the premise that the micro-injection pump provides the dynamic fluid environment.
The use method of the device comprises the following steps: a micro-injection pump is connected with a cell membrane potential detection device to form a haemodynamic system,the system is erected in a 37 ℃ thermostat to maintain stable temperature, PBS is used as fluid in the experiment, and 5% CO is introduced in the whole experiment2To maintain the ph of the fluid environment.
The HEMT device applied to the biosensor has the structure that: the bottom layer is made of sapphire materials, the bottom layer is upwards sequentially provided with a GaN buffer layer of about 1.6 mu m, an AlN insert layer of 1.2nm, an AlGaN barrier layer of about 8-15 nm and a GaN cap layer of 1.5nm, and the aluminum component of the barrier layer is between 25% and 40%. Si above the cap layer3N4And a rectangular groove-shaped bare gate area is etched in the middle of the passivation layer, and the bare gate area is led out without electrodes and has the size within the range of 10 mu m-10 mm.
The manufacturing process of the HEMT device comprises the following steps:
(1) etching the mesa, namely etching the heterojunction material by adopting an ICP (inductively coupled plasma) method to form mesa isolation of the device;
(2) depositing source and drain electrode ohmic metal, obtaining an ohmic metal layer by adopting an electron beam evaporation method, wherein the ohmic metal adopts a Ti/Al/Ni/Au four-layer structure, and annealing at 830 ℃ to form alloy so as to obtain good source and drain electrode ohmic contact;
(3) si deposition by PECVD method3N4The material is used as a passivation layer;
(4) photoetching and etching Si by adopting ICP method3N4Exposing the bare gate area.
The structure of the micro-flow chamber in the invention is as follows: a groove-shaped pattern which is made of high polymer materials and is the same as a bare grid is covered above the HEMT device, and a micro-flow chamber is arranged in the groove. The material of the micro flow chamber is Polydimethylsiloxane (PDMS), and other materials such as polymethyl methacrylate (PMMA), polyimide, etc. may be used. The micro-flow chamber reaction chamber size design is consistent with the device grid electrode pattern. The microfluidic chamber has sealing ports on both sides for liquid flow and cell injection. The micro-flow chamber is used for culturing cells, and a micro-injection pump is connected after the culture is finished so as to provide accurate flow control. Two small holes with the diameter of about 0.1 mm-0.5 mm are arranged at two ends of the micro-flow chamber and can be connected with the outside. And sealing the microfluidic chamber component and the HEMT component to obtain a closed channel, wherein the solution can flow in the channel to form the cell membrane potential detection device under fluid dynamics.
The manufacturing process of the micro-flow chamber comprises the following steps: firstly, a photoetching method or a Reactive Ion Etching (RIE) method is adopted to manufacture a convex male die of the microfluidic chamber, then PDMS is cast on the male die, and PDMS is peeled off from the male die after curing at the temperature of about 50 ℃, thus the microfluidic chamber component is manufactured. The small holes at the two ends of the micro-flow chamber are obtained by drilling or other methods. The sealing process of the micro flow chamber part and the HEMT chip adopts a hot pressing method or an adhesion method and the like.
The method for separating living cells comprises the steps of adding 15mL of mixed solution of 1g/L collagenase and 2.5g/L trypsin (1:1) (V/V) into fresh human umbilical vein blood obtained under aseptic conditions, incubating the mixture in a 37 ℃ water bath tank for 8min, collecting digestive juice in a centrifuge tube, flushing the umbilical vein for 2 times by Phosphate Buffer Solution (PBS), collecting flushing liquid in the centrifuge tube, centrifuging the flushing liquid for 10min at 1000r/min, taking supernatant, adding complete culture solution to resuspend cells, taking 0.1mL of cell suspension, staining the cell suspension by 4g/L trypan blue, counting viable cells, inoculating 2 × 104/L of cells in a 24-well plate, adding 1mL of complete culture solution into each well, placing the mixture at 37 ℃ and 950mL/L O, and inoculating the viable cells into the 24-well plate2、50mL/L CO2Standing and culturing in an incubator, uniformly mixing 0.1mL of uniformly mixed endothelial cell suspension and 0.9mL of 4g/L of trypan blue by adopting a trypan blue exclusion method, and taking 1 drop to count 100 cells under a microscope. When the cell survival rate in the cell suspension is more than 95 percent, the method can be used for primary cell culture.
The method for culturing the living cells comprises the following steps: after sterilizing the HEMT device with alcohol, treatment with fibronectin solution (fibronectin) was performed for 30min to enhance the adhesion of cells. Washing with PBS, inoculating cells to reach cell density of 5000-12000 cells/mm2And ensures that the cells are placed in the medium containing 5% CO in the whole process2In a 37 ℃ incubator.
The shear force to which the cells cultured on the bare grid of the device of the invention are subjected is deduced from the following formula:
wherein tau is the shear force applied to the cell and has the unit of dyne/cm2η is the viscosity (viscosity) of the culture solution as a fluid in g/(cm. multidot.s), Q is the flow rate of the fluid per second in cm3S; w is the gate width of the device and h is the height of the inner wall of the microfluidic chamber. By adjusting the flow rate of the pump per second, the required shear force can be obtained.
The device adopts a semiconductor testing analytical instrument, such as Keithley4200SCS, to test the source-drain current of the HEMT device, and can be converted into a corresponding cell membrane potential through the following formula.
Wherein,to the cell membrane potential, (g)m)VDSFor an applied drain-source voltage VDSLower and gate voltage VGSAnd the transconductance of the HEMT device is correspondingly arranged at the position of-70 mV to 0V. Since the change of the gate voltage is small, it can be said thatIs a constant and is obtained by pre-measurement. The pre-measurement method comprises the following steps: adding the same drain-source voltage V to the conventional three-terminal HEMT device on the same wafer and adopting the same layoutDSTesting its transfer curve (V)G-ID) And obtaining the transconductance value of the device corresponding to the grid voltage of-70 mV to 0V.
Drawings
FIG. 1 is a schematic structural diagram of the present invention.
FIG. 2 is a measurement schematic diagram of the present invention
Detailed Description
The invention will be further explained with reference to the drawings. Embodiments of the present invention include, but are not limited to, the following cases.
Example 1
As shown in figures 1 and 2, the device for accurately measuring the potential of the living multicellular membrane influenced by the shearing force comprises a HEMT device sensor, a micro-flow chamber, a micro-injection pump and a liquid storage bottle.
In this example, living cells were isolated from human umbilical veins, cultured and propagated, and then injected into a microfluidic chamber for culturing and bonding. The device was placed in a 37 ℃ incubator and the liquid was injected into the microfluidic chamber using a micro syringe pump. PBS was used as the fluid in the experiment and 5% CO was introduced throughout the experiment2To maintain the ph of the fluid environment. And measuring the viscosity of the fluid by using the HEMT device, and calculating by using the following formula to obtain the relation between the cell membrane potential and the time.
The shear force to which the cells cultured on the bare grid of the device of the invention are subjected is deduced from the following formula:
wherein tau is the shear force applied to the cell and has the unit of dyne/cm2η is the viscosity (viscosity) of the culture solution as a fluid in g/(cm. multidot.s), Q is the flow rate of the fluid per second in cm3S; w is the gate width of the device,h is the microfluidic chamber height. By adjusting the flow rate of the pump per second, the required shear force can be obtained.
The device adopts a semiconductor testing analytical instrument, such as Keithley4200SCS, to test the source-drain current of the HEMT device, and can be converted into a corresponding cell membrane potential through the following formula.
Wherein,is the potential of the cell membrane,for an applied drain-source voltage VDSLower and gate voltage VGSAnd the transconductance of the HEMT device is correspondingly arranged at the position of-70 mV to 0V. Since the change of the gate voltage is small, it can be said thatIs a constant and is obtained by pre-measurement. The pre-measurement method comprises the following steps: adding the same drain-source voltage V to the conventional three-terminal HEMT device on the same wafer and adopting the same layoutDSTesting its transfer curve (V)G-ID) And obtaining the transconductance value of the device corresponding to the grid voltage of-70 mV to 0V.
Although the illustrative embodiments of the present invention have been described for illustrative purposes, those skilled in the art will appreciate that various modifications, additions and substitutions are possible, without departing from the scope and spirit of the invention as disclosed in the accompanying claims.
Claims (8)
1. An apparatus for dynamically measuring cell membrane potential in real time, comprising: the device comprises a gallium nitride (GaN) High Electron Mobility Transistor (HEMT) device used as a biosensor, a micro flow chamber, a micro injection pump and a liquid storage bottle, and is characterized in that the micro flow chamber, the micro injection pump and the liquid storage bottle are connected in sequence to form a closed circulating system; the microfluidic chamber is used for liquid flowing and cell injection, liquid in the microfluidic chamber is transmitted to the liquid storage bottle through the micro injection pump, the HEMT device used as the biosensor is coupled with the microfluidic chamber and used for detecting real-time cell membrane potential of cells in the microfluidic chamber under a dynamic condition, and the real-time cell membrane potential is calculated according to source leakage current of the HEMT device; in the micro-flow chamber, according to the detected real-time flow data, the shearing force under the flow is calculated, and finally, the detection of the cell membrane potential under the actual biological blood vessel fluid environment is simulated by combining the flow data with the real-time monitored cell membrane potential.
2. The apparatus of claim 1, wherein the HEMT device comprises a sapphire substrate layer, a GaN buffer layer, an AlN insert layer, an AlGaN barrier layer, a GaN cap layer and a Si layer in sequence from a bottom layer to an upper layer3N4A passivation layer; wherein the thickness of the GaN buffer layer is 1.6 mu m; the AlN insert layer is 1.2nm thick; the thickness of the AlGaN barrier layer is 8-15 nm; the thickness of the GaN cap layer is 1.5 nm; the barrier layer of the HEMT device has the aluminum component of 25-40%; a rectangular groove-shaped bare gate region is arranged in the middle of the passivation layer of the HEMT device; the bare grid region of the HEMT device is not subjected to electrode extraction, and the size is within the range of 10 mu m-10 mm; the covering above the HEMT device is made of high polymer materials, and the shape of the covering is a groove-shaped pattern the same as that of the bare gate.
3. The apparatus of claim 2, wherein the microfluidic chamber is mounted in a trench-shaped space formed between the HEMT device and the polymeric material, the microfluidic chamber reaction chamber being dimensioned to conform to the device gate pattern.
4. The device of claim 3, wherein the microfluidic chamber is made of a material selected from any one of the following: polydimethylsiloxane, polymethyl methacrylate, and polyimide; the microflow chamber comprises sealing ports on two sides of the microflow chamber, and the microflow chamber is connected with the micro injection pump.
5. A HEMT device for use in the apparatus of claim 1, constructed by the steps of:
1) mesa etching, namely etching the heterojunction material by adopting an ICP method to form mesa isolation of the device;
2) depositing source and drain electrode ohmic metal, obtaining an ohmic metal layer by adopting an electron beam evaporation method, wherein the ohmic metal adopts a Ti/Al/Ni/Au four-layer structure, and annealing at 830 ℃ to form alloy so as to obtain good source and drain electrode ohmic contact;
3) si deposition by PECVD method3N4The material is used as a passivation layer;
4) photoetching and etching Si by adopting ICP method3N4Exposing the bare gate area.
6. A microfluidic chamber for use in the device of claim 1, constructed by the steps of:
1) manufacturing a convex male die of the microfluidic chamber by adopting a photoetching method or a reactive ion etching method, then casting PDMS (polydimethylsiloxane) on the male die, and stripping the PDMS from the male die after curing at about 50 ℃ to obtain the microfluidic chamber component;
2) the small holes at the two ends of the micro-flow chamber are obtained by a drilling method;
3) the sealing process of the micro flow chamber part and the HEMT chip adopts a hot pressing method or an adhesion method.
7. The method of measuring cell membrane potential according to the device of claim 1, the method comprising the steps of:
1) adding 15mL of mixed solution of 1g/L collagenase and 2.5g/L trypsin in an equal volume ratio into the obtained fresh human umbilical vein blood under an aseptic condition, and placing the mixture in a water bath tank at 37 ℃ for incubation for 8 min;
2) collecting the liquid obtained in the step 1) into a centrifuge tube, washing the umbilical vein for 2 times by using a phosphate buffer solution, collecting the washed liquid into the centrifuge tube together, centrifuging for 10min at 1000r/min, taking supernatant, and adding complete culture solution to resuspend cells;
3) taking 0.1mL of cell suspension, staining the cell suspension with 4g/L trypan blue, and counting the living cells;
4) 2 × 104the/L cells were seeded in 24-well plates, each wellAdding 1mL of complete culture medium, and standing at 37 deg.C and 950mL/L O2、50mL/L CO2Standing and culturing in an incubator, uniformly mixing 0.1mL of uniformly mixed endothelial cell suspension with 0.9mL of 4g/L of trypan blue by adopting a trypan blue exclusion method, and taking 1 drop to count 100 cells under a microscope;
5) disinfecting an HEMT device by using alcohol, treating the HEMT device for 30min by using fibronectin solution, washing the HEMT device by using phosphate buffer solution, and then inoculating cells to ensure that the cell density reaches 5000-12000 cells/mm2And ensures that the cells are placed in the medium containing 5% CO in the whole process2In a 37 ℃ incubator;
6) the hemodynamics system is formed by connecting a micro injection pump and a cell membrane potential detection device, the system is erected in a 37 ℃ thermostat to maintain stable temperature, phosphate buffer solution is used as fluid in the experiment, and 5% CO is introduced in the whole experiment2To maintain the ph of the fluid environment;
7) the shear force to which the cells cultured on the bare grid of the device are subjected is deduced from the following formula:
wherein tau is the shear force applied to the cell and has the unit of dyne/cm2η is the viscosity (viscosity) of the culture solution as a fluid in g/(cm. multidot.s), Q is the flow rate of the fluid per second in cm3S; w is the gate width of the device, and h is the height of the inner wall of the microfluidic chamber;
8) the source-drain current of the HEMT device is tested by adopting a semiconductor testing analyzer, and is converted into corresponding cell membrane potential through the following formula:
wherein,is the potential of the cell membrane,for an applied drain-source voltage VDSLower and gate voltage VGSThe transconductance of the HEMT device corresponding to the range from-70 mV to 0V can be considered as small in gate voltage variationIs a constant and is obtained by pre-measurement.
8. The method of measuring cell membrane potential of claim 7, wherein the prior measurement is: adding the same drain-source voltage V to the conventional three-terminal HEMT device on the same wafer and adopting the same layoutDSTesting its transfer curve (V)G-ID) And obtaining the transconductance value of the device corresponding to the grid voltage of-70 mV to 0V.
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