Microdevice for measuring the hydraulic permeability coefficient of single cell membrane

Abstract

The present invention provides a microdevice for measuring the water permeability coefficient of a single cell membrane, comprising an upper chip, a film and a lower chip stacked in sequence; the front judgment channel of the upper chip, the front judgment electrode and the film of the lower chip constitute the front judgment area, and the single cell capture channel of the upper chip, the differential electrode pair and the film of the lower chip constitute the cell membrane water permeability coefficient measurement area. This microdevice based on single cell capture and cell impedance spectroscopy can measure the water permeability coefficient of a single cell membrane more accurately, quickly and efficiently, which is conducive to reducing the loss of rare cells in the measurement.

Description

Technical Field

[0001] The invention relates to the field of medical device technology and cell cryopreservation technology, and in particular to a microdevice for measuring the water permeability coefficient of a single cell membrane based on single cell capture and cell impedance spectroscopy. Background Art

[0002] Cryopreservation is an important technology for preserving precious cells in the fields of reproductive medicine, tissue regeneration and cell therapy. For some relatively rare, expensive cells with limited proliferation capacity, cryopreservation can significantly enhance their tissue availability for clinical applications. However, the current cryopreservation technology has the problem of low recovery efficiency, and the related cryopreservation technology is not very effective. In order to improve the success rate of cryopreservation, the optimization of the cryopreservation process is the primary consideration. Among them, the accurate determination of the cell membrane water permeability coefficient (Lp) is extremely important for the optimization of cryopreservation. For precious and rare cells, the Lp determination method at the single cell level is beneficial to reduce cell loss during the determination process. Therefore, it is of great significance to develop a fast, efficient and accurate method for determining Lp at the single cell level.

[0003] In the past few decades, researchers have adopted a variety of methods to measure Lp. In general, the known methods mainly include: microscopic stopped-flow method, microperfusion chamber method, perfusion microscopy method, microdiffusion chamber method and micropipette perfusion method. The microscopic stopped-flow method controls the concentration change of the extracellular solution by mixing two fluids, so that the instantaneous volume change of the cell can be recorded by microscopy, but the two-phase flow also makes it impossible to observe the reverse recovery process of the cell. The microperfusion chamber method is a conventional method for measuring cell Lp; in this method, a group of cells can be captured along the edge of an obstacle. The device cannot keep the captured cells separate, and the cells may interact with each other, resulting in errors in the measurement process. The perfusion microscopy method captures cells in a tiny structure, which improves the deficiency of the microperfusion chamber method that the captured cells cannot be kept separate, but the calculation of cell volume by microscopy requires the assumption that the cells are spherical, which introduces errors in the results. The change in the osmotic pressure of the extracellular fluid in the microdiffusion chamber method is through diffusion, but this method has two potential problems: (a) the dialysis membrane is easily damaged, and (b) additional mathematical complexity is introduced due to the presence of the dialysis membrane. The micropipette perfusion method uses a micropipette to perfuse anisotropic solutions into fixed oocytes. However, this method is only applicable to cell types with shells. In addition, the above methods all use optical microscopes to record the changes in cell volume. Due to insufficient optical resolution and differences in manual focusing, errors will occur in the measurement results. In short, the existing methods usually have shortcomings such as inaccurate measurements, complex operations, and a small scope of application; therefore, they are limited in the application of cell Lp measurement.

[0004] Therefore, there is an urgent need for a single-cell Lp determination method that is accurate, widely applicable, and simple to operate. Summary of the invention

[0005] The technical problem solved by the present invention is to provide a method for determining single cell Lp with accurate measurement and wide application range.

[0006] In view of this, the present application provides a microdevice for measuring the hydraulic permeability coefficient of a single cell membrane, comprising an upper chip, a thin film and a lower chip stacked in sequence;

[0007] The upper chip is provided with a through liquid storage tank and a waste liquid outlet;

[0008] From the upper chip end, a microchannel is arranged on the lower surface of the upper chip, and the microchannel is arranged between the liquid storage tank and the waste liquid outlet;

[0009] The microchannel includes a main channel, a front-end judgment channel and a single cell capture channel, wherein the main channel is used to connect the liquid storage tank, the front-end judgment channel, the single cell capture channel and the waste liquid outlet; the single cell capture channel includes two single cell capture microstructures arranged symmetrically;

[0010] From the upper chip end, the upper surface of the lower chip is provided with a detection electrode with a convex structure;

[0011] The detection electrodes include a front-end judgment electrode pair and a differential electrode pair, and the differential electrode pair includes a measurement electrode pair and a reference electrode pair;

[0012] The front-end judgment channel is arranged opposite to the front-end judgment electrode pair, and the single-cell capture channel is arranged opposite to the differential electrode pair;

[0013] The front-end judgment channel, the front-end judgment electrode and the film constitute the front-end judgment area; the single cell capture channel, the differential electrode pair and the film constitute the cell membrane water permeability coefficient measurement area;

[0014] The film is used to realize the bonding between the upper chip and the lower chip.

[0015] Preferably, the measuring electrode pair and the reference electrode pair are symmetrically distributed about the common input electrode.

[0016] Preferably, the measuring electrode pair is disposed opposite to one single-cell capture microstructure of the single-cell capture channel, and the reference electrode pair is disposed opposite to another single-cell capture microstructure of the single-cell capture channel.

[0017] Preferably, the single cell capture microstructure is a dual-path microcapture structure, which consists of a short and straight path and a long and annular bypass channel.

[0018] Preferably, the materials of the upper chip and the lower chip are independently selected from polydimethylsiloxane or organic glass, and the film is a polydimethylsiloxane film.

[0019] Preferably, the area of ​​the upper chip is independently 5 cm 2 ~10cm 2 , the thickness is independently 0.5cm to 3cm; the area of ​​the lower chip is independently 10cm 2 ~40cm 2 , the thickness is independently 0.1cm to 0.4cm.

[0020] Preferably, the cross-section of the microchannel is in the shape of a rectangular groove.

[0021] The present application also provides the application of the microdevice for measuring the water permeability coefficient of cells in single-cell drug screening.

[0022] The present application provides a microdevice for measuring the water permeability coefficient of a single cell membrane, which includes an upper chip with a microchannel, a thin film, and a lower chip with a detection electrode; the front-end judgment channel of the upper chip, the front-end judgment electrode of the lower chip, and the thin film constitute the front-end judgment area, and the single cell capture channel of the upper chip, the differential electrode pair of the lower chip, and the thin film constitute the water permeability coefficient measurement area of ​​the cell membrane; when the microdevice of the present invention is running, the cell suspension is injected into the chip, and the cell first passes through the front-end judgment area, and the judgment area will judge whether there is a cell passing according to the impedance output value; then, the cell is captured in the measurement area, and the isotonic extracellular fluid is quickly replaced with a hypertonic solution, and the cell loses water and shrinks, and the impedance change information related to the cell volume is output by the differential electrode, and then the water permeability coefficient of the cell membrane is obtained. This microdevice based on single cell capture and cell impedance spectrum can measure the water permeability coefficient of single cell membrane more accurately, quickly, and efficiently, which is conducive to reducing the loss of rare cells in measurement. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 A schematic diagram of a single unit structure and a schematic diagram of the overall structure of a microdevice for measuring the hydraulic permeability coefficient of a single cell membrane provided in an embodiment of the present invention;

[0024] Figure 2 It is an enlarged schematic diagram of judging the channel structure and the single cell capturing structure in a microdevice for measuring the hydraulic permeability coefficient of a single cell membrane provided in an embodiment of the present invention. DETAILED DESCRIPTION

[0025] In order to further understand the present invention, preferred embodiments of the present invention are described below in conjunction with examples. However, it should be understood that these descriptions are only for further illustrating the features and advantages of the present invention, rather than limiting the claims of the present invention.

[0026] The impedance method of the dielectric properties of cells is an effective method to evaluate the state of cells. Cell impedance signals of various frequencies can provide parameter information directly related to the physiological status of single cells, such as cell volume (<1MHz), membrane capacitance (1-4MHz) and cytoplasmic conductivity (4-10MHz). Microfluidic platforms for single-cell impedance flow cytometry have been reported, in which cells are driven to the sensing area of ​​a microchannel in a continuous flow manner, and their impedance is measured by microelectrodes embedded in the channel. However, such systems usually operate in continuous flow mode, and it is difficult to measure the time-dependent response of single cells in a flow system. The determination of single-cell Lp usually requires monitoring single cells over an important time course in order to study their dynamic response characteristics. Therefore, combining label-free impedance spectroscopy with long-term single-cell capture schemes can meet this requirement.

[0027] The main advantages of single-cell capture / impedance spectroscopy technology are as follows: (a) Due to the single-cell capture technology used, the separation between cells is maintained, solving the influence of cell-to-cell interactions on the measurement results; (b) It allows the observation of impedance changes over time, which can more easily obtain information related to cell morphological changes; (c) It has a wide range of applications and can be applied to cells of different sizes and types by simply modifying the size of the capture structure; (d) It can measure very detailed impedance spectra, which makes the process of extracting cell morphology more reliable and Lp measurement more accurate, and can well solve the problems of insufficient optical resolution and artificial focusing errors in the process of recording cell volume by microscope photography. In summary, the current scheme for fast, efficient, and accurate measurement of cell Lp using single-cell capture / impedance spectroscopy technology has not been reported.

[0028] Therefore, the present application proposes a cell Lp determination device based on microfluidic single cell capture and impedance spectroscopy scheme; Figure 1 As shown, Figure 1Schematic diagram of a single unit structure and a schematic diagram of the overall structure of a microdevice for measuring the hydraulic permeability coefficient of a single cell membrane provided in an embodiment of the present invention, wherein (a) is a schematic diagram of a single unit structure, 1 is an upper chip with a microchannel, 2 is an intermediate polydimethylsiloxane film, and 3 is a lower chip with a detection electrode; (b) is a schematic diagram of the overall structure of the device; (c) is a front view of the upper chip 1 with a microchannel, wherein 4 is a liquid storage tank, 5 is a waste liquid outlet, 6 is a front judgment channel, 7 and 8 are two single cell capture microstructures, and 7 and 8 constitute a single cell capture channel; (d) is a front view of the polydimethylsiloxane film; (e) is a front view of the lower chip 3 with a detection electrode, which includes a judgment electrode pair (composed of electrodes 9 and 10) and a differential electrode pair, and the differential electrode pair includes a measuring electrode pair (composed of electrodes 11 and 12) and a reference electrode pair (composed of electrodes 12 and 13).

[0029] Specifically, the microdevice for measuring the hydraulic permeability coefficient of a single cell membrane of the present invention comprises an upper chip, a thin film and a lower chip which are stacked in sequence;

[0030] The upper chip is provided with a through liquid storage tank and a waste liquid outlet;

[0031] From the upper chip end, a microchannel is arranged on the lower surface of the upper chip, and the microchannel is arranged between the liquid storage tank and the waste liquid outlet;

[0032] The microchannel includes a main channel, a front-end judgment channel and a single cell capture channel, wherein the main channel is used to connect the liquid storage tank, the front-end judgment channel, the single cell capture channel and the waste liquid outlet; the single cell capture channel includes two single cell capture microstructures arranged symmetrically;

[0033] From the upper chip end, the upper surface of the lower chip is provided with a detection electrode with a convex structure;

[0034] The detection electrodes include a front-end judgment electrode pair and a differential electrode pair, and the differential electrode pair includes a measurement electrode pair and a reference electrode pair;

[0035] The front-end judgment channel is arranged opposite to the front-end judgment electrode pair, and the single-cell capture channel is arranged opposite to the differential electrode pair;

[0036] The single cell capture channel, the differential electrode pair, and the thin film constitute a cell membrane water permeability coefficient measurement area; the front-end judgment channel, the front-end judgment electrode, and the thin film constitute a front-end judgment area;

[0037] The film is used to realize the bonding between the upper chip and the lower chip.

[0038] The present invention provides a micro device for measuring the water permeability coefficient of a single cell membrane, which comprises an upper chip with a microchannel and a lower chip with a detection electrode, wherein the depth of the channel is less than the thickness of the chip, the microchannel is located on the lower surface of the upper chip, and the detection electrode is located on the upper surface of the lower chip. Figure 1 c and e are conceptual diagrams of the upper and lower chips of the working platform for measuring the hydraulic permeability coefficient of a single cell membrane provided by the embodiment of the present invention. The present invention has no special restrictions on the shape and material of the upper and lower chips, as long as they meet the actual operating conditions.

[0039] In an embodiment of the present invention, the upper and lower chips are rectangular in shape, and the upper chip material is polydimethylsiloxane (PDMS), with a length, width and height of 3.5cm×2cm×1cm. The lower chip material is organic glass with an indium tin oxide (ITO) layer, with a length, width and height of 6cm×3.4cm×0.1cm; in other embodiments, the upper chip and the lower chip may be circular or square in shape, the upper chip material may be organic glass (PMMA), and the lower chip material may be a printed circuit board (PCB). In a specific embodiment, the size of the upper chip can be adjusted by changing the channel depth and width, and the area of ​​the upper chip is independently 5cm 2 ~10cm 2 , the thickness is independently 0.5cm to 3cm; the size of the lower chip can be adjusted by changing the length and width of the electrode, and the area of ​​the lower chip is independently 10cm 2 ~40cm 2 , the thickness is independently 0.1cm to 0.4cm.

[0040] Figure 2 (a) is a schematic diagram of the microchannel structure in the working platform for measuring the hydraulic permeability coefficient of a single cell membrane provided in an embodiment of the present invention, wherein the microchannel comprises a liquid storage tank 4, a waste liquid outlet 5, a front-end judgment channel 6, and two single cell capture microstructures 7 and 8. The two single cell capture microstructures constitute a single cell capture channel. In an embodiment of the present invention, the main channel is 300 μm wide and 20 μm high; the length, width and height of the microchannel in the front-end judgment channel are all 20 μm. Figure 2 (a) shown.

[0041] The single cell capture channel contains two identical single cell capture microstructures. Figure 2 (b) Figure 2(b) A schematic diagram of a single cell capture microstructure in a working platform for measuring the hydraulic permeability coefficient of a single cell membrane provided in an embodiment of the present invention. In an embodiment of the present invention, the single cell capture microstructure is designed as a short and straight path, which contains a small capture gap smaller than the size of a cell so as to mechanically fix the cell; and a long and annular bypass channel. Due to the difference in length and geometry between the two paths, the flow resistance along the short channel can be designed to be smaller than the flow resistance along the bypass channel, which leads to a greater flow rate in the short channel, which can effectively capture the cell in the trap position of the short channel. When an annular channel is designed, after the cell is captured, the flow resistance of the short straight channel will increase, and the fluid will flow more to the annular channel, which reduces the squeezing of the fluid on the cell when there is only a short straight channel, and reduces the result error; after the cell is captured, if other cells or cell fragments move toward the capture structure, they will flow through the loop channel to prevent multiple cells from being captured at the same time.

[0042] In this embodiment, the cells are driven into the microchannel through the liquid reservoir, first pass through the front-end judgment channel, and then are captured in the first single-cell capture channel, and the cell-free solution flows out from the waste liquid outlet.

[0043] In a specific embodiment, the lower chip is made of organic glass, and the detection electrode is made of indium tin oxide material, such as Figure 2 As shown in (b); every two electrodes constitute a group of electrode pairs, electrodes 9 and 10 are front-end judgment electrode pairs, electrodes 11 and 12 are measuring electrode pairs, electrodes 12 and 13 are reference electrode pairs, and the measuring electrode pairs and the reference electrode pairs constitute differential electrode pairs. The judgment electrode pair is below the front-end judgment channel, and the differential electrode pair is below the capture channel, wherein the measuring electrode pair is below the first single-cell capture microstructure 7, and the reference electrode pair is below the second single-cell capture microstructure 8. The differential electrode pairs are symmetrically distributed about the common input electrode 12. In an embodiment of the present invention, the electrode sizes are as follows: L1, L2, and L3. In the embodiment of the present invention, the sizes of indium tin oxide electrodes are all in the millimeter level. The use of millimeter-sized electrodes is more conducive to processing and manufacturing, reduces manufacturing costs, and is easier to align during bonding with the upper chip using oxygen plasma technology.

[0044] Capture structure 7 is used to capture cells, and only one is captured at a time, and capture structure 8 is not used to capture cells. The measuring electrode pair and the reference electrode pair constitute a differential electrode structure, the measuring electrode is located below position 7, and the reference electrode pair is located below position 8, and the two parts are symmetrically distributed. The above-mentioned symmetrical setting method is to eliminate the influence of the baseline current and voltage drift caused by the solution electrolysis reaction and external interference on the test results during the experiment. Before the cell is captured, although the electrolysis reaction or external interference occurs, because the two structures are completely symmetrical, the influence of this electrolysis reaction or external interference on the output voltage of the two pairs of electrodes is the same, so the output voltage of the two pairs of electrodes is always the same (the manufacturing process is very high, and the two structures are exactly the same) or the ratio of the two output voltages is always a constant (the manufacturing process is not perfect, and there are differences between the two structures). When the 7 structure captures the cell, the output voltage of the measuring electrode pair will change due to the capture of the cell, but because the 8 structure does not capture the cell, the output voltage of the reference electrode will not change. Using the reference electrode output voltage as a reference, the baseline of the measuring electrode output voltage can be calculated (the voltage baseline refers to the voltage value output when no cells are captured. Because their output voltages are the same or the ratio is a constant before cells are captured, the output voltage value of one of them can be calculated based on the other output voltage value). The result of subtracting the baseline voltage value of the measuring electrode pair from the output voltage value of the measuring electrode pair is the voltage change caused by the cells.

[0045] In the present invention, a judgment electrode pair is arranged in front of the differential electrode. The introduction of the judgment electrode pair is conducive to improving the single cell capture efficiency. When the judgment electrode pair detects that a cell has passed through, it will feedback control to reduce the fluid speed, so that the cell speed is reduced, and the success rate of being captured in the capture channel is increased. Each pair of electrodes includes an input electrode and an output electrode. The input electrode inputs an excitation signal of a fixed frequency, and the output electrode outputs the impedance information between the two electrodes. When a cell enters the microchannel between the electrode pair, it will cause the impedance between the electrodes to change. Information related to the cell volume can be extracted from the collected impedance value.

[0046] In the present invention, the method of combining the front-end judgment electrode pair and the differential electrode pair can more accurately measure the water permeability coefficient of the single cell membrane, reduce the error introduced in the optical measurement method, and reduce the loss of rare cells in the measurement. In other embodiments, a gold electrode with higher conductivity can be used to improve the output signal-to-noise ratio and improve the measurement accuracy of the water permeability coefficient of the single cell membrane.

[0047] In an embodiment of the present invention, the cross-section of the microchannel is in the shape of a rectangular groove, the depth of the groove is fixed, and the width is variable. In an embodiment of the present invention, the width of the rectangular groove channel can be 0.1mm to 0.4mm, and the depth can be 0.02mm; in other embodiments, the width of the channel can be 0.1mm to 2mm, and the depth can be 0.02mm to 0.06mm. In the present invention, the use of a smaller channel can be beneficial to driving cells, can more effectively capture single cells, improve detection sensitivity, and improve signal-to-noise ratio.

[0048] The present invention provides a microdevice for measuring the water permeability coefficient of a single cell membrane, comprising a polydimethylsiloxane (PDMS) film disposed between the upper chip and the lower chip, such as Figure 1 a(2). In the present invention, there is no special restriction on the shape of the polydimethylsiloxane film, and the size can be used to completely cover the detection electrode. The purpose of using the PDMS film is to promote the bonding of the upper chip and the lower chip using plasma technology, so that the chip bonding is more firm and the reuse rate of the lower chip is improved. The film is obtained by spin coating a mixture of polydimethylsiloxane and toluene on the lower chip and then curing. The thickness of the film is not fixed. In this embodiment, the thickness of the film can be 5μm to 10μm, and the thinner the film, the higher the sensitivity of cell impedance detection.

[0049] In an embodiment of the present invention, the preparation method of the microdevice is as follows: the process of making the upper chip with microchannels can be described as follows: photoresist SU-82050 is coated on a 3-inch silicon wafer with a thickness of 20 μm, exposed to a direct laser etcher for etching, and then the patterned silicon wafer is rinsed with SU-8 developer and deionized water and hard baked; finally, the polydimethylsiloxane premix is ​​poured on the top of the SU-8 mold in the tray and cured in an 80°C oven for 1 hour to form the upper chip; in an embodiment of the present invention, the process of making the lower chip with detection electrodes can be described as follows: the electrode pattern is designed using AutoCAD 2013; indium tin oxide electrodes are prepared on a 0.1 cm thick glass surface using laser etching technology. The process of making the polydimethylsiloxane film can be described as follows: a mixed solution of polydimethylsiloxane and toluene is flattened on the lower chip by spin coating, and then cured to form a film. In an embodiment of the present invention, the upper chip with microchannels and the lower chip with the film are treated with oxygen plasma for 40 seconds and combined together to form a microdevice. The chip was then placed at 80°C for at least 30 minutes.

[0050] In another aspect of the present invention, the microdevice can also be expanded for single-cell drug screening.

[0051] When the microdevice of the present invention is in operation, a cell suspension is injected into the microfluidic chip, and the cells first pass through the front judgment area, which determines whether a cell has passed according to the impedance output value; then, the cells are captured in the measurement area, and the isotonic extracellular fluid is quickly replaced with a hypertonic solution, and the cells lose water and shrink, and the differential electrode outputs impedance change information related to the cell volume, thereby obtaining the cell membrane water permeability coefficient. This microdevice based on single cell capture and cell impedance spectroscopy can measure the single cell membrane water permeability coefficient more accurately, quickly, and efficiently, which is conducive to reducing the loss of rare cells in the measurement.

[0052] In order to further understand the present invention, the microdevice for measuring the hydraulic permeability coefficient of a single cell membrane provided by the present invention is described in detail below in conjunction with the examples. The protection scope of the present invention is not limited by the following examples.

[0053] Example 1

[0054] have Figure 1 The schematic diagram of the structure of a device for measuring the water permeability coefficient of a single cell membrane and the conceptual diagram of the upper, middle and lower chips of the micro-device are shown, including:

[0055] The upper, middle and lower chips of the microdevice for measuring the hydraulic permeability coefficient of single cell membrane are rectangular in shape. The upper chip material is polydimethylsiloxane (PDMS), with a length, width and height of 3.5cm×2cm×1cm; the lower chip material is organic glass with an indium tin oxide (ITO) layer, with a length, width and height of 6cm×3.4cm×0.1cm; the microchannel on the lower surface of the upper chip includes a cylindrical liquid storage tank (radius of 0.25cm, depth of 1cm), a main channel, a front-end judgment channel, a single cell capture channel and a cylindrical waste liquid outlet The main channel is 300 μm wide and 20 μm high. The length, width and height of the microchannel of the front judgment channel are all 20 μm. The single-cell capture channel contains two identical single-cell capture microstructures, each of which is designed as a short and straight path and a long and annular bypass channel. A short and straight path contains a small capture gap, which is 4 μm × 4 μm × 20 μm in length, width and height. A long and annular bypass channel has a total length of 200 μm, a width of 10 μm and a height of 20 μm. In this embodiment; Figure 1 In the middle, 4 is a reservoir, which is the inlet of cell suspension and hypertonic solution, and 5 is the outlet of waste solution;

[0056] The detection electrodes on the upper surface of the lower chip include a front-end judgment electrode pair and a differential electrode pair, wherein the differential electrode pair is composed of a measuring electrode pair and a reference electrode pair, the measuring electrode pair and the reference electrode pair have a common input electrode, and the two pairs of electrodes are symmetrical about the common input electrode; electrodes 9, 11, and 13 have the same size, with a length and width of 3.8 mm and 1.5 mm respectively; electrode 10 has a length and width of 2 mm×1.5 mm; electrode 12 has a length and width of 1.5 mm×1 mm;

[0057] The polydimethylsiloxane film disposed between the upper and lower chips has a thickness of 5 μm and a size that completely covers the detection electrode;

[0058] The upper chip and the lower chip are bonded and fixed by oxygen plasma treatment.

[0059] Example 2

[0060] The microdevice for measuring the water permeability coefficient of a single cell membrane provided in Example 1 is used to measure the water permeability coefficient of induced pluripotent stem cells. The specific process is as follows:

[0061] Add 6.4 g of mannitol to 30 mL of PBS to prepare a hypertonic solution. Use a conductivity meter to measure the conductivity of the solution. Use NaCl solution to titrate the solution to keep the conductivity of the hypertonic solution the same as that of the original PBS solution.

[0062] A sinusoidal signal with a fixed frequency of 500kHz is input to the input electrode ends of the judgment electrode pair and the differential electrode pair, and a cell suspension of induced pluripotent stem cells with a density of 1000 cell / mL is injected into the reservoir using a syringe pump. The cell suspension is extracted at a flow rate of 10 μL / min using a syringe pump connected to the outlet, and the cells are driven from the reservoir into the chip; when the first cell is detected passing through the judgment electrode, the extraction speed of the outlet pump is reduced to 0 μL / min until the cell is captured in the capture microstructure in the single-cell capture channel, and the extraction of the outlet pump is stopped; next, the remaining cell suspension in the reservoir is emptied using a syringe, and the hypertonic solution is injected therein, and the hypertonic solution is driven to quickly replace the extracellular fluid using a syringe pump at the outlet at a flow rate of 10 μL / min, the cells lose water and shrink, and the differential electrode outputs impedance change information related to the cell volume, and finally, the water permeability coefficient of the cell membrane is obtained.

[0063] The test results are as follows: the cell membrane water permeability coefficient of induced pluripotent stem cells was measured by using the cell membrane water permeability measurement microdevice based on the principle of single cell capture and cell impedance spectroscopy provided in Example 1 of the present invention, and the measurement was completed efficiently in a short time, and the measurement result was 0.48±0.05μm / atm / min. This result shows that the single cell cell membrane water permeability coefficient measurement microdevice can be used to accurately, quickly and efficiently measure the single cell cell membrane water permeability coefficient.

[0064] The present invention provides a working platform for measuring the water permeability coefficient of a single cell membrane, comprising: an upper chip with a microchannel, the upper chip comprising a liquid storage tank, a main channel, a judgment channel, a single cell capture channel and a waste liquid outlet; a lower chip with a detection electrode, the lower chip comprising a judgment electrode pair and a differential electrode pair, wherein the differential electrode pair consists of a measuring electrode pair and a reference electrode pair; a polydimethylsiloxane film arranged between the upper chip and the lower chip; a cell membrane water permeability coefficient measurement area composed of the single cell capture channel of the upper chip, the differential electrode pair of the lower chip and the polydimethylsiloxane film arranged between the upper chip and the lower chip; and a front-end judgment channel, a front-end judgment electrode and a polydimethylsiloxane film of the upper chip forming a front-end judgment area.

[0065] The upper chip is used for the passage of cell suspension and cell capture; the lower chip is used for measuring impedance information when cells pass through the channel between electrode pairs; the front judgment area completes the detection of whether there are cells passing through; the cell membrane water permeability coefficient measurement area completes the capture of single cells and the measurement of impedance information related to cell morphological changes.

[0066] The microdevice for measuring the water permeability coefficient of a single cell membrane provided by the present invention drives the cell suspension from the reservoir into the chip, first passes through the front judgment area, detects that a cell has passed, and then controls the driving speed to capture the cell in the single cell capture channel; drives the hypertonic solution from the reservoir into the chip, the osmotic pressure of the extracellular fluid rises rapidly, the captured cells lose water and shrink, and the differential electrode outputs impedance change information related to the cell volume, thereby calculating the water permeability coefficient of the cell membrane. The technology combining single cell capture and cell impedance spectroscopy can accurately measure the water permeability coefficient of the cell membrane, which helps to reduce the loss of precious cells in the measurement.

[0067] The above embodiments are only used to help understand the method and core idea of ​​the present invention. It should be noted that, for those skilled in the art, several improvements and modifications can be made to the present invention without departing from the principles of the present invention, and these improvements and modifications also fall within the scope of protection of the claims of the present invention.

[0068] The above description of the disclosed embodiments enables one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A microdevice for measuring the hydraulic permeability coefficient of a single cell membrane, comprising an upper chip, a thin film and a lower chip stacked in sequence; The upper chip is provided with a through liquid storage tank and a waste liquid outlet; From the upper chip end, a microchannel is arranged on the lower surface of the upper chip, and the microchannel is arranged between the liquid storage tank and the waste liquid outlet; The microchannel includes a main channel, a front-end judgment channel and a single-cell capture channel, wherein the main channel is used to connect the liquid storage tank, the front-end judgment channel, the single-cell capture channel and the waste liquid outlet; the single-cell capture channel includes two single-cell capture microstructures that are symmetrically arranged; the single-cell capture microstructure is a dual-path microcapture structure, which is composed of a short and straight path and a long and annular bypass channel; From the upper chip end, the upper surface of the lower chip is provided with a detection electrode with a convex structure; The detection electrodes include a front-end judgment electrode pair and a differential electrode pair, and the differential electrode pair includes a measurement electrode pair and a reference electrode pair; The front-end judgment channel is arranged opposite to the front-end judgment electrode pair, and the single-cell capture channel is arranged opposite to the differential electrode pair; The front-end judgment channel, the front-end judgment electrode and the film constitute the front-end judgment area; the single cell capture channel, the differential electrode pair and the film constitute the cell membrane water permeability coefficient measurement area; The film is used to realize the bonding between the upper chip and the lower chip.

2. The microdevice according to claim 1, characterized in that The measuring electrode pair and the reference electrode pair are symmetrically distributed about a common input electrode.

3. The microdevice according to claim 1 or 2, characterized in that: The measuring electrode pair is disposed opposite to one single cell capturing microstructure of the single cell capturing channel, and the reference electrode pair is disposed opposite to another single cell capturing microstructure of the single cell capturing channel.

4. The microdevice according to claim 1 or 2, characterized in that: The materials of the upper chip and the lower chip are independently selected from polydimethylsiloxane or organic glass, and the film is a polydimethylsiloxane film.

5. The microdevice according to claim 1 or 2, characterized in that: The area of ​​the upper chip is independently 5cm 2 ~10cm 2 , the thickness is independently 0.5cm to 3cm; the area of ​​the lower chip is independently 10cm 2 ~40cm 2 , the thickness is independently 0.1cm to 0.4cm.

6. The microdevice according to claim 1 or 2, characterized in that: The cross section of the microchannel is in the shape of a rectangular groove.

7. Use of the microdevice for measuring the water permeability coefficient of cells as claimed in any one of claims 1 to 6 in single cell drug screening.

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