A low-voltage cell disruption microfluidic chip and its manufacturing method

Abstract

The present invention discloses a low-voltage cell disruption microfluidic chip and a manufacturing method thereof, which includes a PDMS substrate and a PDMS cover sheet. The PDMS substrate is provided with a mixing section including two liquid inlet pools and a T-shaped micro mixer, a disruption section with a plurality of trapezoidal electrodes inserted therein, a sorting section with an enlarged cross-sectional area and including two columns of columnar obstacles in the channel, a secondary disruption section for processing the remaining cells that have not been completely disrupted, a staining section, and a final counting and observation section. The present invention can be used for low-voltage cell disruption, and through a variety of sorting / separation techniques, cell contents and cell organelles of different qualities can be separated after staining, which is convenient for observation and statistics to calculate the cell disruption rate. The present invention has high safety and controllability in experiments and low experimental costs.

Description

Technical Field

[0001] The present invention relates to a low-voltage cell disruption microfluidic chip and a method for fabricating the same. Background Art

[0002] Cell disruption technology is a technology that uses a certain method to destroy the selective permeability of cell membranes, including bead milling disruption, centrifugal disruption, chemical substance dissolution disruption, pulsed electric field disruption, etc. As a prerequisite technology for extracting non-secretory biochemical substances from animal and plant cells, it is widely used in fields such as the food industry, drug manufacturing, and extraction.

[0003] The principle of pulsed electric field disruption method is the electroporation effect of biological membranes. This effect refers to the phenomenon that when the transmembrane voltage exceeds the threshold (about 1V), the hydrophobic pores generated by biological membranes rearrange into hydrophilic pores, resulting in the temporary or permanent loss of selective permeability of biological membranes. According to whether the biological membrane can recover after electroporation, it can be divided into reversible electroporation and irreversible electroporation. The former is mainly applied to the fields of cell fusion and genetic engineering; the latter is mainly applied to the extraction of non-secretory substances and the disinfection of pathogens, etc.

[0004] The pulsed electric field (Pulsed Electric Field) disruption method has been widely applied in fields such as biochemical substance extraction, pathogen disinfection, and cancer treatment due to its characteristics of high disruption rate, high controllability, and selective disruption.

[0005] A microfluidic chip, also known as a "lab-on-a-chip", is to use microelectromechanical technology to fabricate structural elements such as micropumps, microvalves, microelectrodes, microfilters, and microreactors on a chip with a small area, realizing the miniaturized integration of the functions of each laboratory unit at the macroscopic level, and finally realizing the functions of a chip laboratory. Due to its characteristics of greatly reducing the consumption of experimental materials brought about by its integration and its low requirements for energy and experimental sites, microfluidic technology has been gradually promoted and used in the field of cell disruption, and has very important application value.

[0006] Currently, the methods mainly adopted for microfluidic chips used in cell disruption include chemical disruption, mechanical force disruption, and electric pulse disruption. Among them, the electric pulse disruption method has an extremely high disruption rate and has the characteristics of high controllability and observable throughout the process.

[0007] The Reynolds number of the fluid in this microfluidic chip is relatively low, so the fluid mixing structure adopted is an inclined groove chaotic convection micromixer. This structure can effectively enhance the mixing effect of cell suspension and dye at low Reynolds numbers.

[0008] The present invention uses three sorting / separation methods: physical structure sorting, dielectrophoresis sorting, and centrifugal sorting.

[0009] The physical structure sorting used in the present invention uses a plurality of cylindrical structures with intervals smaller than the cell diameter to sort the cell contents after the first disruption from the intact cells that have not been disrupted, so as to perform a secondary treatment on the cells that have not been successfully disrupted.

[0010] Dielectrophoresis sorting and centrifugal sorting are used simultaneously to separate non-secretory substances and organelles that escape from the cell structure due to the destruction of selective permeability after disruption, so as to achieve subsequent observation and counting.

[0011] In electromagnetism, dielectric molecules are equivalent to charged systems composed of positive and negative point charges. The locations of positive and negative point charges are called the centers of positive and negative charges. Molecules whose centers of positive and negative charges coincide are called non-polar molecules, and molecules whose centers of positive and negative charges do not coincide are called polar molecules.

[0012] In an external electric field, the positive and negative point charges that make up the non-polar molecules undergo opposite displacements, and the centers of positive and negative charges no longer coincide, which is called displacement polarization; the electric dipoles that make up the polar molecules are acted upon by torque and turn in the direction of the external electric field, which is called orientation polarization.

[0013] Dielectrophoresis sorting technology utilizes the above two polarization phenomena. First, displacement polarization is used to convert the dielectric medium (intact cells and organelles) in the channel from non-polar to polar. Then, orientation polarization is used to assist centrifugal sorting to separate substances of different masses.

[0014] In addition, the polarization of the medium will reduce the energy required for fragmentation by providing a polarization electric field. Therefore, before fragmentation, the overall relative dielectric constant of the solution can be lowered by mixing it with a solution with a lower relative dielectric constant, thereby enhancing the polarization effect and reducing the power consumption of the chip during operation. Summary of the invention

[0015] The technical problem to be solved by the present invention is to provide a low-voltage cell disruption microfluidic chip and a manufacturing method. The voltage required for cell disruption is low, and the chip has a sorting function, which can separate the cell structures after successful disruption from the cells that have not been successfully disrupted, and perform a secondary disruption on the intact cells. After the secondary disruption, the different cell structures are stained and separated for observation through a microscope, thereby improving the safety and operability of the experiment and reducing the experimental cost.

[0016] The technical solutions of the invention are as follows:

[0017] A low-voltage cell disruption microfluidic chip, comprising a PDMS substrate and a PDMS cover;

[0018] It includes a PDMS substrate and a PDMS cover sheet. The PDMS substrate is divided into six parts along the flow direction of the main channel according to the main functions of the main channel and the channels connected to it:

[0019] Two liquid inlets, a mixing section of a T-shaped micro mixer, a fragmentation section (also known as the primary fragmentation section) with a plurality of trapezoidal electrodes inserted, a sorting section with an enlarged cross-sectional area and containing two rows of columnar obstacles in the channel, a secondary fragmentation section for processing the remaining cells that have not been completely fragmented, and a staining section and a final counting and observation section;

[0020] The PDMS substrate also includes 6 branch channels connected to the main channel. The 6 branch channels are respectively connected to 1 cell suspension inlet pool, 1 low relative dielectric constant solution inlet pool, 3 cleaning inlet pools and 1 dye inlet pool.

[0021] Both the PDMS substrate and the PDMS cover are square sheets, with the bottom surface being a rectangle of 6 cm * 3 cm and the thickness being 3 mm.

[0022] Mixing section: At a distance of 1 cm from the left edge of the chip, two cylindrical liquid inlets with a diameter of 3 mm and a depth of 40 μm are provided. The two liquid inlets are spaced 1 cm apart and parallel to the side edge; A rectangular liquid inlet channel with a length of 1 cm, a width of 30 μm, and a depth of 40 μm connects the two liquid inlets. At the midpoint of the channel, a mixing channel with a length of 0.5 cm, a width of 60 μm, and a depth of 40 μm extends to the right;

[0023] Fragmentation section: On both sides of the end of the mixing channel (i.e., Figure 1 the upper and lower sides), it contracts from 60 μm to 40 μm at a 45-degree angle (see Figure 1 , Figure 1 for the top view). On both sides of the front end of the fragmentation section channel, 8 trapezoidal electrodes are provided on each side (a total of 16). The cross-sectional dimensions of the trapezoidal electrodes are: the upper base length is 30 μm, the lower base length is 50 μm, the height is 10 μm, and the thickness of the trapezoidal electrodes is 40 μm; The lower bottom edge is set close to the side wall of the channel (i.e., Figure 2 the upper and lower sides), and in the extending direction of the channel (i.e., the left-right direction), the trapezoidal electrodes are spaced 30 μm apart from each other. The total length of the fragmentation section channel is 0.5 cm (see Figure 2 , Figure 2 for the top view);

[0024] Sorting section: The width of the end of the fragmentation section channel expands from 40 μm to 80 μm to the right at a 30-degree angle. With the axis at 55 μm behind the starting point of the channel expansion, a cylinder with a bottom diameter of 20 μm and a height of 40 μm is set;

[0025] In the sorting section, another row of 14 cylinders is provided along the extending direction of the channel. The axes of the cylinders are set on the center line of the channel, that is, the axis is 40 μm away from both side walls of the channel, and the adjacent cylinder spacing is 10 μm (in fact, the distance between the axes of two adjacent cylinders is 30 μm);

[0026] The 15 cylinders are used to separate the unbroken cells; See the channel structure inFigure 5 ;

[0027] On one side of the sorting section channel (specifically, it can be the right side in the channel extension direction, see Figure 5 ), three channels with a width of 40 microns, a length of 0.5 cm, and a height of 40 microns, with a spacing of 110 microns between each pair, are vertically connected. Each of the three channel ends is connected to a cylindrical liquid inlet pool with a diameter of 3 mm and a height of 40 microns;

[0028] The end of the sorting section channel is connected to two straight channels: both channels are 40 microns wide. One of the channels, i.e., the straight channel, has no angular offset, and the other channel, i.e., the deflected channel, has an angle of 30 degrees with the extension direction;

[0029] Secondary crushing section: It has 2 channels connected to the end of the sorting section channel, namely the first channel and the second channel. These 2 channels converge at the end. There are 4 trapezoidal electrodes on each side of the first channel. The cross-sectional dimensions of the trapezoidal electrodes are: the upper base length is 30 microns, the lower base length is 50 microns, the height is 10 microns, and the thickness of the trapezoidal electrode is 40 microns; the lower bottom edge is set close to the side wall of the channel (i.e., Figure 2 the upper and lower sides), in the extension direction of the channel (i.e., the left - right direction), the trapezoidal electrodes are spaced 30 microns apart from each other, and the total length of the channel is 500 microns;

[0030] Staining section: The first channel and the second channel are connected at the end of the secondary crushing section. The connection point continues to extend 200 microns to the right and is perpendicularly connected to a vertical channel of the same size on one side. The length of the vertical channel is 0.5 cm, and the end is connected to a cylindrical liquid inlet pool with a diameter of 3 mm and a height of 40 microns;

[0031] The connection point between the end of the secondary crushing section channel and the staining agent channel extends 0.1 cm to the right and then connects to an embedded oblique - linear structure. The width of the groove - type structure is 20 microns, the depth is 20 microns, the inclination angle is 450 (the inclination angle refers to the angle inclined relative to the channel extension direction), and the spacing between each pair is 40 microns, with a total of 6 (the inclination angle is the angle inclined relative to the channel extension direction), and it continues to extend 0.4 cm to the right (the oblique - linear structure is used to increase the mixing rate of the fluid by creating turbulent flow, and the structure is shown in Figure 3 );

[0032] Counting and observation section: The embedded oblique - linear structure is connected to an annular channel with a width of 40 microns, a height of 40 microns, and a diameter of 0.5 cm, with a total angle of 180 degrees (i.e., the end is 1 cm directly above the starting section), and a pair of patch electrodes is placed on both sides of the channel every 30 degrees, with a total of 6 pairs;

[0033] The width of the annular channel expands to 120 microns at an angle of 30 degrees to both sides at the end, then extends 0.5 cm, and the end is connected to a cylindrical liquid storage pool with a diameter of 3 mm and a height of 40 microns.

[0034] A method for fabricating a microfluidic separation chip, comprising the following steps:

[0035] a) Using a silicon wafer lithography process, all the patterns of the chip substrate are obtained on the silicon wafer, and the silicon wafer is etched using ICP plasma etching technology (Inductively Couple Plasma Etch) to obtain the structural shapes of the substrate liquid inlet pool, mixing channel, fragmentation channel, separation channel, trapezoidal electrode, patch electrode, annular separation channel, substrate liquid storage pool, and counting and observation area;

[0036] b) Using the silicon wafer as a mold, PDMS material is selected and cast on the silicon wafer, and transfer molding is performed to obtain a PDMS (polydimethylsiloxane) substrate;

[0037] c) Fabricate a cuboid paper box with a bottom of 6 cm * 3 cm rectangle and a height of 3 mm, the same size and shape as the PDMS substrate. Glue a solid cylinder model with a diameter of 3 mm and a height of 1 mm at the positions corresponding to the 6 PDMS substrate liquid inlet pools. Select PDMS material and inject it into the paper box, cure it, and peel it off to obtain a PDMS cover sheet;

[0038] d) Bond the PDMS substrate and the PDMS cover sheet: Place the PDMS substrate and the PDMS cover sheet 3 cm away from a 6-watt low-pressure mercury lamp and irradiate for 3 hours. Then, drop deionized water on the PDMS substrate and the PDMS cover sheet. Within 1 minute, fit and close the corresponding surfaces of the substrate liquid inlet pool, mixing channel, fragmentation channel, separation channel, trapezoidal electrode, patch electrode, annular separation channel, and substrate liquid storage pool of the PDMS substrate. Then, place it in a drying oven and store it at 65 degrees for 2 hours. Then take it out and place it in the air for 24 hours for standby; The chip is the aforementioned low-voltage cell disruption microfluidic chip.

[0039] Input a solution (such as ethanol, etc.) and a cell suspension into the low relative dielectric constant solution inlet pool, and utilize the dielectric polarization effect to reduce the power consumption of the chip during operation.

[0040] A low relative dielectric constant solution can be used to input the solution into the liquid inlet cell and mix it with the cell suspension. By utilizing the dielectric polarization effect, the power consumption of the chip during operation can be reduced. The sorting section contains a cylindrical array with a diameter of 20 microns and a spacing of 10 microns, which can physically sort the suspension after the first fragmentation, separating the unbroken cells from the organelles and cell contents that have escaped after fragmentation. By precisely performing secondary fragmentation on the cells that have failed to fragment, the overall cell fragmentation rate can be increased. The sorting section channel is vertically connected to three cleaning channels, through which cleaning solvent can be input after the experiment to prevent experimental residues from affecting the working efficiency and the accuracy of repeated experiments. After the solution after secondary fragmentation is mixed with the staining solvent, it passes through an annular separation channel with a width of 40 microns, a height of 40 microns, and a radius of 0.5 cm, with patch electrodes attached on the outside. By using dielectrophoresis-assisted centrifugal separation technology, organelles and cell contents of different masses are separated and then sent to the counting and observation area for easy observation and counting.

[0041] Advantages:

[0042] The present invention can be used for low-voltage cell fragmentation, and through various sorting / separation techniques, cell contents and organelles of different masses can be separated after staining, facilitating observation and statistics to calculate the cell fragmentation rate. In addition, after use, the present invention can inject a cleaning solution into the cleaning liquid inlet cell to clean the sorting section channel, and then flow to the left and right along the channel and finally be discharged from other liquid inlet cells and drain cells to prevent residues from affecting the working efficiency and the accuracy of repeated experiments. The safety and controllability of the experiment are high, and the experimental cost is low. Description of the Drawings

[0043] Figure 1 It is a schematic diagram of the fragmentation section structure;

[0044] Figure 2 It is the trapezoidal electrode structure for fragmentation;

[0045] Figure 3 It is a schematic diagram of the oblique linear structure;

[0046] Figure 4 It is a structural diagram of the microfluidic chip;

[0047] Figure 5 It is the sorting section and the cylindrical sorting array therein.

[0048] Figure 6 It is the simulation result of the fragmentation section. Among them, Figure 6 (a) is the electric field intensity distribution diagram, Figure 6 (b) is the electric field intensity curve in the axial direction;

[0049] Figure 7 It is the particle tracking result of the separation section

[0050] Figure 8Particle tracking results map of the sorting section;

[0051] Figure 9 It is a process flow chart of photolithography and etching for silicon wafers;

[0052] Figure 10 It is a process flow chart for fabricating a PDMS chip. Specific implementation manners

[0053] The present invention will be further described in detail below in conjunction with the accompanying drawings and specific embodiments:

[0054] Example 1:

[0055] A microfluidic separation chip, as Figure 1 shown, includes a PDMS (polydimethylsiloxane) substrate and a PDMS cover sheet. Both the PDMS substrate and the PDMS cover sheet are cuboids, with the bottom being a rectangle with side lengths of 6 cm * 3 cm and a height of 3 mm. The PDMS substrate and the PDMS cover sheet are bonded.

[0056] This microfluidic chip can be divided into five sections: including a mixing section of two liquid inlet pools and a T-shaped micro mixer, a fragmentation section with a plurality of trapezoidal electrodes inserted, a sorting section with an enlarged cross-sectional area and two columns of columnar obstacles in the channel, a secondary fragmentation section for processing the remaining cells that have not been completely fragmented, as well as a staining section and a final counting and observation section.

[0057] Mixing section: Two cylindrical liquid inlet pools with a diameter of 3 mm and a height of 40 μm are set at a distance of 1 cm from the left edge of the chip. The two liquid inlet pools are spaced 1 cm apart and parallel to the side edge. A cuboid-shaped liquid inlet channel with a length of 1 cm, a width of 30 μm, and a height of 40 μm connects the two liquid inlet pools. At the midpoint of the channel, a mixing channel with a length of 0.5 cm, a width of 60 μm, and a height of 40 μm extends to the right.

[0058] Fragmentation section: The two sides at the end of the mixing channel contract to 40 μm at a 45-degree angle. Then, on each side of the channel, there are trapezoidal electrodes with an upper base length of 30 μm, a lower base length of 50 μm, a height of 10 μm, and a thickness of 40 μm. There are 8 on each side (the lower bottom edge is set close to the channel wall), and they are spaced 200 μm apart in the channel extension direction. The total length of the fragmentation section channel is 0.5 cm.

[0059] Sorting section: The width of the channel at the end of the fragmentation section expands to 80 μm at a 30-degree angle to the right. In the center of the channel, there is also a row of cylinders with a spacing of 10 μm, a bottom diameter of 20 μm, and a height of 40 μm, a total of 11.

[0060] One side of the sorting section channel is vertically connected to 3 channels with a width of 40 μm, a length of 0.5 cm, and a height of 40 μm. Each of the three channel ends is connected to a cylindrical liquid inlet pool with a diameter of 3 mm and a height of 40 μm.

[0061] The end of the sorting section channel is connected to two channels: both channels are 40 microns wide, one channel has no angular offset, and the other channel (i.e., the second channel) is angularly offset 30 degrees clockwise (i.e., the included angle is 30 degrees, see Figure 5 ).

[0062] Secondary crushing section: It has 2 channels docked with the end of the sorting section channel, namely the first channel and the second channel. These 2 channels converge at the end. There are 4 trapezoidal electrodes on each side of the first channel. The cross-sectional dimensions of the trapezoidal electrode are: the upper base length is 30 microns, the lower base length is 50 microns, the height is 10 microns, and the thickness of the trapezoidal electrode is 40 microns; the lower bottom edge is set close to the side wall of the channel (i.e., Figure 2 the upper and lower sides), in the extending direction of the channel (i.e., the left - right direction), the trapezoidal electrodes are spaced 30 microns apart from each other, and the total length of the channel is 500 microns;

[0063] Staining section: The first channel and the second channel are connected at the end of the secondary crushing section. The connection point continues to extend 200 microns to the right and is perpendicularly connected to a vertical channel of the same size on one side. The length of the vertical channel is 0.5 cm, and the end is connected to a cylindrical liquid inlet pool with a diameter of 3 mm and a height of 40 microns;

[0064] The connection point between the end of the secondary crushing section channel and the staining agent channel extends 0.1 cm to the right and then connects to an embedded oblique - linear structure and continues to extend 0.4 cm to the right.

[0065] Counting and observation section: The embedded oblique - linear structure is connected to an annular channel with a width of 40 microns, a height of 40 microns, and a diameter of 0.5 cm. The total angle is 180 degrees (i.e., a semi - circle, i.e., the end is located 1 cm from the starting section on one side, i.e., Figure 4 directly above the center at 1 cm), and a pair of patch electrodes is placed on both sides of the channel every 30 degrees, for a total of 6 pairs.

[0066] The width of the annular channel expands to 120 microns at an angle of 30 degrees to both sides at the end, then extends 0.5 cm, and the end is connected to a cylindrical liquid storage pool with a diameter of 3 mm and a height of 40 microns.

[0067] Bonding the PDMS substrate and the PDMS cover sheet includes the following steps: The substrate liquid inlet pool, mixing channel, crushing channel, separation channel, trapezoidal electrode, patch electrode, annular separation channel, and substrate liquid storage pool on the PDMS substrate are correspondingly aligned and closed.

[0068] A method for fabricating a microfluidic separation chip includes the following steps:

[0069] a) Using the silicon wafer lithography process, all the patterns of the chip substrate are obtained on the silicon wafer. The silicon wafer is etched using ICP plasma etching technology (Inductively Couple Plasma Etch) to obtain the structural shapes of the substrate liquid inlet pool, mixing channel, fragmentation channel, separation channel, trapezoidal electrode, patch electrode, annular separation channel, and substrate liquid storage pool of the PDMS substrate.

[0070] As Figure 2 shown, the lithography and etching process flow of the silicon wafer is as follows:

[0071] Cleaning: The silicon wafer is ultrasonically cleaned with acetone, alcohol, and deionized water for 5 minutes each in sequence, dried with nitrogen, and then baked on a hot plate at 150 °C for 2 - 5 minutes.

[0072] Spin coating: Spin coating is carried out in two steps. In the first step, it rotates at 500 r / min for 15 s - 20 s, and in the second step, it rotates at 4000 r / min for 30 - 35 s.

[0073] Pre - exposure: Ultraviolet exposure for 90 s,

[0074] Post - bake: Bake on a hot plate at 120 °C for 120 s,

[0075] Post - exposure: Ultraviolet exposure for 160 s,

[0076] Development: Develop in the developer for 40 - 45 s,

[0077] Sputtering: The radio - frequency power is 120 W, the vacuum degree is 4×10 4 Pa, the argon gas pressure is 5 mTorr, and the substrate cooling method is water cooling. The sputtering rate is about 16 nm / min, and the sputtering time is 12 min. The thickness of the Cr film is about 100 nm.

[0078] Then, the photoresist is stripped, and an etching machine with the model Oxford Instruments Plasma - lab system 100 is selected for etching using the built - in Bosch process in the system. The built - in inductively coupled plasma coupling parameters are shown in the following table:

[0079]

[0080] b) Using the silicon wafer as a mold, PDMS material is selected for casting on the silicon wafer, and transfer molding is carried out to obtain a PDMS (polydimethylsiloxane) substrate. The structure of the PDMS substrate is as follows: The PDMS substrate is a cuboid, the bottom is a rectangle with side lengths of 6 cm * 3 cm, and the height is 3 mm.

[0081] Mixing section: Two cylindrical liquid inlet pools with a diameter of 3 mm and a height of 40 μm are set at a distance of 1 cm from the left edge of the chip. The two liquid inlet pools are spaced 1 cm apart and parallel to the side edge. A rectangular liquid inlet channel with a length of 1 cm, a width of 30 μm, and a height of 40 μm connects the two liquid inlet pools. At the midpoint of the channel, a mixing channel with a length of 0.5 cm, a width of 60 μm, and a height of 40 μm extends to the right.

[0082] Fragmentation section: The upper and lower sides of the end of the mixing channel contract to 40 μm at a 45-degree angle. Then, there are trapezoidal electrodes on the upper and lower sides of the channel, each with an upper base length of 30 μm, a lower base length of 50 μm, a height of 10 μm, and a thickness of 40 μm. There are 8 on each of the upper and lower sides (the lower base is set close to the channel wall), and the left and right are spaced 200 μm apart. The total length of the fragmentation section channel is 0.5 cm.

[0083] Sorting section: The width of the end of the fragmentation section channel expands downward at an angle of 30 degrees to 80 μm. At 40 μm below the center of the original channel, there is a row of cylinders with a center distance of 10 μm, a bottom diameter of 20 μm, and a height of 40 μm, a total of 11.

[0084] On the lower sides of the sorting section channel, 3 channels with a width of 40 μm, a length of 0.5 cm, and a height of 40 μm are vertically connected. Each of the three channel ends is connected to a cylindrical liquid inlet pool with a diameter of 3 mm and a height of 40 μm.

[0085] Secondary fragmentation section: There are 2 channels connected to the end of the sorting section channel, namely the first channel and the second channel. The 2 channels converge at the end. There are 4 trapezoidal electrodes on each side of the first channel. The cross-sectional dimensions of the trapezoidal electrodes are: an upper base length of 30 μm, a lower base length of 50 μm, a height of 10 μm, and the thickness of the trapezoidal electrode is 40 μm; the lower base is set close to the side wall of the channel (i.e., Figure 2 the upper and lower sides), in the extending direction of the channel (i.e., the left and right direction), the trapezoidal electrodes are spaced 30 μm apart, and the total length of the channel is 500 μm;

[0086] Staining section: The first channel and the second channel are connected at the end of the secondary fragmentation section. At the connection point, it continues to extend 200 μm to the right and is perpendicularly connected to a vertical channel of the same size on one side - the length of the vertical channel is 0.5 cm, and the end is connected to a cylindrical liquid inlet pool with a diameter of 3 mm and a height of 40 μm;

[0087] After extending 0.1 cm to the right from the connection point of the two channels, it is connected to an embedded oblique linear structure and continues to extend 0.4 cm to the right.

[0088] Counting and observation section: The embedded oblique linear structure is connected to an annular channel with a width of 40 μm, a height of 40 μm, and a diameter of 0.5 cm. The total angle is 180 degrees (i.e., the end is 1 cm directly above the starting section), and a pair of patch electrodes is placed on both sides of the channel every 30 degrees, a total of 6 pairs.

[0089] The width of the annular channel expands to 120 microns at an angle of 30 degrees to both sides at the end, then extends for 0.5 cm, and the end is connected to a cylindrical liquid storage pool with a diameter of 3 mm and a height of 40 microns.

[0090] c) Make a cuboid paper box with a bottom of 6 cm * 3 cm rectangle and a height of 3 mm, which has the same size and shape as the PDMS substrate. Glue a solid cylinder model with a diameter of 3 mm and a height of 1 mm at the positions corresponding to the liquid inlet pools of 6 PDMS substrates. Select PDMS material, inject it into the paper box, cure it, and peel it off to obtain the PDMS cover sheet.

[0091] d) Bond the PDMS substrate and the PDMS cover sheet: Place the PDMS substrate and the PDMS cover sheet 3 cm away from a 6-watt low-pressure mercury lamp. After irradiating for 3 hours, drop deionized water on the PDMS substrate and the PDMS cover sheet. Within 1 minute, fit and close the corresponding surfaces of the liquid inlet pool of the PDMS substrate, the separation fulcrum, the substrate separation liquid storage pool, the liquid inlet pool of the PDMS cover sheet, the glass beads, and the cover sheet separation liquid storage pool. After closing, place it in a drying oven and store it at 65 degrees for 2 hours, then take it out and place it in the air for 24 hours for standby.

[0092] The manufacturing process of the PDMS chip is as Figure 3 shown.

[0093] In the present invention, copper wires with a diameter of 50 microns are glued at the positions of 12 breaking electrodes. The length of the copper wires protrudes 1 cm from the edge of the PDMS substrate and is connected to the output pins of the external multi-channel analog control switch chip CD4051.

[0094] Combined with the image acquisition and sensing technology, the cell breaking and sorting / separation conditions in the channel can be monitored in real time, the organelles and cell contents that have been stained and separated inside the counting observation section can be observed more intuitively, and the digital image processing technology can be used to compare the identified images with the database data stored in the computer.

[0095] Calculate the cell breaking rate and determine the types of organelles and cell contents.

[0096] The T-shaped passive mixing structure of the present invention uses a solution inlet pool with a low relative dielectric constant to input the solution and mix it with the cell suspension, and utilizes the dielectric polarization effect to reduce the power consumption when the chip performs the crushing operation. The cylindrical PDMS filtration array can physically sort the suspension after the first crushing, separate the unbroken cells from the organelles and cell contents that escape after crushing, and rely on precisely re-crushing the cells that have failed to be crushed to improve the overall cell crushing rate. The annular separation channel with externally attached patch electrodes uses dielectrophoresis-assisted centrifugal separation technology to separate organelles and cell contents of different masses, facilitating more intuitive observation and statistics. The trapezoidal electrode spacing in the crushing section channel is short, and a voltage of only ~20V can meet the PEF cell crushing requirements, and the flow velocity dead zone caused by the trapezoidal structure is smaller, avoiding the situation of excessive temperature rise. The experiment has high safety and controllability, and low experimental cost.

[0097] Simulation verification results

[0098] Through the COMSOL multi-physics simulation software, each structure of the designed microfluidic chip is segmented and simulated to verify its feasibility.

[0099] Figure 6 (a) shows the simulation result of the electric field intensity distribution in the crushing section when the terminal electrode voltage is 4V. The red area in the figure is the area where the electric field intensity is higher than 1.9 kV / cm, that is, the transmembrane voltage provided by this part of the area can reach the threshold requirement of electroporation and meet the cell crushing requirement. Figure 6 (b) shows the electric field intensity in the axial direction, indicating that when the fluid passes through the crushing section at a certain rate, it is equivalent to being affected by a periodic pulsed electric field, which can effectively reduce the risk of electrolysis of the suspension and damage to the electrodes.

[0100] Figure 7 The particle tracking simulation results in the separation section are shown. It can be seen from the figure that particles with a diameter less than 10 μm can freely pass through from above and below, while particles with a diameter greater than 10 μm will not be able to flow out through the lower channel, verifying the screening effect of the separation section.

[0101] The particle trajectories in the sorting section are as Figure 8 shown. It can be seen that the particles in the figure flow out from the upper outlet relatively uniformly, which can effectively reduce the cell density in the observation area and reduce the overlap of cells under the microscope.

Claims

1. A low-voltage cell disruption microfluidic chip, characterized in that It includes a PDMS substrate and a PDMS cover sheet; It includes a PDMS substrate and a PDMS cover sheet; The PDMS substrate is divided into six parts along the flow direction of the main channel according to the main functions of the main channel and the channels connected thereto: Two liquid inlet pools, a mixing section of a T-shaped micro mixer, a fragmentation section with a plurality of trapezoidal electrodes inserted, a sorting section with an enlarged cross-sectional area and including two rows of columnar obstacles in the channel, a secondary fragmentation section for processing the remaining cells that have not been completely fragmented, and a staining section and a final counting and observation section; The PDMS substrate also includes 6 branch channels connected to the main channel, and the 6 branch channels are respectively connected to 1 cell suspension liquid inlet pool, 1 low relative dielectric constant solution inlet pool, 3 cleaning liquid inlet pools and 1 dye inlet pool; Both the PDMS substrate and the PDMS cover sheet are square sheets, with a bottom surface being a rectangle of 6 cm * 3 cm and a thickness of 3 mm; Mixing section: At a distance of 1 cm from the left edge of the chip, two cylindrical liquid inlet pools with a diameter of 3 mm and a depth of 40 μm are provided. The two liquid inlet pools are spaced 1 cm apart and parallel to the side edge; A cuboid liquid inlet channel with a length of 1 cm, a width of 30 μm and a depth of 40 μm connects the two liquid inlet pools, and a mixing channel with a length of 0.5 cm, a width of 60 μm and a depth of 40 μm extends to the right from the midpoint of the channel; Fragmentation section: The two sides at the end of the mixing channel shrink from 60 μm to 40 μm at a 45-degree angle. On each side of the front end of the fragmentation section channel, 8 trapezoidal electrodes are provided. The cross-sectional dimensions of the trapezoidal electrodes are: the upper base length is 30 μm, the lower base length is 50 μm, the height is 10 μm, and the thickness of the trapezoidal electrodes is 40 μm; The lower bottom edge is closely attached to the side wall of the channel. In the extending direction of the channel, the trapezoidal electrodes are spaced 30 μm apart from each other, and the total length of the fragmentation section channel is 0.5 cm; Sorting section: The width of the end of the fragmentation section channel expands from 40 μm to 80 μm to the right at a 30-degree angle. Taking a point 55 μm behind the starting point of the channel expansion as the axis, a cylinder with a bottom diameter of 20 μm and a height of 40 μm is set; Another row of 14 cylinders is provided along the extending direction of the channel in the sorting section, and the adjacent cylinders are spaced 10 μm apart; The 15 cylinders are used to separate the unbroken cells; One side of the sorting section channel is vertically connected with 3 channels with a width of 40 μm, a length of 0.5 cm and a height of 40 μm, and the three channels are spaced 110 μm apart from each other. Each end of the three channels is connected to a cylindrical liquid inlet pool with a diameter of 3 mm and a height of 40 μm; The end of the sorting section channel is connected to two straight channels: both channels have a width of 40 μm. One of the channels, i.e., the straight channel, has no angular offset, and the other channel, i.e., the deflection channel, has an angle of 30 degrees with the extending direction; Secondary fragmentation section: It has 2 channels that dock with the end of the sorting section channel, namely the first channel and the second channel. These 2 channels converge at the end. There are 4 trapezoidal electrodes on each side of the first channel. The cross-sectional dimensions of the trapezoidal electrode are: the upper base length is 30 microns, the lower base length is 50 microns, the height is 10 microns, and the thickness of the trapezoidal electrode is 40 microns; the lower bottom edge is closely attached to the side wall of the channel. In the extending direction of the channel, the trapezoidal electrodes are spaced 30 microns apart from each other, and the total length of the channel is 500 microns. Dyeing section: The first channel and the second channel are connected at the end of the secondary fragmentation section. The joint continues to extend 200 microns to the right and is perpendicularly connected to a vertical channel of the same size on one side. The length of the vertical channel is 0.5 cm, and the end is connected to a cylindrical liquid inlet pool with a diameter of 3 mm and a height of 40 microns. The joint of the end of the secondary fragmentation section channel and the dyeing agent channel extends 0.1 cm to the right and then connects to an embedded oblique linear structure. The width of the groove structure is 20 microns, the depth is 20 microns, the inclination angle is 45°, the spacing between each two is 40 microns, and there are 6 in total, and it continues to extend 0.4 cm to the right. Counting and observation section: The embedded oblique linear structure is connected to an annular channel with a width of 40 microns, a height of 40 microns, and a diameter of 0.5 cm. The total angle is 180 degrees, and a pair of patch electrodes are placed on both sides of the channel every 30 degrees, with a total of 6 pairs. The width of the annular channel expands to 120 microns at an angle of 30 degrees to both sides at the end, and then extends 0.5 cm. The end is connected to a cylindrical liquid storage pool with a diameter of 3 mm and a height of 40 microns.

2. A manufacturing method of a microfluidic separation chip, characterized in that, It includes the following steps: a) Using silicon wafer lithography technology, all the patterns of the chip substrate are obtained on the silicon wafer, and the silicon wafer is etched using ICP plasma etching technology to obtain the structural shapes of the substrate liquid inlet pool, mixing channel, fragmentation channel, separation channel, trapezoidal electrode, patch electrode, annular separation channel, substrate liquid storage pool, and counting and observation area. b) Using the silicon wafer as a mold, selecting PDMS material to pour on the silicon wafer and perform transfer molding to obtain the PDMS substrate. c) Making a cuboid paper box with a bottom of 6 cm * 3 cm rectangle and a height of 3 mm, and gluing a solid cylinder model with a diameter of 3 mm and a height of 1 mm at the positions corresponding to a total of 6 PDMS substrate liquid inlet pools respectively. Selecting PDMS material to inject into the paper box, curing, and peeling to obtain the PDMS cover sheet. d) Bonding the PDMS substrate and the PDMS cover sheet: Placing the PDMS substrate and the PDMS cover sheet 3 cm away from a 6-watt low-pressure mercury lamp and irradiating for 3 hours. Then dropping deionized water on the PDMS substrate and the PDMS cover sheet. Within 1 minute, the corresponding surfaces of the substrate liquid inlet pool, mixing channel, fragmentation channel, separation channel, trapezoidal electrode, patch electrode, annular separation channel, and substrate liquid storage pool of the PDMS substrate are fitted and closed, and then placed in a drying oven at 65 degrees for 2 hours, and then taken out and placed in the air for 24 hours for standby. The chip described above is the low-voltage cell fragmentation microfluidic chip described in claim 1.

3. The manufacturing method of the microfluidic separation chip according to claim 2, characterized in that Input the solution and cell suspension into the mixing chamber using a low relative dielectric constant solution inlet pool, and utilize the dielectric polarization effect to reduce the power consumption of the chip during operation.

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