Micro-fluidic chip for accurately controlling and sorting cells based on bipolar electrode and use method of micro-fluidic chip

Abstract

The invention discloses a micro-fluidic chip for accurately controlling and sorting cells based on a bipolar electrode and a use method of the micro-fluidic chip, and relates to a micro-fluidic chip for accurately controlling and sorting cells and a use method of the micro-fluidic chip. The invention aims to solve the problem that the regulation and control of cells and microparticles by the existing dielectrophoresis depends on a multi-electrode array and complex driving. The micro-fluidic chip consists of a PDMS (Polydimethylsiloxane) cover plate and an ITO (Indium Tin Oxide) glass substrate, a cavity structure is arranged in the center of the PDMS cover plate, and a square suspension electrode is arranged in the center of the ITO glass substrate; the four vertex angles of the square suspension electrode are respectively connected with the four curve electrodes, and the four excitation electrodes are arranged around the square suspension electrode. The use method comprises the following steps: continuously adjusting the voltage and phase of the sine alternating current signal applied to the square suspension electrode, continuously adjusting the frequencies of the sine alternating current signal applied to the square suspension electrode and the four paths of same-frequency sine signals, and keeping the frequencies consistent, so as to realize cell or microparticle control in the square suspension electrode area.

Description

TECHNICAL FIELD

[0001] The present application relates to microfluidic chips for precise manipulation and sorting of cells and methods of using the same. BACKGROUND

[0002] Achieving precise and predictable manipulation (including capture, transport, positioning, and trajectory calibration) of single cells or microparticles has fundamental significance for understanding cell heterogeneity and intercellular interactions. The core of particle manipulation lies in the precise regulation of the motion behavior of particles at the microscale and their microfluidic environment. In recent years, it has become a hot interdisciplinary research field. With the continuous progress of micro-nano processing and fluid mechanics theory, this technology has achieved a leap from passive screening to active control, enabling the capture, migration, rotation, and arrayed arrangement of single cells or microparticles without macroscopic intervention, with extremely high spatial resolution and operation throughput, significantly improving the automation level of biological analysis processes. By integrating complex manipulation functions into a miniaturized platform, researchers can perform highly reproducible experiments in microliter or even nanoliter samples, greatly reducing reagent consumption and improving detection sensitivity, while supporting biomedical applications such as targeted therapy, drug delivery, and organ chips.

[0003] The manipulation strategies for cells and microparticles can be broadly divided into passive manipulation and active manipulation. Passive manipulation does not require external field excitation and mainly relies on the coupling of microchannel geometry and fluid flow to guide particles to migrate and rearrange in the channel. For example, by using structures such as spiral channels and wave-shaped channels, inertial lift, secondary flow, or oscillatory strain effects can be used to achieve differential sorting and position control of particles. This method is relatively simple in structure and easy to process, but the manipulation effect is highly dependent on the given geometric parameters, and once the design is determined, the flexibility is insufficient, and the scope of application is also easily limited.

[0004] Active manipulation uses external excitation fields to generate controllable external forces, achieving more efficient, precise, and real-time switchable manipulation. Common external fields include chemical, optical, acoustic, magnetic, and electrical fields. Chemical driving can achieve propulsion or self-electrophoresis by using reaction bubbles, but it is difficult to terminate immediately after the reaction is started, and the reversibility and real-time scheduling capability are weak. Optical manipulation relies on optical gradient force to achieve non-contact fine control, with high precision, but the system integration is complex, the cost is high, and local photo-thermal effects may damage cell activity. Acoustic manipulation uses ultrasonic standing waves to generate acoustic radiation force, which can achieve fast and label-free manipulation with relatively small impact on activity, but it usually requires precise design of transducer and channel structure and strict matching of sound field parameters, which has a high threshold for device implementation. Magnetic driving has the advantages of fast response and low damage, but it is difficult to directly act on most non-magnetic cells and conventional particles, often requiring magnetic labeling, which increases cost and sample preparation cycle.

[0005] In contrast, electrically driven manipulation has the characteristics of external power supply, on-demand switching, and easy adjustment of parameters, and can be flexibly controlled by adjusting the voltage, frequency and phase of the electric signal. Existing researches realize electrically controlled capture and scheduling based on induced charge electrodynamics and dielectric effect, but some schemes rely on ferroelectric materials or Janus particles, which are complex to prepare. As a typical label-free electric manipulation technology, dielectrophoresis drives particles to migrate using the polarization effect in a non-uniform electric field, and has been verified in capture, release and single particle transmission. However, in terms of real-time programmable control, many dielectrophoretic force systems still rely on multi-electrode arrays and complex driving schemes, thereby increasing the system cost and implementation difficulty. SUMMARY

[0006] The present application aims to solve the problem that existing dielectrophoresis relies on multi-electrode arrays and complex driving for the control of cells and microparticles, and further provides a microfluidic chip for precise manipulation and sorting of cells based on bipolar electrodes and a method for using the same.

[0007] A microfluidic chip for precise manipulation and sorting of cells based on bipolar electrodes, which is composed of a PDMS cover sheet and an ITO glass substrate.

[0008] The center of the PDMS cover sheet is provided with a chamber structure penetrating through the PDMS cover sheet.

[0009] The center of the ITO glass substrate is provided with a square suspension electrode; the four corners of the square suspension electrode are connected with a first curved electrode, a second curved electrode, a third curved electrode and a fourth curved electrode, respectively.

[0010] A first excitation electrode is arranged behind the square suspension electrode, a second excitation electrode is arranged on the left side of the square suspension electrode, a third excitation electrode is arranged in front of the square suspension electrode, and a fourth excitation electrode is arranged on the right side of the square suspension electrode.

[0011] The distance between the front end of the first, second, third and fourth excitation electrodes and the side of the square suspension electrode is 100-110 μm.

[0012] The width of the front end of the first, second, third and fourth excitation electrodes is 20-30 μm.

[0013] The side of the ITO glass substrate provided with the electrodes is sealed against the lower surface of the PDMS cover sheet, and the square suspension electrode is arranged at the center of the chamber structure, which contains the front end of the first, second, third and fourth excitation electrodes.

[0014] The application relates to a method for using a bipolar electrode-based microfluidic chip for precise cell manipulation and sorting, which is performed in the following steps:

[0015] ① Turn on the computer connected with the microscope, the signal generator, the signal amplifier, the oscilloscope, the microscope, the CCD and the fluorescent lamp switch, observe whether the equipment is normal, then turn on the ImageView image acquisition software on the computer, and observe the microscope carrier table in real time; the signal generator comprises a first signal generator, a second signal generator and a third signal generator;

[0016] ② Fix the bipolar electrode-based microfluidic chip for precise cell manipulation and sorting on the carrier table, and adjust the chip position and focal length;

[0017] ③ The first signal generator and the second signal generator provide four same-frequency sinusoidal alternating signals for the first excitation electrode, the second excitation electrode, the third excitation electrode and the fourth excitation electrode; the third signal generator provides a single sinusoidal alternating signal for the square suspension electrode;

[0018] ④ Observe under the microscope, and inject a cell / particle suspension into the chamber structure through a pipette;

[0019] ⑤ Start the first signal generator and the second signal generator, so that the surface of the first excitation electrode, the second excitation electrode, the third excitation electrode and the fourth excitation electrode is subjected to a counterclockwise rotating four-phase electric field, and the phase values are 0°, 90°, 180° and 270° respectively; when the cell or the micro-particle in the chip is stable, the third signal generator is started;

[0020] The cell / particle suspension is one or a combination of several of PS microsphere suspension, yeast cell suspension, 293T cell suspension and red blood cell suspension; the conductivity of the cell / particle suspension is 8 mS / m-48 mS / m;

[0021] ⑥ Observe under the microscope, and adjust the chip position and focal length again until the cell or the micro-particle is clear, and then detect and record the video at a stable height;

[0022] ⑦ Repeat steps ④ to ⑥, and continuously adjust the voltage and phase of the sinusoidal alternating signal applied to the square suspension electrode, continuously adjust the frequency of the sinusoidal alternating signal applied to the square suspension electrode and the four same-frequency sinusoidal alternating signals, and the frequency is kept consistent, so that the cell or the micro-particle in the square suspension electrode area is manipulated, the experimental phenomenon is observed and recorded;

[0023] ⑧ Process and analyze experimental data.

[0024] The application has the beneficial effects that:

[0025] This invention proposes a rotating electric field microfluidic manipulation and sorting chip based on bipolar electrodes. A rotating electric field is formed by applying four-phase signals with a 90-degree phase difference around the microchamber, and a square levitation electrode is integrated at the center. Cells or microparticles on the surface of the levitation electrode are simultaneously subjected to the synergistic effect of a dielectric potential trap and an induced current induced by electroosmosis, achieving contactless, label-free capture, stationary positioning, and directional transport. Furthermore, by adjusting the phase, voltage, and frequency of the AC signal on the levitation electrode, the position of the stationary point and the migration direction can be reconstructed, thereby achieving programmable trajectory control. Sorting areas are delineated on the levitation electrode, and regional sorting is achieved by utilizing the velocity / positioning differences caused by the dielectric properties of different objects. The feasibility of this scheme, combined with simulation analysis using COMSOL 6.2 multiphysics coupling software, has been verified in PS microspheres, yeast cells, erythrocytes, and 293T cell systems. The device fabrication is compatible with standard photolithography processes, the electrode structure is simple and reusable, and it is easy to scale up in parallel to increase throughput. Attached Figure Description

[0026] Figure 1 This is a schematic diagram of the microfluidic chip for precise cell manipulation and sorting based on bipolar electrodes according to the present invention.

[0027] Figure 2 The preparation process of the ITO glass substrate in Example 1 is as follows;

[0028] Figure 3 The image shows a physical diagram of the microfluidic chip for precise cell manipulation and sorting based on bipolar electrodes, as well as a microscopic diagram of the electrode structure (scale bar: 160µm).

[0029] Figure 4 The diagrams show the trajectory migration of PS microspheres or yeast cells controlled in Example 1. (a) A schematic diagram and overlay of the trajectory migration of PS microspheres under the conditions of f=30kHz, σ=0.008S / m, levitation electrode voltage of 5V, and sequential changes in levitation electrode phase to 335°, 225°, 30° and 135°. (b) A schematic diagram and overlay of the trajectory migration of yeast cells under the conditions of f=50kHz, σ=0.008S / m, levitation electrode voltage of 4V, and sequential changes in levitation electrode phase to 180°, 90°, 0°, 270° and 180° (scale bar: 55µm).

[0030] Figure 5Schematic diagram for controlling the trajectory migration of PS microspheres and yeast cells, 293T cells and red blood cells in Example 1, (a) PS microspheres and yeast cells were sorted under the conditions of f = 100 kHz, σ = 0.008 S / m, the suspension electrode voltage was 5 V, and the suspension electrode phase was changed to 0° and 180° in turn; (b) 293T cells and red blood cells were sorted under the conditions of f = 40 kHz, σ = 0.008 S / m, the suspension electrode voltage was 4 V, and the phase was changed to 180° and 0° in turn (scale bar: 55 µm). DETAILED DESCRIPTION

[0031] DETAILED DESCRIPTION Figure 1 The specific embodiment is described as follows: a microfluidic chip for precise manipulation and sorting of cells based on bipolar electrodes, which is composed of a PDMS cover sheet 3 and an ITO glass substrate 11.

[0032] The center of the PDMS cover sheet 3 is provided with a chamber structure 7 penetrating through the PDMS cover sheet 3.

[0033] The center of the ITO glass substrate 11 is provided with a square suspension electrode 1; the four corners of the square suspension electrode 1 are connected with a first curved electrode 4, a second curved electrode 6, a third curved electrode 9 and a fourth curved electrode 12, respectively.

[0034] A first excitation electrode 2 is arranged behind the square suspension electrode 1; a second excitation electrode 10 is arranged on the left side of the square suspension electrode 1; a third excitation electrode 8 is arranged in front of the square suspension electrode 1; and a fourth excitation electrode 5 is arranged on the right side of the square suspension electrode 1.

[0035] The distance between the front end of the first excitation electrode 2, the second excitation electrode 10, the third excitation electrode 8 and the fourth excitation electrode 5 and the side of the square suspension electrode 1 is 100 µm~110 µm.

[0036] The width of the front end of the first excitation electrode 2, the second excitation electrode 10, the third excitation electrode 8 and the fourth excitation electrode 5 is 20 µm~30 µm.

[0037] The side of the ITO glass substrate 11 provided with the electrodes is sealed against the lower surface of the PDMS cover sheet 3, and the square suspension electrode 1 is arranged at the center of the chamber structure 7, which contains the front end of the first excitation electrode 2, the second excitation electrode 10, the third excitation electrode 8 and the fourth excitation electrode 5.

[0038] The embodiment uses the principle of induced charge electroosmosis of fixed potential and the principle of dielectrophoresis to design a microfluidic chip for cell or microparticle manipulation and sorting under a bipolar electrode. Compared with other microfluidic manipulation chips based on bipolar electrodes, the chip design based on induced charge electroosmosis of fixed potential changes the flow field by introducing a suspended electrode, and can realize the manipulation of cells or microparticles without external contact and labeling, and the manipulation is convenient. The chip has low cost, simple processing, and small applied voltage, and can not only realize efficient manipulation but also has biological adaptability, which is beneficial to cell delivery and other medical applications. The simulation comparison and the experiment have good consistency.

[0039] The embodiment has the following advantages:

[0040] The embodiment proposes a rotating electric field microfluidic manipulation and sorting chip based on a bipolar electrode: four-phase signals with a phase difference of 90 degrees are applied around the microcavity to form a rotating electric field, and a square suspended electrode is integrated in the center, so that the cells or microparticles are simultaneously subjected to the synergistic action of dielectrophoresis potential well and induced charge electroosmosis induced flow on the surface of the suspended electrode, realizing contactless and label-free capture, fixed-point parking and directional transportation; further, by adjusting the phase, voltage and frequency of the suspended electrode AC signal, the position of the stagnation point and the migration direction can be reconfigured, so that programmable trajectory control can be realized, and a sorting area is defined on the suspended electrode, and regional sorting is realized by using the speed / position difference caused by the dielectric properties of different objects. The scheme has been verified in PS microspheres, yeast cells, red blood cells and 293T cell systems through COMSOL 6.2 multi-physical field coupling software simulation analysis to verify the feasibility of manipulation and sorting; the device preparation is compatible with standard photolithography process, the electrode structure is simple and reusable, and it is easy to parallel and enlarge to improve the throughput.

[0041] Specific embodiment two: the difference between the embodiment and specific embodiment one is that the square suspended electrode 1, the first curve electrode 4, the second curve electrode 6, the third curve electrode 9, the fourth curve electrode 12, the first excitation electrode 2, the second excitation electrode 10, the third excitation electrode 8 and the fourth excitation electrode 5 are obtained by etching the ITO conductive film on the surface of the ITO glass substrate 11. The others are the same as specific embodiment one.

[0042] Specific embodiment three: the difference between the embodiment and specific embodiment one or two is that the thickness of the square suspended electrode 1, the first curve electrode 4, the second curve electrode 6, the third curve electrode 9, the fourth curve electrode 12, the first excitation electrode 2, the second excitation electrode 10, the third excitation electrode 8 and the fourth excitation electrode 5 is 150nm-200nm. The others are the same as specific embodiment one or two.

[0043] Specific embodiment four: the difference between this embodiment and one of the specific embodiments one to three is that the side length of the square suspension electrode 1 is 200 μm ~ 300 μm; the width of the front end of the first curved electrode 4, the second curved electrode 6, the third curved electrode 9 and the fourth curved electrode 12 is 20 μm ~ 30 μm. The others are the same as specific embodiments one to three.

[0044] Specific embodiment five: the difference between this embodiment and one of the specific embodiments one to four is that the thickness of the PDMS cover sheet 3 and the depth of the chamber structure 7 are both 5 mm ~ 10 mm. The others are the same as specific embodiments one to four.

[0045] Specific embodiment six: the difference between this embodiment and one of the specific embodiments one to five is that the bottom surface of the chamber structure 7 is circular, square or elliptical; the bottom surface area is 0.8 cm 2 ~1.6 cm 2 . The others are the same as specific embodiments one to five.

[0046] Specific embodiment seven: a method for using a microfluidic chip based on bipolar electrode cell precise manipulation and sorting, which is carried out in the following steps:

[0047] ① Turn on the computer connected with the microscope, signal generator, signal amplifier, oscilloscope, microscope, CCD and fluorescent lamp switch, observe whether the equipment is running normally, then turn on the ImageView image acquisition software on the computer, and observe the microscope stage in real time; the signal generator includes a first signal generator, a second signal generator and a third signal generator;

[0048] ② Fix the microfluidic chip based on bipolar electrode cell precise manipulation and sorting on the stage, adjust the chip position and focal length;

[0049] ③ The first signal generator and the second signal generator provide 4-way same frequency sinusoidal alternating current signals for the first excitation electrode 2, the second excitation electrode 10, the third excitation electrode 8 and the fourth excitation electrode 5, and the third signal generator provides a single sinusoidal alternating current signal for the square suspension electrode 1;

[0050] ④ Observe under the microscope, inject cell / particle suspension into the chamber structure 7 through the pipette;

[0051] ⑤ Start the first signal generator and the second signal generator, so that the surface of the first excitation electrode 2, the second excitation electrode 10, the third excitation electrode 8 and the fourth excitation electrode 5 applies a counterclockwise rotating four-phase electric field, and the phase values are 0°, 90°, 180° and 270° respectively, and when the cell or micro-particle in the chip stays stable, start the third signal generator;

[0052] The cell / particle suspension is one or a combination of several of PS microsphere suspension, yeast cell suspension, 293T cell suspension and red blood cell suspension; the conductivity of the cell / particle suspension is 8 mS / m to 48 mS / m;

[0053] ⑥Under a microscope, the chip position and focal length are adjusted again until the cells or micro-particles are clear, and the video detection and recording are performed at a stable height;

[0054] ⑦Steps ④ to ⑥ are repeated, and the voltage and phase of the sinusoidal alternating signal applied to the square suspension electrode 1 are continuously adjusted, and the frequency of the sinusoidal alternating signal applied to the square suspension electrode 1 and the 4-way sinusoidal alternating signal of the same frequency is continuously adjusted and kept consistent, so as to realize the manipulation of the cells or micro-particles in the square suspension electrode 1 region, and the experimental phenomena are observed and recorded;

[0055] ⑧Processing and analysis of experimental data.

[0056] In this embodiment, four excitation electrodes are symmetrically arranged along the periphery of the chamber structure 7, and a square suspension electrode 1 is arranged in the center region of the electric field. The square suspension electrode 1 is symmetrically connected to external lead wires at four corners for applying a sinusoidal alternating signal. By adjusting the voltage, frequency and phase of the sinusoidal alternating signal of the suspension electrode, the charging and discharging process of the induced ζ potential on the surface of the suspension electrode can be actively changed, and then a controllable asymmetric induced charge electroosmotic micro-vortex is generated in the neighborhood of the electrode, which cooperates with the dielectrophoresis force induced by the non-uniform electric field. By changing the phase signal of the suspension electrode, the migration direction of the cells or micro-particles can be controlled, and by changing the voltage signal of the suspension electrode, the migration distance of the cells or micro-particles can be controlled. The combination of the two can realize the free movement of the cells or micro-particles on the surface of the suspension electrode.

[0057] In this embodiment, all signal generators are connected through an external clock signal to realize synchronization. The 10MHz Out of the host and the 10MHz In of the slave are connected together, and the Aux In / Out of the three signal generators are connected together, which are used for synchronous clock signal, to ensure the coherence of the alternating signals applied to the excitation electrodes and the suspension electrode in the time domain.

[0058] In this embodiment, the first signal generator, the second signal generator and the third signal generator in step ③ are synchronous double-output signal generators. The other steps are the same as those in embodiment seven.

[0059] Specific implementation nine: the difference between this embodiment and one of specific implementation seven or eight is that: the concentration of the PS microsphere suspension in step 5 is 500-1000 / μL; the concentration of the yeast cell suspension in step 5 is 500-1000 / μL; the concentration of the 293T cell suspension in step 5 is 200-500 / μL; the concentration of the red blood cell suspension in step 5 is 200-500 / μL. The others are the same as specific implementation seven or eight.

[0060] Specific implementation ten: the difference between this embodiment and one of specific implementation seven to nine is that: the conductivity of the cell / particle suspension in step 5 is adjusted by adding KCl. The others are the same as specific implementation seven to nine.

[0061] The beneficial effects of the present application are verified by the following examples:

[0062] Example one:

[0063] A microfluidic chip for precise manipulation and sorting of cells based on bipolar electrodes, which is composed of a PDMS cover sheet 3 and an ITO glass substrate 11;

[0064] The center of the PDMS cover sheet 3 is provided with a chamber structure 7 penetrating through the PDMS cover sheet 3;

[0065] The center of the ITO glass substrate 11 is provided with a square suspension electrode 1; the four corners of the square suspension electrode 1 are connected with a first curved electrode 4, a second curved electrode 6, a third curved electrode 9 and a fourth curved electrode 12, respectively;

[0066] A first excitation electrode 2 is arranged behind the square suspension electrode 1; a second excitation electrode 10 is arranged on the left side of the square suspension electrode 1; a third excitation electrode 8 is arranged in front of the square suspension electrode 1; and a fourth excitation electrode 5 is arranged on the right side of the square suspension electrode 1;

[0067] The distance between the front end of the first excitation electrode 2, the second excitation electrode 10, the third excitation electrode 8 and the fourth excitation electrode 5 and the side of the square suspension electrode 1 is 100 μm;

[0068] The width of the front end of the first excitation electrode 2, the second excitation electrode 10, the third excitation electrode 8 and the fourth excitation electrode 5 is 200 μm;

[0069] The ITO glass substrate 11 is sealed with the side provided with electrodes and the lower surface of the PDMS cover 3, and the square suspended electrode 1 is placed in the center of the chamber structure 7, which contains the front end of the first excitation electrode 2, the second excitation electrode 10, the third excitation electrode 8 and the fourth excitation electrode 5.

[0070] The square suspended electrode 1, the first curved electrode 4, the second curved electrode 6, the third curved electrode 9, the fourth curved electrode 12, the first excitation electrode 2, the second excitation electrode 10, the third excitation electrode 8 and the fourth excitation electrode 5 are all obtained by etching the ITO conductive film on the surface of the ITO glass substrate 11.

[0071] The thickness of the square suspended electrode 1, the first curved electrode 4, the second curved electrode 6, the third curved electrode 9, the fourth curved electrode 12, the first excitation electrode 2, the second excitation electrode 10, the third excitation electrode 8 and the fourth excitation electrode 5 is 200 nm.

[0072] The length of the side of the square suspended electrode 1 is 200 μm; the width of the front end of the first curved electrode 4, the second curved electrode 6, the third curved electrode 9 and the fourth curved electrode 12 is 30 μm.

[0073] The thickness of the PDMS cover 3 and the depth of the chamber structure 7 are both 10 mm.

[0074] The bottom surface of the chamber structure 7 is elliptical; the area of the bottom surface is 0.8 cm 2 .

[0075] The specific preparation method of the above-mentioned microfluidic chip based on bipolar electrode cell precise manipulation and sorting is as follows:

[0076] I. Preparation method of the PDMS cover 3: first, pretreat the glass, wash the surface of the glass with IPA, then wash the residual IPA with deionized water, then blow off the residual deionized water on the surface with a nitrogen gun, then heat the glass on a 100℃ electric heating plate for 15 min, take the glass off the electric heating plate, and cool it at room temperature for 1 min to obtain a glass substrate; use a clean glass substrate as a mold, then pour the PDMS. Mix the PDMS and the curing agent at a mass ratio of 10:1, stir them evenly, then pour them into a culture dish containing the mold, place the culture dish in a vacuum drying box, and vacuumize it to-0.07 MPa, wait for 30 min to remove the bubbles. Then place the culture dish in an electric heating air drying oven, heat it at 75℃ for 4 h to obtain the cured PDMS. After curing, use a scalpel to peel off the whole PDMS, and trim it into a regular shape according to the experimental requirements, then use a scalpel to cut through the central part of the PDMS block to obtain the PDMS cover 3.

[0077] II. ITO glass substrate 11 refers to the glass with tin oxide ITO film plated on the surface of the glass. Although its conductivity is slightly lower than that of gold electrode, it is lower in cost, so it will be used for electric field manipulation with ITO electrode, such as Figure 2 As shown in the specific process is as follows:

[0078] (1) ITO glass pretreatment and uniform coating: first, the ITO glass is pretreated, the surface is washed with IPA, then the residual IPA is washed with deionized water, then the surface residual deionized water is blown dry with nitrogen gun, and finally the ITO glass is placed on the 100℃ electric heating plate for heating for 15min. After drying the moisture, the ITO glass is taken off from the electric heating plate and cooled to room temperature; use a dropper to drop AZ4620 photoresist on the glass center, then place the glass on the center of the rotor of the uniform coating machine, first run at 500rpm for 10s, then run at 4000rpm for 30s, to form a photoresist layer with a thickness of about 6μm~10μm.

[0079] (2) pre-baking and exposure: when pre-baking, the ITO glass is placed on the 100℃ electric heating plate for heating for 1.5min, then taken off and cooled to room temperature. When exposure, the ITO glass is fixed on the platform of the photoetch machine and fixed with vacuum suction, the film mask is covered on the surface of the ITO glass, and the clean electronic grade glass is lightly pressed to ensure that the mask is tightly attached to the photoresist layer, then the exposure time is set to 8s~9s to complete the exposure.

[0080] (3) development, cleaning and post-baking: the developer for AZ4620 needs to be diluted with deionized water according to the volume ratio of 1:3, the prepared developer is poured into the culture dish with ITO glass, and the culture dish is shaken gently or the glass is shaken with tweezers for about 1.5min. After development, the ITO glass is taken out, the surface residual developer is washed with deionized water, and dried with nitrogen. Finally, the ITO glass is placed on the 120℃ hot plate for heating for 1min to make the photoresist stable, and then taken off and cooled to room temperature.

[0081] (4) ITO glass etching and photoresist stripping: first, prepare the etching solution, dilute hydrochloric acid to 60%(v / v) concentration with deionized water, and add 1mg iron oxide powder as catalyst to promote etching reaction. Put the ITO glass into the culture dish with prepared etching solution and etch for 30min; after etching, take it out, wash it thoroughly with deionized water and dry it with nitrogen; finally, immerse the etched ITO glass in IPA solution for 2min and shake it gently, take it out, wash it with deionized water and dry it with nitrogen, to obtain ITO glass substrate 11. At this time, only the electrode pattern part is conductive, and the rest is not conductive.

[0082] III. After the PDMS cover 3 and ITO glass substrate 11 are prepared, oxygen plasma treatment is used to form irreversible covalent bonds between PDMS and glass to form a firm bond. The specific process is as follows:

[0083] (1) Surface treatment: Put the PDMS cover 3 and ITO glass substrate 11 into the plasma cleaning machine, close the bonding machine valve and open the air pump until it is close to vacuum. Open the oxygen bottle switch and introduce appropriate amount of oxygen into the bonding machine for 30s, and the red glow appears in the cavity for 1.5min~2min. After treatment, the hydrophilicity of PDMS and substrate will be significantly enhanced.

[0084] (2) Move the bonding: After taking out, place the ITO glass under the microscope, add a small amount of water on the electrode surface and align the electrode with the chamber. After waiting for the water to evaporate, place the chip on the 60℃ hot plate for 12h to complete the bonding. After heating, observe the bonding effect. If the effect is poor, apply AB glue around the PDMS to ensure that the suspension does not leak.

[0085] Finally, use conductive tape to connect the ITO electrode with the lead wire, and use an external table to test the conductivity of all electrodes, and the four pins of the suspended electrode ensure that they are conductive to each other, ensuring that the electric field distribution formed is symmetrical.

[0086] The above method for using the microfluidic chip for precise manipulation and sorting of cells based on bipolar electrodes is carried out according to the following steps:

[0087] ① Turn on the computer connected to the microscope, signal generator, signal amplifier, oscilloscope, microscope, CCD and fluorescent lamp switch, observe whether the equipment is running normally, then turn on the ImageView image acquisition software on the computer, and observe the microscope stage in real time; the signal generator includes a first signal generator, a second signal generator and a third signal generator;

[0088] ② Fix the microfluidic chip for precise manipulation and sorting of cells based on bipolar electrodes on the stage, adjust the chip position and focal length;

[0089] ③ The first signal generator and the second signal generator provide 4-way same frequency sinusoidal alternating current signals for the first excitation electrode 2, the second excitation electrode 10, the third excitation electrode 8 and the fourth excitation electrode 5, and the third signal generator provides a single sinusoidal alternating current signal for the square suspended electrode 1;

[0090] ④ Observe under the microscope, inject the particle suspension into the chamber structure 7 by the pipette until the height of the particle suspension is 100μm;

[0091] ⑤ Start the first signal generator and the second signal generator so that the electrical signal applied to the surface of the first excitation electrode 2, the second excitation electrode 10, the third excitation electrode 8 and the fourth excitation electrode 5 is a four-phase electric field rotating counterclockwise, with phase values ​​of 0°, 90°, 180° and 270° respectively. After the position of the particle suspension in the chip stabilizes, start the third signal generator.

[0092] The particulate suspension is a mixture of PS microsphere suspension, yeast cell suspension, PS microsphere suspension and yeast cell suspension, and 293T cell suspension and red blood cell suspension; the conductivity of the particulate suspension is 0.008 S / m.

[0093] ⑥ Observe under a microscope, and readjust the chip position and focus until the cells or microparticles are clear and the height is stable for video detection and recording;

[0094] ⑦ Repeat steps ④ to ⑥, and continuously adjust the voltage and phase of the sinusoidal AC signal applied to the square suspended electrode 1, and continuously adjust the frequency of the sinusoidal AC signal applied to the square suspended electrode 1 and the four sinusoidal AC signals of the same frequency, and keep the frequency consistent, so as to achieve cell or microparticle manipulation in the area of ​​the square suspended electrode 1, observe the experimental phenomena and record them.

[0095] ⑧ Processing and analysis of experimental data.

[0096] In step ③, the first signal generator, the second signal generator, and the third signal generator are synchronous dual-output signal generators.

[0097] The concentration of the PS microsphere suspension mentioned in step ⑤ is 500 particles / μL to 1000 particles / μL. Specifically, it is prepared according to the following steps: potassium chloride is added to deionized water to obtain a solution with a conductivity of 8 mS / m. Then, the solution with a conductivity of 8 mS / m is mixed with 10 μm PS microspheres and shaken for 5 minutes using an ultrasonic cleaner.

[0098] The concentration of the yeast cell suspension mentioned in step ⑤ is 500 cells / μL to 1000 cells / μL. Specifically, it is prepared according to the following steps: 50 mg of bread yeast powder is poured into a culture dish, 10 mL of deionized water is added, and the mixture is activated at 30 °C for 30 min. After activation, 2 mL of the yeast suspension is transferred to a centrifuge tube and washed three times. Then, the yeast cells are transferred to a solution with a fixed conductivity of 8 mS / m. The conductivity of the solution is adjusted by adding KCl to the deionized water.

[0099] The mixture of the PS microsphere solution and the yeast cell suspension in step ⑤ is specifically a mixture of the PS microsphere solution with a concentration of 500-1000 / μL and the yeast cell suspension with a concentration of 500-1000 / μL in a volume ratio of 1:1.

[0100] The concentration of the 293T cell suspension in step ⑤ is 200-500 / μL, and it is prepared by the following steps: culturing 293T cells in a DEME medium at 37°C and in a 5% carbon dioxide environment; the DEME medium is specifically a mixture of DEME medium, 10% fetal bovine serum, 1.5 g / L sodium pyruvate, 4.5 g / L glutamine, 1% penicillin-streptomycin double antibody, and 0.05 mM β-mercaptoethanol, and then washing with a buffer solution composed of 8.5% (w / v) sucrose, PBS solution, and deionized water, and finally transferring to a solution with a fixed conductivity of 8 mS / m; and the solution conductivity is adjusted by adding KCl to deionized water.

[0101] The concentration of the red blood cell suspension in step ⑤ is 200-500 / μL, and it is prepared by the following steps: taking 2 mL of blood into a centrifuge tube and washing it with PBS buffer solution for 3 times to obtain a red blood cell suspension, and directly adding KCl to the suspension to adjust the conductivity.

[0102] The mixture of the 293T cell suspension and the red blood cell suspension in step ⑤ is specifically a mixture of the 293T cell suspension with a concentration of 200-500 / μL and the red blood cell suspension with a concentration of 200-500 / μL in a volume ratio of 1:1.

[0103] In order to measure the activity of the cells, it is also necessary to use fluorescein diacetate FDA to stain the living yeast cells; in order to prevent particles from adhering to the wall and the substrate, 10 μL of 0.1% w / w Tween 20 is added to the above four solutions.

[0104] Figure 3 The physical map of the microfluidic chip for precise manipulation and sorting of cells based on bipolar electrodes in Example 1 and the electrode structure under a microscope (scale: 160 µm). As can be seen from the figure, V0 corresponds to the square suspension electrode 1, V3 corresponds to the first excitation electrode 2, V4 corresponds to the second excitation electrode 10, V1 corresponds to the third excitation electrode 8, and V2 corresponds to the fourth excitation electrode 5.

[0105] Figure 4Schematic diagram of trajectory migration of PS microspheres or yeast cells in Example 1, (a) Schematic diagram and superimposed image of trajectory migration of PS microspheres under the conditions of f = 30 kHz, σ = 0.008 S / m, suspension electrode voltage of 5 V, and sequentially changing the suspension electrode phase to 335°, 225°, 30°, and 135°, (b) Schematic diagram and superimposed image of trajectory migration of yeast cells under the conditions of f = 50 kHz, σ = 0.008 S / m, suspension electrode voltage of 4 V, and sequentially changing the suspension electrode phase to 180°, 90°, 0°, 270°, and 180° (scale bar: 55 µm); as can be seen from the figure, it shows that the chip can freely control particles and cells.

[0106] Figure 5 Schematic diagram of trajectory migration of PS microspheres and yeast cells, 293T cells, and red blood cells in Example 1, (a) PS microspheres and yeast cells are sorted under the conditions of f = 100 kHz, σ = 0.008 S / m, suspension electrode voltage of 5 V, and sequentially changing the suspension electrode phase to 0° and 180°, (b) 293T cells and red blood cells are sorted under the conditions of f = 40 kHz, σ = 0.008 S / m, suspension electrode voltage of 4 V, and sequentially changing the phase to 180° and 0° (scale bar: 55 µm); as can be seen from the figure, there is a clear separation band between two different cells or micro-particles on the surface of the suspension electrode, verifying the sorting function of the chip on particles and cells.

Claims

1. A microfluidic chip for precise cell manipulation and sorting based on bipolar electrodes, characterized in that... It consists of a PDMS cover sheet (3) and an ITO glass substrate (11); A cavity structure (7) is provided in the center of the PDMS cover (3) that penetrates the PDMS cover (3); A square floating electrode (1) is provided at the center of the ITO glass substrate (11); the four vertices of the square floating electrode (1) are respectively connected to the first curved electrode (4), the second curved electrode (6), the third curved electrode (9) and the fourth curved electrode (12); A first excitation electrode (2) is provided behind the square suspended electrode (1), a second excitation electrode (10) is provided on the left side of the square suspended electrode (1), a third excitation electrode (8) is provided in front of the square suspended electrode (1), and a fourth excitation electrode (5) is provided on the right side of the square suspended electrode (1). The distance between the front ends of the first excitation electrode (2), the second excitation electrode (10), the third excitation electrode (8) and the fourth excitation electrode (5) and the side of the square suspended electrode (1) is 100μm~110μm; The width of the front ends of the first excitation electrode (2), the second excitation electrode (10), the third excitation electrode (8) and the fourth excitation electrode (5) is 20μm~30μm; The ITO glass substrate (11) has an electrode on one side and the lower surface of the PDMS cover (3) sealed relative to each other, and the square suspended electrode (1) is placed in the center of the chamber structure (7). The chamber structure (7) encloses the front ends of the first excitation electrode (2), the second excitation electrode (10), the third excitation electrode (8) and the fourth excitation electrode (5).

2. The microfluidic chip for precise cell manipulation and sorting based on bipolar electrodes according to claim 1, characterized in that... The square suspended electrode (1), the first curved electrode (4), the second curved electrode (6), the third curved electrode (9), the fourth curved electrode (12), the first excitation electrode (2), the second excitation electrode (10), the third excitation electrode (8), and the fourth excitation electrode (5) are all obtained by etching the ITO conductive film on the surface of the ITO glass substrate (11).

3. A microfluidic chip for precise cell manipulation and sorting based on bipolar electrodes according to claim 1, characterized in that... The thickness of the square suspended electrode (1), the first curved electrode (4), the second curved electrode (6), the third curved electrode (9), the fourth curved electrode (12), the first excitation electrode (2), the second excitation electrode (10), the third excitation electrode (8), and the fourth excitation electrode (5) is 150nm~200nm.

4. A microfluidic chip for precise cell manipulation and sorting based on bipolar electrodes according to claim 1, characterized in that... The square suspended electrode (1) has a side length of 200μm~300μm; the width of the front end of the first curved electrode (4), the second curved electrode (6), the third curved electrode (9) and the fourth curved electrode (12) is 20μm~30μm.

5. A microfluidic chip for precise cell manipulation and sorting based on bipolar electrodes according to claim 1, characterized in that... The thickness of the PDMS cover (3) and the depth of the chamber structure (7) are both 5mm to 10mm.

6. A microfluidic chip for precise cell manipulation and sorting based on bipolar electrodes according to claim 1, characterized in that... The bottom surface of the chamber structure (7) is circular, square, or elliptical; the area of ​​the bottom surface is 0.8 cm². 2 ~1.6cm 2 .

7. The method of using a microfluidic chip for precise cell manipulation and sorting based on bipolar electrodes as described in claim 1, characterized in that... It is done in the following steps: ① Turn on the computer, signal generator, signal amplifier, oscilloscope, microscope, CCD, and fluorescent lamp connected to the microscope, and observe whether the equipment is operating normally. Then, open the ImageView image acquisition software on the computer and observe the microscope stage in real time. The signal generator includes a first signal generator, a second signal generator, and a third signal generator. ② Fix the microfluidic chip for precise cell manipulation and sorting based on bipolar electrodes on the stage and adjust the chip position and focal length; ③ The first signal generator and the second signal generator provide four sinusoidal AC signals of the same frequency to the first excitation electrode (2), the second excitation electrode (10), the third excitation electrode (8) and the fourth excitation electrode (5), and the third signal generator provides a single sinusoidal AC signal to the square floating electrode (1); ④ Observe under a microscope and inject cell / particle suspension into the chamber structure (7) using a pipette; ⑤ Start the first signal generator and the second signal generator so that the electrical signal applied to the surface of the first excitation electrode (2), the second excitation electrode (10), the third excitation electrode (8) and the fourth excitation electrode (5) is a four-phase electric field rotating counterclockwise, with phase values ​​of 0°, 90°, 180° and 270° respectively. After the position of the cell or microparticle in the chip is stable, start the third signal generator. The cell / particle suspension is one or a combination of several of the following: PS microsphere suspension, yeast cell suspension, 293T cell suspension, and erythrocyte suspension; the conductivity of the cell / particle suspension is 8 mS / m to 48 mS / m. ⑥ Observe under a microscope, and readjust the chip position and focus until the cells or microparticles are clear and the height is stable for video detection and recording; ⑦ Repeat steps ④ to ⑥, and continuously adjust the voltage and phase of the sinusoidal AC signal applied to the square suspended electrode (1), continuously adjust the frequency of the sinusoidal AC signal applied to the square suspended electrode (1) and the frequency of the four sinusoidal AC signals of the same frequency, and keep the frequency consistent, so as to realize the manipulation of cells or microparticles in the area of ​​the square suspended electrode (1), observe the experimental phenomena and record them. ⑧ Processing and analysis of experimental data.

8. The method of using a microfluidic chip for precise cell manipulation and sorting based on bipolar electrodes according to claim 7, characterized in that... In step ③, the first signal generator, the second signal generator, and the third signal generator are synchronous dual-output signal generators.

9. A method for using a microfluidic chip for precise cell manipulation and sorting based on bipolar electrodes according to claim 7, characterized in that... The concentration of the PS microsphere suspension in step ⑤ is 500 cells / μL to 1000 cells / μL; the concentration of the yeast cell suspension in step ⑤ is 500 cells / μL to 1000 cells / μL; the concentration of the 293T cell suspension in step ⑤ is 200 cells / μL to 500 cells / μL; and the concentration of the erythrocyte suspension in step ⑤ is 200 cells / μL to 500 cells / μL.

10. A method for using a microfluidic chip for precise cell manipulation and sorting based on bipolar electrodes according to claim 7, characterized in that... The conductivity of the cell / particle suspension described in step ⑤ is adjusted by adding KCl.

Images