CN217324123U - Cell capturing and pairing microfluidic packaging chip - Google Patents

Abstract

The utility model provides a cell is caught and is paird micro-fluidic packaging chip, catch and pair micro-fluidic chip including the cell, and with the cell is caught and is paird the packaging structure of micro-fluidic packaging chip bonding, packaging structure includes inlet and liquid outlet, the cell is caught and is paird and establish in the micro-fluidic chip and set up at least one and have the cell to the unit, the inlet forms two branching runners respectively to flow to same cell to the unit at least uniformly. The cell capturing and matching microfluidic packaging chip can realize the flow of cell suspension uniformly from the structure, prevent the unstable flow rate caused by the non-uniform suspension, and simultaneously can capture three cells on the chip successively to realize a large-scale triple cell array; one of the three cells in the array is selected, and the three cells are arranged in a group in a matching way, so that great convenience and possibility are provided for researching the interaction between cells such as three-cell paracrine and the like or cell fusion.

Description

Cell capturing and pairing microfluidic packaging chip

Technical Field

The utility model relates to a receive the alternately technical field of processing and life science a little, especially relate to a cell is caught and is paird micro-fluidic packaging chip.

Background

After long-term research, it has been found that even homogeneous cells have individual differences in cell biology, a property referred to as cellular heterogeneity. In traditional biological cell experiments, population analysis can mask differences between individual cells. Therefore, the study of cell heterogeneity at the single cell level can make the cell more clear and study the life history of various organisms. For example, cancer is a highly heterogeneous disease, and studying the heterogeneity of cancer cells may better understand the development and progression of cancer for accurate treatment of patients.

The heterogeneity of the cells widely exists in the processes of differentiation, immunity, metabolism and other cell life activities, and the problem that the individual difference in an averaging experiment is covered can be avoided by adopting single cell analysis, so that the heterogeneity of the cells is researched. The cell is a life unit containing abundant information, the research on the cell at present comprises the fields of genomics, transcriptomics, metabonomics, secreomics, intercellular interaction and the like, and with the deepening of the cell research, the wide research objects in the cell require that the experimental conditions of single cell analysis have high-throughput analysis capability.

A microfluidic chip is an experimental chip with the capability of manipulating and analyzing a minute amount of fluid over an area of several square centimeters or less. The microfluidic technology is adopted for biological or chemical analysis, so that the research scale can be reduced, the experimental error can be reduced, the detection sensitivity can be improved, and the loss of reagents or samples can be reduced. For single cell and even subcellular level analysis, the microfluidic chip is an ideal experimental operating platform.

For single cell analysis on a microfluidic chip, the cells are first captured. Currently, there are two methods, active and passive, for single cell capture on microfluidic chips: the passive method comprises the means of microstructure filtration, fluid shear force method and the like, and the principle is that the cells are captured to a specific area through the difference of microstructures or cells under different fluid power; active methods include dielectrophoresis, optical tweezers, acoustic tweezers, magnetic bead sorting, antigen-antibody labeling, etc., and some exogenous force or biological markers are artificially applied to control the movement of cells. The passive cell capturing mode has low sensitivity, the required cell amount is larger, and the cells can also cause certain extrusion deformation or damage due to special microstructures; the active cell capture mode usually needs to add a specific marker, and can cause certain influence on downstream single cell analysis.

Dielectrophoresis (DEP) is a technique developed on the basis of electrophoretic techniques, a special type of electrophoresis, also known as Dielectrophoresis. Dielectrophoresis refers to the phenomenon in which neutral particles move relative to a liquid in the presence of a non-uniform electric field. By utilizing dielectrophoresis, different structures can be designed, and a high-frequency electric field is applied to ensure that cells move directionally in the microfluidic chip, so that the method is a cell capture method which has high flux, does not need special marks and has no contact and no damage to the cells.

At present, more and more researches arrange cells on a microfluidic chip into a single cell array for single cell analysis. Multiple cell arrays formed on the chip can perform multiple analyses on multiple cells, the contrast is enhanced in parallel experiments, and experimental errors caused by differences among chips in sample loading of multiple chips are avoided.

At present, no means can realize a multiple cell array, so that a cell capturing and matching microfluidic packaging chip is needed.

SUMMERY OF THE UTILITY MODEL

An object of the utility model is to provide a can catch three kinds of cells and can provide very big possibility for the heterogeneity of research homogeneous cell and catch and pair micro-fluidic packaging chip, can realize comparatively stable cell suspension velocity of flow, realize good cell capture effect.

The utility model provides a cell is caught and is paird micro-fluidic packaging chip, catch and pair micro-fluidic chip including the cell, and with the cell is caught and is paird the packaging structure of micro-fluidic packaging chip bonding, packaging structure includes inlet and liquid outlet, the cell is caught and is paird and establish in the micro-fluidic chip and set up at least one and have the cell to the unit, the inlet forms two branching runners respectively to flow to same cell to the unit on average at least.

The cell pair units in the cell capturing and pairing microfluidic chip are arranged in an array mode, a large micro-well, three small micro-wells which are located in the large micro-well and are arranged in a sinking interval mode, three groups of electrode pairs and shielding electrodes located between the adjacent small micro-wells are arranged in each cell pair unit, and the small micro-wells are located between the corresponding electrode pairs.

Further, the shape of the big micro-trap is elliptical.

Furthermore, the cell capturing and matching microfluidic chip also comprises buffer areas positioned on two sides of the cell capturing area.

Furthermore, the three small micro-wells are respectively a first small micro-well, a second small micro-well and a third small micro-well which are sequentially arranged; the shielding electrodes include a first shielding electrode located between the first and second mini-microwells and a second shielding electrode located between the second and third mini-microwells.

Furthermore, the three groups of electrode pairs are respectively a first group of electrode pairs, a second group of electrode pairs and a third group of electrode pairs, and the first group of electrode pairs comprise a first upper electrode and a first lower electrode which are respectively positioned at two sides of the first small micro-trap; the second group of electrode pairs comprise a second upper electrode and a second lower electrode which are respectively positioned at two sides of the second small micro-trap; the third group of electrode pairs comprise a third upper electrode and a third lower electrode which are respectively positioned at two sides of the third small micro-well; the first shielding electrode is positioned between the first group of electrode pairs and the second group of electrode pairs, and the second shielding electrode is positioned between the second group of electrode pairs and the third group of electrode pairs.

Further, three sets of electrode pairs and two shielding electrodes constitute one electrode unit, and each electrode unit has a plurality of large micro-wells in the transverse direction.

Furthermore, N electrode units are arranged on the cell capturing and pairing micro-fluidic chip in the longitudinal direction, the N electrode units form an electrode pair array, and N is a positive integer; the cell capturing and matching microfluidic chip also comprises four groups of metal structure pairs which are respectively positioned at two sides of the cell capturing and matching microfluidic chip, wherein the four groups of metal structure pairs comprise a first group of metal structure pairs connected with the first group of electrode pairs, a second group of metal structure pairs connected with the two shielding electrodes, a third group of metal structure pairs connected with the second group of electrode pairs and a fourth group of metal structure pairs connected with the third group of electrode pairs.

Furthermore, the cell capturing and matching microfluidic chip also comprises a glass substrate, and the electrode unit is positioned on the glass substrate.

Furthermore, the large microtrap and the small microtrap are used as a microtrap structure with nested sizes for fixing cells.

The cell capturing and matching microfluidic packaging chip can realize the flow of cell suspension uniformly from the structure, prevent the unstable flow rate caused by the non-uniform suspension, and simultaneously can capture three cells on the chip successively to realize a large-scale triple cell array; one of the three cells in the array is selected, and the three cells are arranged in a group in a pairing way, so that great convenience and possibility are provided for researching the interaction between the cells such as three-cell paracrine and the like or cell fusion.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the embodiments or the prior art descriptions will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the drawings without creative efforts.

Fig. 1 is a front view within a microfluidic chip of an embodiment of the present invention;

fig. 2 is a schematic structural diagram of a single cell pair unit in a microfluidic chip according to an embodiment of the present invention;

fig. 3 is a partial schematic view of a microfluidic chip according to an embodiment of the present invention;

fig. 4 is a schematic diagram of steps S1 to S5 of a manufacturing process of a microfluidic chip of an embodiment of the present invention;

fig. 5 is a schematic diagram of steps S6 to S8 of a manufacturing process of a microfluidic chip of an embodiment of the present invention; fig. 6 is a top view of the manufacturing process of the microfluidic chip according to the embodiment of the present invention and the through holes are reserved;

fig. 7 is a top view of copper leaked out of a process of manufacturing a microfluidic chip according to an embodiment of the present invention;

fig. 8 is a schematic diagram of a cell experiment of the microfluidic chip according to an embodiment of the present invention;

fig. 9 is a schematic diagram comparing a natural light and a fluorescence synthesis map of a triple cell array captured by a microfluidic chip according to an embodiment of the present invention.

Detailed Description

Reference will now be made in detail to embodiments of the present invention, examples of which are illustrated in the accompanying drawings, wherein like reference numerals refer to the same or similar elements or elements having the same or similar function throughout. The embodiments described below with reference to the drawings are exemplary and intended to be used for explaining the present invention, and should not be construed as limiting the present invention.

In the description of the present invention, it is to be understood that the terms "length", "width", "upper", "lower", "front", "rear", "left", "right", "vertical", "horizontal", "top", "bottom", "inner", "outer", and the like indicate orientations or positional relationships based on those shown in the drawings, and are merely for convenience of description and simplicity of description, and do not indicate or imply that the device or element being referred to must have a particular orientation, be constructed and operated in a particular orientation, and thus, should not be construed as limiting the present invention. In addition, in the description of the present invention, "a plurality" means two or more unless specifically limited otherwise.

The utility model discloses a cell is caught and is paired micro-fluidic packaging chip, as shown in figure 1 and figure 2, it includes glass substrate 100, is located electrode layer, little microwell, big microwell 10 and the encapsulated layer on glass substrate 100, and wherein the electrode layer includes three electrode pairs of group and two shielding electrode, and wherein three electrode pairs of group set up according to the preface, and little microwell is located between every electrode pair of group, and every shielding electrode is located between the adjacent electrode pair of group. The cell packaging chip comprises a cell capturing and matching microfluidic chip and a packaging structure bonded with the cell capturing and matching microfluidic packaging chip, the packaging structure comprises a liquid inlet and a liquid outlet (not shown in the figure), at least one cell pair unit is arranged in the cell capturing and matching microfluidic chip, and the liquid inlet uniformly forms at least two branched flow channels which respectively flow to the same cell pair unit.

Through the design of import branching runner, can realize the even feed liquor of cell suspension, prevent the unstable condition of cell suspension velocity of flow, when the cell suspension velocity of flow is unstable, the circular telegram snatchs the cell and probably leads to the unsuccessful problem of snatching, carries out the letter sorting and the screening of cell pair after the velocity of flow is stable, can realize better cell capture effect.

The schematic diagram of the internal macrostructure of the cell capture and mating microfluidic chip is shown in fig. 3, and it includes a buffer region 1 and a cell capture region 2, wherein the width of the buffer region 1 is 4mm to 6mm (preferably 5mm), and the length and width of the cell capture region 2 are 0.3cm to 0.7cm (preferably 0.5cm) and 0.8cm to 1.2cm (preferably 1cm), respectively.

The buffer area is used as a reserved area in front of and behind the cell capturing and matching microfluidic chip and is positioned on two sides of the cell capturing area 2 so as to ensure the stable sampling flow rate.

The cell capture region 2 has M x N cell pair units (M and N are positive integers, M can be 72, and N can be 28) arranged in an array, each cell pair unit is spaced from 0.3mm to 0.6mm (preferably 0.4mm) up and down, and each cell pair unit is spaced from 0.1mm to 0.3mm (preferably 0.2mm) left and right.

Each cell pair unit is internally provided with an elliptic big micro-trap 10, three small micro-traps 11, 12 and 13 which are positioned in the big micro-trap 10 and sink at intervals, three groups of electrode pairs and a shielding electrode positioned between the adjacent small micro-traps, and the cells are captured by the three small micro-traps.

Wherein the length of the big micro-well 10 is 250 μm to 270 μm (preferably 260 μm), the width is 110 μm to 130 μm (preferably 120 μm) and the depth is 20 μm to 35 μm (preferably 27 μm), the diameter of the small micro-well is 13 μm to 17 μm (preferably 15 μm) and the depth is 1 μm to 6 μm (preferably 1.2 μm), the size of the micro-well is adjusted according to the average particle size of the experimental cell types, and the size of the micro-well is determined according to the human lung cancer epithelial cell A549 of the embodiment of the present invention.

The three micro wells are respectively a first micro well 11, a second micro well 12 and a third micro well 13 which are arranged in sequence.

The shielding electrodes comprise a first shielding electrode 51 located between the first microtrap 11 and the second microtrap 12 and a second shielding electrode 52 located between the second microtrap 12 and the third microtrap 13. The shielding electrodes can avoid the mutual inductance effect between each group of electrodes when the electrodes are electrified to generate dielectrophoresis force to capture cells (namely, the condition that the electrodes are electrified and then an induced electric field is generated between adjacent pairs of non-electrified electrodes to capture the cells).

The three groups of electrode pairs are respectively a first group of electrode pairs 20, a second group of electrode pairs 30 and a third group of electrode pairs 40, wherein the first group of electrode pairs 20 comprise a first upper electrode 21 and a first lower electrode 22 which are respectively positioned at two sides of the first small micro-trap 11; the second group of electrode pairs 30 comprises a second upper electrode 31 and a second lower electrode 32 which are respectively positioned at two sides of the second small micro-well 12; the third group of electrode pairs 40 includes a third upper electrode 41 and a third lower electrode 42 respectively located at both sides of the third small microtrap 13. The spacing between each electrode pair is 4 μm to 8 μm (preferably 6 μm).

Three groups of electrode pairs and two shielding electrodes form an electrode unit, namely 8 parallel electrodes form an electrode unit. Each electrode unit has M large micro wells 10 (specifically 72) in the transverse direction, the length of a single electrode in each electrode unit is 2.3cm to 2.6cm (preferably 2.5cm), and the distance between two adjacent electrode units is 4 μ M to 8 μ M (preferably 6 μ M).

The embodiment of the utility model provides an adopt human lung cancer epithelial cell A549, the width of single electrode is 7 microns in the embodiment, and the interval between shielding electrode and the electrode that corresponds is 6 microns. Among them, the human lung cancer cell line a549 was established in 1972 by GiardDJ through lung cancer tissue transplant culture and originated from a 58-year-old white human male. The human lung cancer cell line A549 can synthesize lecithin rich in unsaturated fatty acid through a cytidine diphosphatidylcholine pathway; keratin is positive.

The cell capturing and matching microfluidic chip is provided with N electrode units (28 in number) in the longitudinal direction, and the N electrode units form an electrode pair array.

The cell capturing and pairing microfluidic chip further comprises four groups of metal structure pairs respectively located at two sides of the chip, specifically, the four groups of metal structure pairs comprise a first group of metal structure pairs connected with the first group of electrode pairs 20, a second group of metal structure pairs connected with the shielding electrodes, a third group of metal structure pairs connected with the second group of electrode pairs 30, and a fourth group of metal structure pairs connected with the third group of electrode pairs 40.

As shown in fig. 7, the first group of metal structure pairs includes a first left metal structure 611 located at the left side of the cell capture and mating microfluidic chip and connected to the first upper electrode 21, and a first right metal structure 612 located at the right side of the cell capture and mating microfluidic chip and connected to the first lower electrode 22; the second group of metal structure pairs comprises a second left metal structure 621 which is positioned on the left side of the cell capturing and matching microfluidic chip and connected with the first shielding electrode 51, and a second right metal structure 622 which is positioned on the right side of the cell capturing and matching microfluidic chip and connected with the second shielding electrode 52; the third group of metal structure pairs comprises a third left metal structure 631 located on the left side of the cell-capturing and mating microfluidic chip and connected to the second upper electrode 31, a third right metal structure 632 located on the right side of the cell-capturing and mating microfluidic chip and connected to the second lower electrode 32, and the fourth group of metal structure pairs comprises a third left metal structure 641 located on the left side of the cell-capturing and mating microfluidic chip and connected to the third upper electrode 41, and a fourth right metal structure 642 located on the right side of the cell-capturing and mating microfluidic chip and connected to the third lower electrode 42.

The N electrode units form an integrated control structure through four groups of metal structures on two sides, namely the metal structures on the two sides connect the electrode pair array; in the embodiment, the first group of metal structure pairs is made of ITO, and the two groups of metal structure pairs respectively connected with the other two groups of electrode pairs are made of copper or other conductive metals (copper is selected in the example), so that the cost can be saved.

As shown in FIG. 3, the large microwell 10 and the small microwells 11, 12, 13 are used as a structure of nested microwells of different sizes for fixing cells, when in operation, the cells enter the cell capture region 2 from the inlet, the electrode units in the same layer can be simultaneously electrified to generate positive dielectrophoresis force to capture the cells and downwards enter the small microwells 11, 12, 13 to form a cell array, and the cells which are not captured leave the cell capture region 2 from the outlet.

Since the PDMS encapsulation material has good biocompatibility and is easy to observe, the PDMS encapsulation material is adopted in the large micro-well 10 for encapsulating the flow channel, and the height of the flow channel is 45 μm to 65 μm (preferably 55 μm).

The utility model discloses still disclose a cell capture and mate micro-fluidic chip's manufacturing method, including following step:

s1: as shown in part (a) of fig. 4, an ITO electrode layer 101 is deposited on a glass substrate 100, and then the glass substrate 100 with the ITO electrode layer 20 is cleaned TO ensure that the surface of the TO electrode layer 101 is clean and has a certain adhesion;

with respect to step S1, a specific method for cleaning the glass substrate 100 having the ITO electrode layer 101 is: placing a square ITO electrode layer 101 with the square resistance of 20 omega and the side length of 8cm and a glass substrate 100 in a cleaning frame, soaking the square ITO electrode layer and the glass substrate in a mixed solution of hydrogen peroxide, ammonia water and deionized water, wherein the ratio of the hydrogen peroxide to the ammonia water to the deionized water is 1, 1 and 6 respectively, heating the square ITO electrode layer and the glass substrate in a water bath at 70 ℃ for 40 minutes, and taking out the square ITO electrode layer and the glass substrate. And taking out, spraying the surface of the ITO electrode layer 101 by using a water gun, drying moisture by using nitrogen, and then putting into a silicon wafer box for storage. The purpose of surface cleaning is to remove various particles and impurities on the surface of the ITO electrode layer 101 and increase a certain surface energy to ensure that the subsequent spin-on photoresist is formed flat and has sufficient adhesion.

S2: after the cleaning, Ultraviolet (UV) irradiation is performed on the surface of the glass substrate 100 of the ITO electrode layer 101 for 30 minutes to remove the impurity dangling bonds on the surface of the ITO electrode layer 101 and further increase the surface energy of the ITO electrode layer 101 at the same time, so that the photoresist has sufficient adhesion to the surface of the ITO electrode layer 101.

S3: as shown in fig. 4 (b) to (d), a positive photoresist 102 is spin-coated on the ITO electrode layer 101, and an electrode-like photoresist pattern with a certain height is manufactured as a wet etching protection layer;

before spin-coating the positive photoresist 102, the glass substrate 100 having the ITO electrode layer 101 is first baked on a hot plate at 180 ℃ for 20 minutes to remove residual moisture on the surface of the ITO electrode layer 101, and then the positive photoresist 102 is spin-coated in a spin coater with the rotation parameters of table 1.

TABLE 1 spin coating parameters for positive photoresists

S4: as shown in parts (c) and (f) of fig. 4, the pattern formed in S3 is exposed to light together with the reticle 200 in a photolithography machine (not shown), and the electrode layer pattern 103 is formed;

after the spin coating is finished, firstly, pre-drying the ITO electrode layer 102 on a hot plate for 2 minutes at 95 ℃; the ITO electrode layer 102 is then fixed to an alignment sheet (not shown), as shown in parts (c) and (d) of FIG. 3, and the ITO layer is electrically connectedThe pole layer 102 and the mask 200 are put into a photoetching machine and adjusted to a fixed position for exposure, and the exposure dose is 40mJ/cm ² (ii) a As shown in fig. 4 (e) and (f), after the exposure is finished, placing the substrate into a proper amount of developing solution for developing for 30 seconds, removing the positive photoresist 103, cleaning the substrate with deionized water, and drying the substrate with nitrogen; and finally, placing the substrate on a hot plate at 150 ℃ for heating for 2 minutes to harden the substrate, and forming an electrode layer pattern 103, wherein the electrode layer pattern 103 specifically comprises three groups of electrode pairs and two shielding electrodes, and the electrode layer pattern 103 is specifically shown in fig. 1 and 2.

Specifically, after the electrode layer pattern 103 is formed, wet etching is performed using an ITO etching solution in order to etch the ITO electrode layer 101 without the photoresist protection region clean, leaving the desired electrode layer pattern 103. Specifically, the photoresist is washed out by acetone to obtain an electrode layer pattern, and the specific method comprises the following steps: firstly, heating the ITO etching solution to 35 ℃ in a water bath, then placing the electrode layer pattern into the ITO etching solution for etching for 150s, preferably quickly transferring the ITO etching solution into a beaker filled with deionized water to dilute the etching solution, and finally washing with water, drying and storing.

After etching is completed, the positive photoresist 102 needs to be removed to obtain the exposed electrode. The positive photoresist 102 can be removed by directly drying the photoresist after the photoresist is placed into an acetone solution for ultrasonic treatment for 1 minute. After the electrode layers are obtained, a universal meter is needed to test the resistance between each group of integrated electrode pairs, if each group of integrated electrode pairs is conducted, the electrode preparation fails, and if the electrode pairs are not conducted, the electrode preparation can be used for the next step of manufacturing. Before the next step of photoetching, the sample still needs to be subjected to ultraviolet irradiation for 30 minutes and baked at 180 ℃ for 20 minutes to ensure that the adhesion between the photoresist and the sample is sufficient.

S5: as shown in part (g) of fig. 4, spin-coating a first negative photoresist 104 on the electrode layer pattern 103 using a hard contact photolithography process, as shown in fig. 1 and 2, to fabricate small micro wells 11, 12, 13;

specifically, first, a first negative photoresist 104 was spin-coated on the electrode layer pattern 103 according to table 2, and then pre-baked on a hot plate at 95 ℃ for 5 minutes, and after the pre-baking, the first negative photoresist 104 was left to stand for 20 minutes to be completely driedThe ultraviolet light is reflected in the glass substrate 100 to cause unnecessary overexposure, so a layer of black wallpaper (not shown) is adhered to the back surface of the glass substrate 100 for light absorption; as shown in part (g) of fig. 4, the mask plate 105 with the structures of circles of different sizes and the electrode layer pattern with the first negative photoresist 104 are sequentially placed into a photoetching machine, and are aligned by using the alignment marks on the mask plate 105 and the electrode layer pattern 103, and then hard-contact exposure is carried out, wherein the exposure dose is 150mJ/cm ² (ii) a When ultraviolet light irradiates the unshielded first negative photoresist 104, the first negative photoresist 104 generates acid to crosslink the macromolecules, and the macromolecules can be left in the developing solution; after the exposure is finished, the film is placed on a hot plate at the temperature of 95 ℃ for 2 minutes for post-baking, and then is kept stand and cooled to room temperature. After the temperature is reduced, developing, specifically, soaking the sample in a developing solution, uniformly shaking the container to enable the developing solution to flow, taking out the sample after 60s, fixing the sample with isopropanol, and drying the sample with nitrogen; finally, the first negative photoresist 104 is fully set and remains stable by hardening with heat on a hot plate at 150 ℃ for 2 minutes.

TABLE 2 first negative photoresist spin coating parameters for small micro well layers

The first negative photoresist of the small microwell layer produced the small microwells at the rotation speed of table 2 with a thickness of about 1.2 microns.

And S5, placing the cell capturing and matching microfluidic chip in a step instrument for testing to obtain that the thickness of the small micro-trap is about 1.2 μm, and measuring whether the aperture of the small micro-trap meets the expectation in an optical microscope.

S6: as shown in part (a) of fig. 5, each set of electrode pairs was integrated using a magnetron sputtering copper plating technique.

Specifically, during the fabrication of the small micro-wells, as shown in fig. 6, vias 201 are reserved at the ends of these electrodes, i.e. there is no first negative photoresist 104 at the ends of the electrodes, and copper can be grown directly on the ITO electrodes. To protect the micro-well region and the regions that have been integrated by the ITO from influence, a high temperature tape is applied to unnecessary regions on the sample to block the growth of copper, and only the copper (metal structure pair) 621, 622, 631, 632, 641, 642 in fig. 7 is exposed. The sputtering process is carried out under the Ar atmosphere, the gas flow is 50sccm, the sputtering power is 100w, and a layer of copper (metal structure pair) 621, 622, 631, 632, 641 and 642 with the thickness of about 370nm can be sputtered, wherein the thickness requirement of the copper is not particularly accurate, and only the part needing to be integrated is ensured to be conductive.

After the sputtering is finished, conducting tests are carried out on the three groups of electrode pairs again, and the next step can be carried out only by ensuring that each pair of electrode pairs are not mutually conducted, so that the smooth production of the dielectrophoresis force in the subsequent cell experiment can be ensured.

S7: as shown in parts (b) and (c) of fig. 5, negative photoresist lithography is performed again on the small micro-wells using a hard contact lithography process to fabricate large micro-wells 10;

specifically, a second negative photoresist was spin coated on the microwell according to Table 3, then pre-baked on a 65 deg.C hotplate for 1min, then transferred to a 95 deg.C hotplate and baked for 5 min. The sample was allowed to stand for 20 minutes after the pre-bake to allow the photoresist to dry completely. Similarly, a black wallpaper is adhered to the back of the glass substrate 100 for absorption. Then, the mask plate with the micro-well structure and the sample are sequentially placed into a photoetching machine, alignment is carried out by utilizing the alignment marks on the mask plate and the electrode, and then hard contact type exposure is carried out, wherein the exposure dose is 180mJ/cm ² (ii) a After exposure, putting the sample on a hot plate at 65 ℃ for post-baking for 1min, and then transferring the sample to a hot plate at 95 ℃ for baking for 6 min; standing and cooling to room temperature, developing, soaking the sample in SU-8 developing solution for 60s, taking out the sample, fixing with isopropanol, and blow-drying with nitrogen; finally, hardening the sample by heating on a hot plate at 150 ℃ for 2 minutes to completely set and keep the second negative photoresist stable; after the steps are completed, the large micro-well 10 with the thickness of about 25 mu m is obtained, microscopic examination is carried out under an optical microscope, and after the size of the micro-well is verified to meet the requirements of the specification, the manufacture of the chip main body microstructure is completed.

TABLE 3 spin coating parameters for second negative photoresist for large microwells

S8: as shown in part (d) of fig. 5, a PDMS (Polydimethylsiloxane) material is formed on the encapsulation layer 301 for encapsulation, so as to form a closed space for fluid to flow in.

Firstly, sequentially performing ultrasonic treatment on a silicon wafer for 2min by using three solutions of acetone, isopropanol and water, then performing ultraviolet irradiation for 30 min, and baking at 180 ℃ for 20 min to ensure that the adhesion between the photoresist and the electrode layer pattern is sufficient; after the silicon wafer returns to room temperature, a third negative photoresist (not shown) is spin-coated on the silicon wafer according to the table 4, then is baked on a hot plate at 65 ℃ for 3min30s, and is baked on a hot plate at 95 ℃ for 9min30 s; standing for 30 minutes after pre-baking to completely dry the third negative photoresist, and performing ultraviolet hard contact exposure by using a film mask plate with exposure dose of 200mJ/cm ² (ii) a After exposure, post-baking, baking on a hot plate at 65 ℃ for 1min30s, and then baking on a hot plate at 95 ℃ for 6min30 s; after the postbaking is finished, standing to normal temperature, then developing in a developing solution for 7 minutes, and then washing with isopropanol and drying; finally, after development, the photolithography manufacturing step is completed by hardening on a hot plate at 150 ℃ for 10min, and then the surface of the encapsulation layer 301 is subjected to hydrophobic treatment by using FDTS (1H,1H,2H, 2H-perfluorodecyl trichlorosilane) to prevent the encapsulation layer 301 from being adhered to the template during curing, so that the template for PDMS casting is obtained, and the height of the flow channel in the encapsulation layer 301 is about 55 μm through a step profiler test.

TABLE 4 spin coating parameters for third negative tone photoresists

The method comprises the following steps of (1) in a ratio of 10: 1, mixing PDMS and a curing agent thereof, stirring for 5 minutes to fully mix the PDMS and the curing agent, and then putting the mixture into a vacuum dish for vacuumizing to remove bubbles in the mixed solution; and after bubbles are completely removed, casting the prepared PDMS into a soft photoetching mold with a channel structure, baking the PDMS in a 65 ℃ oven for 3 hours, taking out the solidified PDMS, demolding, cutting the solidified PDMS to a proper size, and punching a liquid inlet (liquid inlet) and a liquid outlet (outlet) and connecting the PDMS with a conduit.

The treated PDMS encapsulation layer 301 was placed on a microfluidic chip and subjected to oxygen plasma (O) in a plasma cleaner for 1min ₂ Plasma) bonding, and completing the packaging of the microfluidic chip. The microfluidic chip is formed through the steps.

After the encapsulation, the electrodes are attached with conductive tape so that the signal generator can clamp the applied signal, and finally the microfluidic chip is fixed by a clamp (not shown).

The general flow of cell experiments for cell capture and pairing microfluidic chips is shown in fig. 8: in a cell experiment, the shielding electrodes 51 and 52 are always grounded, high-frequency alternating current is applied to the first group of electrode pair array through a signal generator after cell suspension containing low-conductivity buffer solution is introduced at a pulse flow rate, the other electrode pairs are grounded, positive dielectrophoresis force is generated to grab the first cell into the micro-well, then the low-conductivity buffer solution is introduced to empty the unhatched cells, and a single-cell array is formed at the moment. Keeping high-frequency alternating current on the first group of electrode pair arrays to fix the first cells, introducing a second cell suspension containing a buffer solution with low conductivity at a pulse flow rate, electrifying the second group of electrode pair arrays, grounding the other electrodes, capturing the cells into corresponding micro-wells, introducing the same buffer solution as before to empty the cells which are not captured, and forming a double array by the cells captured before and after. Then, two groups of electrodes which capture cells are kept electrified, the last group of electrodes is electrified, the shielding wire is grounded, and the three types of cells can finally form a triple cell array on the chip.

Human lung cancer epithelial cells A549 are selected for cell experiments to verify the cell capturing and the effect of pairing the microfluidic chip. In the experiment, a micro-fluidic experiment platform is required to be built, and the micro-fluidic experiment platform comprises a fluorescence macroscopic zoom microscope, a display screen, two injection pumps, three signal generators and other equipment, and is used for performing autonomous culture and fluorescence dyeing on the selected experimental cells.

During the experiment, a buffer solution with low conductivity (hereinafter referred to as a first micro pump) is carried on one micro pump, a cell suspension containing the buffer solution with low conductivity (hereinafter referred to as a second micro pump) is carried on the other micro pump, the two liquids are connected to the chip through a micro three-way pipe, and then the displacement of the micro pump is regulated and controlled through a program and software for sample introduction.

Firstly, a first micropump is used for introducing pure buffer solution at constant speed to ensure that the liquid in the chip does not contain bubbles, then the micropump is stopped, a second micropump is started, the first cell suspension is introduced into the manufactured microfluidic chip in a pulse mode, and after the cells enter a working area, a signal generator is used for applying high-frequency alternating current signals (V) to the first group of electrode pair arrays _PP 18V, the flow rate of the fluid at the first point is higher, so that the highest voltage is applied to ensure stable grasping), and the rest electrodes are grounded to capture the first cells to form a single cell array.

Stopping pulse displacement of the second micropump, keeping the electrical parameters of the first group of electrode pair arrays all the time, starting the first micropump, introducing a buffer solution to empty redundant uncaptured suspended cells, replacing cells carried on the second micropump, starting the second micropump again, and applying high-frequency alternating current (V) to the second group of electrode pair arrays when a second type of cells enters a capturing area _PP And (4) when the flow velocity is relatively stable at the second point, the influence of the mutual induction effect on the other electrodes is reduced by reducing the voltage), the other electrodes are grounded, and the second type of suspended cells are captured.

Then stopping the pulse displacement of the second micropump, always keeping the electrical parameters of the first and second groups of electrode pair arrays, starting the first micropump to introduce a buffer solution to empty redundant uncaptured suspension cells, replacing cells carried on the second micropump, starting the second micropump again, and applying high-frequency alternating current (V) to the third group of electrode pair arrays when a third cell enters a capture area _PP The third point is at the lowest liquid flow rate and applies the minimum voltage, if the voltage is too high, a plurality of cells are grabbed in one micro-trap, which is not beneficial to the experiment), and the suspended cells are captured.

And finally, stopping pulse displacement of the second micro pump, always keeping the electrical parameters of the first, second and third groups of electrode pair arrays, starting the first micro pump, introducing a buffer solution to empty redundant uncaptured suspension cells, and taking a fluorescence field photo as a record for subsequent experimental analysis.

Through the cell experiment, the cells are stained in three colors of red, green and blue to distinguish different cell arrays, and a triple cell array is successfully captured on the chip, 7 × 8 cells are randomly selected to observe the cell array, for the convenience of observation, the pictures of three fluorescence channels are synthesized, and the comparison of the natural light field and the fluorescence synthesis graph is shown in fig. 9 (the scale bar is 200 microns).

It can be clearly seen that the cells all settled in the small microwell, and most of the cell pairs had only three cells (red-green-blue) within the cell (considered as successful pairings), and a total of 39/56 cell pairs were successfully paired according to a rough calculation of the successful pairing efficiency of the selected region, with a pairing success rate of about 70%. Therefore, the cell capturing and matching microfluidic chip has the construction capacity of a large-scale triple cell array, and the utility model discloses the utility reaches the expectation.

The cell capturing and matching microfluidic chip of the utility model is a microfluidic chip for capturing triple cell arrays by utilizing dielectrophoresis; the utility model discloses a carry out the fluorescence staining of red, green, three kinds of colours of blue to the cell, can clearly see under fluorescence microscope red green blue cell three a set of be array arrangement cell capture and pair micro-fluidic chip on, this has verified the cell capture and has mated micro-fluidic chip function and reach the design expectation, and the cell arrangement of arraying simultaneously can provide very big possibility for studying the heterogeneity of the same kind of cell.

The cells in the form have good contrast and controllability, the positions of the cells in the array are fixed and controllable, and batch operation and batch analysis can be performed on the cells, so that the time and reagent cost generated by parallel operation are reduced, and the experimental error is reduced.

The cell capturing and matching microfluidic chip of the utility model can capture three cells on the chip in sequence, thus realizing large-scale triple cell array; one of the three cells in the array is selected, and the three cells are arranged in a group in a matching way, so that great convenience and possibility are provided for researching the interaction between cells such as three-cell paracrine and the like or cell fusion.

While the invention has been described with reference to a preferred embodiment, it will be understood by those skilled in the art that various changes in form and detail may be made without departing from the spirit and scope of the invention.

Claims (10)

1. The cell capturing and matching microfluidic packaging chip is characterized by comprising a cell capturing and matching microfluidic chip and a packaging structure bonded with the cell capturing and matching microfluidic packaging chip, wherein the packaging structure comprises a liquid inlet and a liquid outlet, at least one cell pair unit is arranged in the cell capturing and matching microfluidic chip, and the liquid inlet uniformly forms at least two branched flow channels which respectively flow to the same cell pair unit.

2. The cell-capturing and mating microfluidic packaged chip according to claim 1, wherein the cell pair units in the cell-capturing and mating microfluidic chip are arranged in an array, each cell pair unit has a large microwell (10), three small microwells (11, 12, 13) that are located in the large microwell (10) and are sinking and spaced, three sets of electrode pairs, and a shielding electrode located between adjacent small microwells, and the small microwells are located between corresponding electrode pairs.

3. The cell-capturing and mating microfluidic packaged chip according to claim 2, wherein the macro-wells (10) are oval in shape.

4. The cell-capturing and mating microfluidic packaged chip of claim 2, further comprising buffer regions on both sides of the cell-capturing region.

5. The cell-capturing and mating microfluidic chip according to claim 2, wherein the three micro wells are a first micro well (11), a second micro well (12) and a third micro well (13) arranged in sequence; the shielding electrodes comprise a first shielding electrode (51) located between the first microtrap (11) and the second microtrap (12), and a second shielding electrode (52) located between the second microtrap (12) and the third microtrap (13).

6. The cell-capturing and mating microfluidic packaged chip according to claim 5, wherein the three sets of electrode pairs are a first set of electrode pairs (20), a second set of electrode pairs (30), and a third set of electrode pairs (40), respectively, the first set of electrode pairs (20) includes a first upper electrode (21) and a first lower electrode (22) respectively located at two sides of the first small micro-well (11); the second group of electrode pairs (30) comprises a second upper electrode (31) and a second lower electrode (32) which are respectively positioned at two sides of the second small micro-trap (12); the third group of electrode pairs (40) comprises a third upper electrode (41) and a third lower electrode (42) which are respectively positioned at two sides of the third small micro-well (13); the first shielding electrode (51) is located between the first set of electrode pairs (20) and the second set of electrode pairs (30), and the second shielding electrode (52) is located between the second set of electrode pairs (30) and the third set of electrode pairs (40).

7. The cell-capturing and mating microfluidic packaged chip according to claim 6, wherein three sets of electrode pairs and two shielding electrodes form an electrode unit, and each electrode unit has a plurality of large micro-wells in the transverse direction.

8. The cell-capturing and mating microfluidic packaged chip of claim 7,

the cell capturing and matching microfluidic packaging chip is provided with N electrode units in the longitudinal direction, the N electrode units form an electrode pair array, and N is a positive integer; the cell capturing and matching microfluidic packaging chip further comprises four groups of metal structure pairs which are respectively positioned at two sides of the cell capturing and matching microfluidic packaging chip, wherein the four groups of metal structure pairs comprise a first group of metal structure pairs connected with the first group of electrode pairs (20), a second group of metal structure pairs connected with the two shielding electrodes, a third group of metal structure pairs connected with the second group of electrode pairs (30) and a fourth group of metal structure pairs connected with the third group of electrode pairs (40).

9. The cell-capturing and mating microfluidic packaged chip of claim 8, wherein the cell-capturing and mating microfluidic packaged chip further comprises a glass substrate, and the electrode unit is located on the glass substrate.

10. The cell-capturing and mating microfluidic packaged chip according to claim 9, wherein the large microwell (10) and the small microwell (11, 12, 13) are used as a large-small nested microwell structure for cell fixation.

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