CN118146947A - Optical stimulation, electrical stimulation and concentration gradient combined regulation and control device and preparation method - Google Patents

Abstract

The invention discloses a light stimulation, electric stimulation and concentration gradient combined regulation and control device and a preparation method thereof, wherein the device comprises a light stimulation light intensity gradient chip layer, a cell capturing culture chip layer and a microelectrode layer; the cell capturing culture chip layer is provided with a plurality of main sample injection sections and a plurality of capturing culture channels; a plurality of groups of capturing culture cavities are arranged on the capturing culture channels, and a plurality of capturing structures are arranged in the capturing culture cavities; the light stimulus light intensity gradient chip layer is provided with a plurality of main liquid inlet sections and a plurality of light intensity adjusting channels, and each light intensity adjusting channel is provided with a light emphasis section; the interdigital electrode region on the microelectrode layer corresponds to the capture culture cavity position. Also comprises the preparation steps of S1-S5, etc. The optical stimulation, electrical stimulation and concentration gradient combined regulation and control device can realize integrated multi-factor combined stimulation based on a microfluidic platform, has the advantages of parallelization experiment, high integration level, simultaneous operation of multiple units, low pollution risk, low reagent loss and the like.

Description

Optical stimulation, electrical stimulation and concentration gradient combined regulation and control device and preparation method

Technical Field

The invention relates to the field of cell culture chips, in particular to a light stimulation, electric stimulation and concentration gradient combined regulation and control device and a preparation method thereof.

Background

Microfluidic chip technology originates from the micro-total analysis system proposed in Manz 1990 in its paper, with the aim of maximally transferring the functions of an analysis laboratory into a portable analysis device, even onto a centimeter-sized chip, through miniaturization and integration of chemical analysis devices. The microfluidic chip can be designed into multiple channels, liquid is split into multiple units through a network structure, the channels are not interfered with each other, and biochemical experiments of the multiple units can be performed simultaneously. The microchip structure is controllable, and control of microscopic objects (e.g., microparticles, cells) such as population cell studies can be achieved through design processing of the microstructures. The two-dimensional cell culture in the microfluidic chip has been widely applied to research on cell response and cell activity, and a cell culture system based on a microfluidic platform provides a relatively stable microenvironment for two-dimensional cell culture in a static and continuous perfusion state.

Although microfluidic chip cell culture techniques have been successfully used in drug screening and pathology research, there are some problems. In particular, in the aspect of microfluidic in-vitro culture of tumor cells, the cells need to be captured and cultured in a positioning way, and meanwhile, various physical and chemical influence factors need to be loaded, so that the growth and proliferation process of target cells is monitored, and subsequent metabolic products and genomics analysis are carried out. In the prior art, when the cells of the microfluidic chip are cultured on the in-vitro sheet, various physical and chemical influence factors cannot be loaded, so that better research on cell body differentiation and behavior response cannot be performed. Therefore, the micro-fluidic chip and a more efficient cell capturing structure are necessary to be combined to realize efficient and stable cell capturing and positioning culture, and a light stimulation, electric stimulation and concentration gradient combined regulation and control device and a preparation method are provided.

Disclosure of Invention

Aiming at the defects of the prior art, the invention provides a light stimulation, electric stimulation and concentration gradient combined regulation and control device and a preparation method thereof, which are used for solving the problems that in the prior art, when cells are cultured on an in-vitro sheet based on a microfluidic chip, multiple factors cannot be loaded and regulated, and research on cell body differentiation and behavior response cannot be realized.

In order to achieve the aim of the invention, the invention adopts the following technical scheme:

The device comprises a light stimulation light intensity gradient chip layer, a cell capturing culture chip layer and a microelectrode layer which are sequentially arranged from top to bottom; the cell capturing culture chip layer is provided with a main sample injection section and a capturing culture channel in series, the main sample injection section is provided with a plurality of capturing culture channels in parallel; each capturing culture channel is provided with a capturing culture section, a plurality of groups of capturing culture cavities are arranged on the capturing culture sections in parallel, each group of capturing culture cavities comprises two capturing culture cavities which are connected in parallel, and a plurality of capturing structures are arranged in each capturing culture cavity; the light stimulation light intensity gradient chip layer is provided with a main liquid inlet section and a light intensity adjusting channel in series, the main liquid inlet section is provided with a plurality of light intensity adjusting channels in parallel, each light intensity adjusting channel is provided with a light emphasis section, and the light emphasis sections correspond to the capturing culture cavity in position; the microelectrode layer is provided with a plurality of first main electrodes and a plurality of second main electrodes, and one ends of the second main electrodes are connected in parallel; the first main electrode is provided with a plurality of groups of first interdigital electrodes, the second main electrode is provided with a plurality of groups of second interdigital electrodes, the plurality of groups of first interdigital electrodes and the plurality of groups of second interdigital electrodes are intersected to form a plurality of interdigital electrode areas, and the interdigital electrode areas correspond to the capturing culture cavity.

Further, catch the structure and include the spill main part, be provided with two places openings on the spill main part, two places openings set up for the symmetry axis symmetry of spill main part.

Further, two sample inlets are arranged on the inlet side of the main sample inlet section in parallel, sample outlets are arranged on the outlet side of the plurality of capturing culture channels in parallel, the sample inlets are positioned at the top of the cell capturing culture chip layer, and the sample outlets are positioned at the bottom of the cell capturing culture chip layer; the inlet side of the main liquid inlet section is provided with two liquid inlets in parallel, and the outlet side of the sub liquid inlet section is provided with a liquid outlet in parallel.

Further, the capturing culture channel further comprises a sample dividing and feeding section and a sample dividing and discharging section, and the sample dividing and feeding section, the capturing culture section and the sample dividing and discharging section are connected in sequence; the light intensity adjusting channel further comprises a liquid separating and feeding section which is communicated with the light intensity adjusting section.

Further, the sample separating and feeding section and the liquid separating and feeding section are all serpentine micro-channels.

Further, the main sample injection section and the main liquid inlet section are both serpentine micro-channels.

Further, the number of main sampling sections is smaller than the number of capturing culture channels, and the number of main liquid inlet sections is smaller than the number of light intensity adjusting channels.

Further, the capturing culture cavity is a rectangular cavity, the inlet and outlet of the capturing culture cavity are arranged at diagonal positions, a plurality of rows of capturing structures are arranged along the width direction of the capturing culture cavity, a plurality of columns of capturing structures are arranged along the length direction of the capturing culture cavity, and capturing structures located in two adjacent columns are staggered.

A preparation method of a light stimulation, electrical stimulation and concentration gradient combined regulation device comprises the following steps:

s1: printing and preparing a light stimulus light intensity gradient chip layer by adopting a 3D printer;

S2, preparing a cell capturing culture chip layer;

S3, preparing a microelectrode layer by adopting a wet etching process;

S4: performing ultrasonic treatment and plasma cleaning on the light-stimulated light intensity gradient chip layer, the microelectrode layer and the cell capturing culture chip layer, wherein the plasma treatment time is 15-20s;

S5: bonding the light-stimulated light intensity gradient chip layer and the cell capturing culture chip layer at the bonding temperature of 100-120 ℃; and then reversely buckling the cell capturing and culturing chip layer on the microelectrode layer, and forming a closed cavity with the cell capturing and culturing chip layer by adopting a thermal bonding process, wherein the bonding temperature is 100-150 ℃.

Further, the preparation of the cell capturing culture chip layer in step S2 specifically includes:

s21, selecting a silicon wafer with the size of 3 inches, and spin-coating SU8-3025 photoresist;

s22, pre-baking the silicon wafer subjected to spin coating and spin coating, and then exposing the silicon wafer under a photoetching machine;

S23, coating a release agent after post-baking and hard-baking the exposed chip;

S24, pouring the PDMS with a certain proportion into a silicon wafer for reverse molding, and then placing the silicon wafer in an oven at 60-100 ℃ for curing;

s25, tearing off the cured PDMS, and adapting and cutting according to the shape of the microelectrode layer;

further, the preparation of the microelectrode layer in step S3 specifically includes:

S31, selecting quartz glass as a substrate of the microelectrode layer;

s32, spin-coating a layer of 5 mu mSU-83005 photoresist on quartz glass subjected to magnetron sputtering of 5-20nmCr and 100-400 nmAu;

S33, engraving an interdigital electrode microarray structure on the microelectrode layer through photoetching;

And S34, etching the interdigital electrode microarray structure on the microelectrode layer in a wet etching mode.

The beneficial effects of the invention are as follows:

the optical stimulation, electrical stimulation and concentration gradient combined regulation and control device can realize integrated multi-factor combined stimulation based on a microfluidic platform, has the advantages of parallelization experiment, high integration level, simultaneous operation of multiple units, low pollution risk, low reagent loss and the like.

The invention can realize the joint regulation and control of the response of the proliferation and differentiation processes of cells under the stimulation of multiple factors of light, electricity and concentration gradient on a microfluidic platform.

The cell to be captured enters the main sample injection section through the sample injection port, the movement track of the cell is controlled to a capturing structure in the capturing culture cavity by utilizing the fluid resistance under the control of the capturing culture channel and the flow, and then pulse signals are applied to perform intermittent electric stimulation; in this process, the cells are prevented from being washed away by the fluid due to the double slit arrangement of the capturing structure.

After the cells to be cultured enter the capturing structure of the capturing culture cavity along the capturing culture channel, external electric signals are introduced to the interdigital electrodes, and electric fields with enough strength are formed between adjacent microelectrodes, so that intermittent electric stimulation on cells cultured on a chip is realized, the growth and differentiation process of target cells is regulated, and the capturing structure can achieve capturing efficiency of more than 80% and capturing uniformity of more than 80%.

The uppermost layer of the invention is composed of light stimulus light intensity gradient chip layers of solutions with different concentrations based on absorbance regulation, the chip layer is prepared by a 3D printer, the upper end of the chip layer is composed of Christmas tree model snake-shaped micro-channels composed of a main liquid inlet section and a sub liquid inlet section, the solutions are passively mixed to generate solutions with four concentration gradients of 0, 1/3C and 2/3C, C, the solutions are introduced into four light emphasis sections, and the light stimulus with different light intensities can be generated in the 4 light emphasis sections based on different absorbance of the solutions with different concentrations.

The middle layer of the invention is composed of a cell capturing culture chip layer, the concentration gradient of 0, 1/3C and 2/3C, C is still generated by a Christmas tree model, the chip of the layer is provided with a 4 multiplied by 4 array capturing culture cavity, and two rows of single cell capturing structures are arranged in a single capturing culture cavity; the intermediate layer chip can realize cell body culture under different drug concentration gradients.

The lower electric field intensity gradient of the invention is composed of a microelectrode layer, a plurality of interdigital electrode groups are arranged on the microelectrode layer, the interdigital electrode groups form interdigital electrode areas, the electric stimulation with different field intensities is realized by a PCB circuit board of an alternating current sine wave electric signal which is externally connected with the microelectrode layer, and the amplitude and the frequency of the electric stimulation to cells are adjustable, corresponding to a 4 multiplied by 4 capturing culture cavity in a cell capturing culture chip layer.

The optical stimulation, electrical stimulation and concentration gradient combined regulation and control device can realize integrated multi-factor combined stimulation based on a microfluidic platform, has the advantages of parallelization experiment, high integration level, simultaneous operation of multiple units, low pollution risk, low reagent loss and the like.

The invention can realize the joint regulation of the response of the proliferation and differentiation processes of the cells under the stimulation of light stimulation, electric stimulation and concentration gradient.

Drawings

FIG. 1 is a schematic diagram of the overall structure of the present invention;

FIG. 2 is a schematic diagram of the structure of a cell capture culture chip layer;

FIG. 3 is a schematic diagram of the structure of a light intensity gradient chip layer;

FIG. 4 is a schematic structural view of a microelectrode layer;

FIG. 5 is a partial enlarged view A of FIG. 2;

FIG. 6 is a schematic structural view of a capture structure;

FIG. 7 is a diagram of a physical experiment of a capture structure;

FIG. 8 is a diagram of a second embodiment of a capture structure;

FIG. 9 is a graph of cell response experiments with four paths of signals applied at different frequencies under the condition of the same stimulation amplitude of 2V in the electrical stimulation module;

FIG. 10 is a graph of intensity gradient experiments;

The main component symbols in the drawings are described as follows:

1. A cell capture culture chip layer; 11. a sample inlet; 12. a main sample injection section; 13. capturing a culture channel; 131. dividing a sample introduction section; 132. separating out sample sections; 133. capturing a culture section; 134. capturing a culture cavity; 135. a capture structure; 1351. a concave body; 1352. a notch; 14. a sample outlet;

2. A light-stimulated light intensity gradient chip layer; 21. a liquid inlet; 22. a main liquid inlet section; 23. a light intensity adjusting channel; 231. a liquid inlet section is divided; 232. a light emphasizing segment; 24. a liquid outlet;

3. a microelectrode layer; 31. a first main electrode; 32. a first interdigital electrode; 33. a second main electrode; 34. a second interdigital electrode; 35. interdigital electrode regions.

Detailed Description

The following description of the embodiments of the present invention is provided to facilitate understanding of the present invention by those skilled in the art, but it should be understood that the present invention is not limited to the scope of the embodiments, and all the inventions which make use of the inventive concept are protected by the spirit and scope of the present invention as defined and defined in the appended claims to those skilled in the art.

As shown in FIG. 1, the optical stimulation, electrical stimulation and concentration gradient combined regulation device and the preparation method thereof comprise an optical stimulation light intensity gradient chip layer 2, a cell capturing and culturing chip layer 1 and a microelectrode layer 3 which are sequentially arranged from top to bottom, and FIG. 2 is a layered diagram of the cell capturing and culturing chip layer and the optical stimulation light intensity gradient chip layer.

As shown in fig. 2, a main sample injection section 12 and a capturing culture channel 13 are arranged on the cell capturing culture chip layer 1 in series, a plurality of main sample injection sections 12 are arranged in parallel, and a plurality of capturing culture channels 13 are arranged in parallel. The number of main injection sections 12 is smaller than the number of capture culture channels 13. In this embodiment, the main sample section 12 is preferably provided with three positions, and the capturing culture channels 13 are preferably provided with four positions, and the three main sample sections 12 and the four capturing culture channels 13 are in a christmas tree structure. The capturing culture channels 13 are provided with capturing culture sections 133, the capturing culture sections 133 are provided with a plurality of groups of capturing culture cavities 134 in parallel, each group of capturing culture cavities 134 comprises two capturing culture cavities 134 which are connected in parallel, and a plurality of capturing structures 135 are arranged in each capturing culture cavity 134. The capturing culture channel 13 further comprises a sample separating section 131 and a sample separating section 132, and the sample separating section 131, the capturing culture section 133 and the sample separating section 132 are sequentially connected. The light intensity adjusting channel 23 further comprises a liquid-separating and feeding section 231, and the liquid-separating and feeding section 231 is communicated with the light intensity adjusting section 232. The main sample section 12, the sub-sample section 131 and the sub-sample section 132 are all serpentine micro-channels.

As shown in fig. 3, a main liquid inlet section 22 and a light intensity adjusting channel 23 are serially arranged on the light stimulation light intensity gradient chip layer 2, a plurality of light intensity adjusting channels 23 are parallelly arranged on the main liquid inlet section 22, a plurality of light intensity adjusting channels 23 are parallelly arranged on each light intensity adjusting channel 23, a light intensity adjusting section 232 is arranged on each light intensity adjusting channel 23, the light intensity adjusting sections 232 correspond to the positions of the capturing culture cavities 134, the light intensity adjusting sections 232 are rectangular micro channels, the length of each rectangular micro channel is 12000 μm, the width is 1000 μm, and the depth is 500 μm; the solution is passively mixed to generate four concentration gradient solutions of 0, 1/3C and 2/3C, C, and the four concentration gradient solutions are introduced into the rectangular micro-channels, so that light stimuli with different light intensities are generated in the 4 rectangular micro-channels based on different absorbance of the solutions with different concentrations. The split intake section 231 and the main intake section 22 are serpentine micro-channels. Two liquid inlets 21 are arranged on the inlet side of the main liquid inlet section 22 in parallel, and a liquid outlet 24 is arranged on the outlet side of the sub liquid inlet section 231 in parallel; the light intensity gradient experimental diagram is shown in fig. 10. Fig. 9 is a graph of cell response experiments of four paths of signals applied with different frequencies under the same amplitude condition after initial verification of the optimal stimulation amplitude of 2V in the electric stimulation module.

In this embodiment, two sample inlets 11 are arranged in parallel on the inlet side of the main sample section 12, and sample outlets 14 are arranged in parallel on the outlet side of the plurality of capturing culture channels 13, the sample inlets 11 are positioned at the top of the cell capturing culture chip layer 1, and the sample outlets 14 are positioned at the bottom of the cell capturing culture chip layer 1. The two sample inlets 11 of the cell capturing culture chip layer 1 are connected with the main sample inlet section 12, the channel width is 100-150 mu m, the main sample inlet section 12 is a serpentine micro-channel, the main sample inlet section 12 can realize drug concentration gradients of 0, 1/3C and 2/3C, C, and the serpentine part of the multiple serpentine channels can realize better solution mixing effect. The diameters of the sample inlet 11 and the sample outlet 14 are 2-5 mm, and the sizes of the sample inlet 11 and the sample outlet 14 are designed mainly for matching with the external pressure pump pipeline required by the test. The number of the main liquid inlet sections 22 is smaller than that of the light intensity adjusting channels 23, the main liquid inlet sections 22 are preferably three, the light intensity adjusting channels 23 are preferably four, and the three main liquid inlet sections 22 and the four light intensity adjusting channels 23 form a Christmas tree structure.

In the present embodiment, the height of the cell-capturing culture chip layer 1 is 20 to 25 μm, and setting the height of the cell-capturing culture chip layer 1 to 20 to 25 μm can ensure smooth flow of cells without occurrence of a phenomenon in which a plurality of cells overlap in the longitudinal direction to affect counting.

As shown in fig. 4, the microelectrode layer 3 is preferably made of gold, platinum, ITO and other materials, the microelectrode layer 3 is externally connected with a PCB board through a conductive adhesive tape, an external electrical signal is introduced to the Au interdigital electrode, an electric field with sufficient strength is formed between adjacent microelectrodes, the gradient loading of the electric field strength of the chip in the on-chip culture process is realized, the main function of the cell capturing and culturing chip layer 1 is to realize the sample injection of a cell buffer in the structure, the gradient loading of the concentration of the drug is realized through a serpentine channel, the solution is discharged from a sample outlet 14 after capturing cells after entering a cavity, and the whole flow path control module comprises the cell capturing and culturing chip layer 1 and a catheter. The microelectrode layer 3 is provided with a plurality of first main electrodes 31 and a plurality of second main electrodes 33, and one ends of the plurality of second main electrodes 33 are arranged in parallel. The first main electrode 31 is provided with a plurality of groups of first interdigital electrodes 32, the second main electrode 33 is provided with a plurality of groups of second interdigital electrodes 34, the plurality of groups of first interdigital electrodes 32 and the plurality of groups of second interdigital electrodes 34 are intersected to form a plurality of interdigital electrode areas 35, and the interdigital electrode areas 35 correspond to the positions of the capturing culture cavities 134. The width of the interdigital electrode is 100-200 mu m, the interdigital distance is 200-400 mu m, and the electric stimulation of different field strengths is realized by connecting the Au microelectrode with the PCB of the alternating current sine wave electric signal, so that the amplitude and the frequency of the electric stimulation to the cells are adjustable. The comb tooth distance of the interdigital electrode array is controlled within the range of 100-200 mu m so as to ensure good conductivity and reliability; the comb tooth width is 50-100 μm, and can be determined according to the density of microelectrode array in practical use.

As shown in fig. 5, 6, 7 and 8, the capturing culture cavity 134 is a rectangular cavity, the inlet and outlet of the capturing culture cavity 134 are arranged at diagonal positions, a plurality of capturing culture cavities 134 are in a 4×4 array structure, two rows of single-cell capturing structures 135 are arranged in a single capturing culture cavity 134, each row of single-cell capturing structures 135 is provided with 5 rows, 4 rows of capturing structures 135 are arranged along the width direction of the capturing culture cavity 134, 4 columns of capturing structures 135 are arranged along the length direction of the capturing culture cavity 134, and capturing structures 135 positioned in two adjacent columns are staggered, and the staggered displacement is 50-60 μm. To ensure that the flow rate of the fluid to each row of chambers is the same, the width of the capture culture channels 13 is preferably set to 100-150 μm, and cells can be uniformly dispersed to the sample inlet of each capture culture chamber 134. The cell capturing culture chip layer 1 can realize in-vitro culture of cells under different drug concentration gradients. The capturing structure 135 comprises a concave main body 1351, wherein two openings 1352 are formed in the concave main body 1351, and the two openings 1352 are symmetrically arranged relative to the symmetry axis of the concave main body 1351. The inclination angle of the opening of the concave main body 1351 is preferably 55-65 degrees, the inclination angle of the opening has an important influence on the cell capturing efficiency, and the cell capturing efficiency is reduced due to the fact that the inclination angle is too large or too small, so that the cell quantity consistency inside each cavity is low during culture, and in addition, the cell can be prevented from escaping from a capturing structure under the action of a flow field through the adjustment of the inclination angle of the opening, so that the stable capturing of the cell is realized. The capturing structures 135 have a transverse spacing of 40-50 μm and a longitudinal spacing of 70-80 μm, and by adjusting the row-column spacing of the capturing structures 135, the capturing structures have a shunting effect in the capturing process, so that the cells can conveniently shuttle to the corresponding capturing structures according to the expected streamline. Two openings 1352 are formed in the concave main body 1351, the width of each opening 1352 is 5-10 μm, the flow resistance of fluid is matched through the openings with the double-slit width of 5-10 μm, the cells can be ensured to smoothly enter the capturing structure 135 and clamped at the positions of the openings 1352, and redundant cells flow to the capturing structure 135 which is emptied next.

In this embodiment, the capturing culture cavity 134 has a diagonal rectangular geometry, the length is set to 1000-1200 μm, the width is set to 600-800 μm, and the inlet and outlet of the capturing culture cavity 134 are designed to be diagonal in and out, so that a capturing load with spatial distribution is provided, the spatial distribution can be more uniformly captured, the spatial distribution of the verified cells after capturing is more uniform, and the capturing uniformity is better.

The specific implementation principle of the optical stimulation, electrical stimulation and concentration gradient combined regulation and control device is as follows: the cells to be captured enter the channel through the two sample inlets 11, enter the capture culture cavity 134 through the main sample inlet section 12 and the capture culture channel 13, capture the cells through fluid resistance, count after the completion of the sample injection process of the cell suspension, add culture medium into the cell capture culture chip layer 1 through a continuous perfusion mode, control the flow rate at 4 mu l/min, and meanwhile, access the bottom microelectrode layer 3 to an electric signal to perform intermittent electric stimulation on the cells, and place the cell capture culture chip layer 1 in an incubator for culture.

A preparation method of a light stimulation, electrical stimulation and concentration gradient combined regulation device comprises the following steps:

S1: the 3D printer is adopted to print and prepare the light-stimulated light intensity gradient chip layer 2, and the photoetching technology can also be adopted to prepare the light-stimulated light intensity gradient chip layer 2;

s2, preparing a cell capturing culture chip layer 1, which is specifically as follows;

s21, selecting a silicon wafer with the size of 3 inches, and spin-coating SU8-3025 photoresist;

s22, pre-baking the silicon wafer subjected to spin coating and spin coating, and then exposing the silicon wafer under a photoetching machine;

S23, coating a release agent after post-baking and hard-baking the exposed chip;

s24, pouring the PDMS with a certain proportion into a silicon wafer for reverse molding, and then placing the silicon wafer into an oven with the temperature of 60-100 ℃ for curing, wherein the temperature of the oven is preferably 60 ℃;

S25, removing the cured PDMS, and adapting and cutting according to the shape of the microelectrode layer 3;

s3, preparing a microelectrode layer 3 by adopting a wet etching process, wherein the preparation method is as follows;

s31, quartz glass is selected as a substrate of the microelectrode layer 3;

s32, spin-coating a layer of 5 mu mSU-83005 photoresist on quartz glass subjected to magnetron sputtering of 5-20 nmCr and 100-400nmAu, wherein the thickness of Cr is preferably 7nmCr, and the thickness of Au is preferably 200nmAu;

S33, engraving an interdigital electrode microarray structure on the microelectrode layer 3 through photoetching;

S34, etching an interdigital electrode microarray structure on the microelectrode layer in a wet etching mode;

S4: performing ultrasonic treatment and plasma cleaning on the light-stimulated light intensity gradient chip layer 2, the microelectrode layer 3 and the cell capturing culture chip layer 1, wherein the plasma treatment time is 15-20s; the plasma cleaning time mainly influences the bonding effect of the cell capturing culture chip layer 1 and the microelectrode layer 3, too tight bonding can be caused if the cleaning time is too long, cell liquid is not easy to enter the structure, and leakage is not easy to occur if the cleaning time is too short; the purpose of respectively carrying out ultrasonic treatment and plasma treatment on the microelectrode layer 3 and the cell capturing culture chip layer 1 before bonding is to introduce hydrophilic-OH groups into the cell capturing culture chip layer 1 to make the cell capturing culture chip layer hydrophilic, and simultaneously change the chemical bonds of the microelectrode layer 3 to enable the microelectrode layer 3 and the cell capturing culture chip layer 3 to complete irreversible bonding, so that the surfaces of an electrode and a chip can be cleaned through ultrasonic treatment; the ultrasonic treatment condition is that three distilled water is utilized for ultrasonic treatment for 10-20 min, nitrogen is used for blow-drying after ultrasonic treatment, the ultrasonic treatment time is the optimal time length through practical verification, if the cleaning time is too short, the cleaning effect is poor, in addition, as the three distilled water is pure water, the interference of other ions can be avoided, and the cleaning effect of chips is ensured;

S5: bonding the light-stimulated light intensity gradient chip layer 2 with the cell capturing culture chip layer 1 at the bonding temperature of 100-120 ℃; and then the cell capturing and culturing chip layer 1 is reversely buckled on the microelectrode layer 3, a thermal bonding process is adopted to form a closed cavity with the cell capturing and culturing chip layer 1, the closed cavity only carries out sample inlet and outlet of cell suspension through the sample inlet 11 and the sample outlet 14, the bonding temperature is 100-150 ℃, and the bonding temperature of 100-150 ℃ is a proper temperature, so that the bonding effect can be ensured to reach the expected effect.

Claims (9)

1. The device is characterized by comprising a light stimulation light intensity gradient chip layer (2), a cell capturing culture chip layer (1) and a microelectrode layer (3) which are sequentially arranged from top to bottom;

The cell capturing and culturing chip layer (1) is provided with a main sample injection section (12) and capturing and culturing channels (13) in series, the main sample injection section (12) is provided with a plurality of parallel connection, and the capturing and culturing channels (13) are provided with a plurality of parallel connection; each capturing culture channel (13) is provided with a capturing culture section (133), a plurality of groups of capturing culture cavities (134) are arranged on the capturing culture sections (133) in parallel, each group of capturing culture cavities (134) comprises two capturing culture cavities (134) which are connected in parallel, and a plurality of capturing structures (135) are arranged in each capturing culture cavity (134);

The light stimulation light intensity gradient chip layer (2) is provided with a main liquid inlet section (22) and light intensity regulating channels (23) in series, the main liquid inlet section (22) is provided with a plurality of light intensity regulating channels (23) in parallel, each light intensity regulating channel (23) is provided with a light emphasis section (232), and the light intensity regulating sections (232) correspond to the capturing culture cavity (134) in position;

A plurality of first main electrodes (31) and a plurality of second main electrodes (33) are arranged on the microelectrode layer (3), and one ends of the second main electrodes (33) are connected in parallel; a plurality of groups of first interdigital electrodes (32) are arranged on the first main electrode (31), a plurality of groups of second interdigital electrodes (34) are arranged on the second main electrode (33), a plurality of groups of first interdigital electrodes (32) and a plurality of groups of second interdigital electrodes (34) are intersected to form a plurality of interdigital electrode areas (35), and the interdigital electrode areas (35) correspond to the positions of the capturing culture cavities (134).

2. The combined light stimulation, electrical stimulation and concentration gradient regulation and control device and the preparation method according to claim 1, wherein the capturing structure (135) comprises a concave main body (1351), two openings (1352) are formed in the concave main body (1351), and the two openings (1352) are symmetrically arranged relative to the symmetry axis of the concave main body (1351).

3. The optical stimulation, electrical stimulation and concentration gradient combined regulation and control device and the preparation method according to claim 1 are characterized in that two sample inlets (11) are arranged on the inlet side of the main sample inlet section (12) in parallel, sample outlets (14) are arranged on the outlet side of the plurality of capturing culture channels (13) in parallel, the sample inlets (11) are positioned at the top of the cell capturing culture chip layer (1), and the sample outlets (14) are positioned at the bottom of the cell capturing culture chip layer (1); two liquid inlets (21) are arranged on the inlet side of the main liquid inlet section (22) in parallel, and a liquid outlet (24) is arranged on the outlet side of the sub liquid inlet section (231) in parallel.

4. The optical stimulation, electrical stimulation and concentration gradient combined regulation and control device and the preparation method according to claim 1, wherein the capturing culture channel (13) further comprises a sample separating section (131) and a sample separating section (132), and the sample separating section (131), the capturing culture section (133) and the sample separating section (132) are sequentially connected; the light intensity adjusting channel (23) further comprises a liquid separating and feeding section (231), and the liquid separating and feeding section (231) is communicated with the light intensity adjusting section (232).

5. The combined light stimulation, electrical stimulation and concentration gradient control device and the preparation method according to claim 4, wherein the sample separating and feeding section (131), the sample separating and feeding section (132) and the liquid separating and feeding section (231) are all serpentine micro-channels.

6. The combined optical stimulation, electrical stimulation and concentration gradient control device and the preparation method according to claim 1, wherein the main sample injection section (12) and the main liquid inlet section (22) are all serpentine micro-channels.

7. The light stimulation, electrical stimulation and concentration gradient combined regulation and control device and the preparation method according to claim 1, wherein the capturing culture cavity (134) is a rectangular cavity, an inlet and an outlet of the capturing culture cavity (134) are arranged at diagonal positions, a plurality of rows of capturing structures (135) are arranged along the width direction of the capturing culture cavity (134), a plurality of columns of capturing structures (135) are arranged along the length direction of the capturing culture cavity (134), and the capturing structures (135) positioned in two adjacent columns are arranged in a staggered mode.

8. A method for preparing the optical stimulation, electrical stimulation and concentration gradient combined regulation and control device according to any one of claims 1-7, which is characterized by comprising the following steps:

s1: printing and preparing a light stimulation light intensity gradient chip layer (2) by adopting a 3D printer;

s2, preparing a cell capturing culture chip layer (1);

s3, preparing a microelectrode layer (3) by adopting a wet etching process;

S4: carrying out ultrasonic treatment and plasma cleaning on the light-stimulated light intensity gradient chip layer (2), the microelectrode layer (3) and the cell capturing culture chip layer (1), wherein the plasma treatment time is 15-20s;

S5: bonding the light-stimulated light intensity gradient chip layer (2) with the cell capturing culture chip layer (1) at the bonding temperature of 100-120 ℃; and then the cell capturing and culturing chip layer (1) is reversely buckled on the microelectrode layer (3), a thermal bonding process is adopted to form a closed cavity with the cell capturing and culturing chip layer (1), and the bonding temperature is 100-150 ℃.

9. The device for combined regulation and control of optical stimulation, electrical stimulation and concentration gradient and the preparation method according to claim 8, wherein the preparation of the cell capturing culture chip layer (1) in step S2 specifically comprises:

s21, selecting a silicon wafer with the size of 3 inches, and spin-coating SU8-3025 photoresist;

s22, pre-baking the silicon wafer subjected to spin coating and spin coating, and then exposing the silicon wafer under a photoetching machine;

S23, coating a release agent after post-baking and hard-baking the exposed chip;

S24, pouring the PDMS with a certain proportion into a silicon wafer for reverse molding, and then placing the silicon wafer in an oven at 60-100 ℃ for curing;

S25, removing the cured PDMS, and adapting and cutting according to the shape of the microelectrode layer (3);

The preparation of the microelectrode layer (3) in the step S3 specifically comprises the following steps:

s31, quartz glass is selected as a substrate of the microelectrode layer (3);

s32, spin-coating a layer of 5 mu mSU-83005 photoresist on quartz glass subjected to magnetron sputtering of 5-20nmCr and 100-400 nmAu;

S33, engraving an interdigital electrode microarray structure on the microelectrode layer (3) through photoetching;

And S34, etching the interdigital electrode microarray structure on the microelectrode layer in a wet etching mode.