A light stimulation, electrical stimulation, and concentration gradient combined regulation device and a preparation method thereof
By designing a device that combines photostimulation, electrostimulation, and concentration gradient regulation in a microfluidic chip, the problem of microfluidic chips being unable to load multiple factors during in vitro cell culture was solved, achieving efficient and integrated cell differentiation regulation, and improving cell capture efficiency and the ability to regulate multiple factors in experiments.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- CHONGQING UNIV
- Filing Date
- 2024-03-21
- Publication Date
- 2026-08-04
AI Technical Summary
Existing microfluidic chips cannot load various physical and chemical influencing factors during cell in vitro culture, thus hindering effective research on cell differentiation and behavioral responses.
Design a device for the combined regulation of photostimulation, electrical stimulation, and concentration gradient, comprising a photostimulation intensity gradient chip layer, a cell capture culture chip layer, and a microelectrode layer, which is fabricated by 3D printing and wet etching processes to achieve the combined regulation of multiple factors.
This enables efficient and integrated control of cell proliferation and differentiation processes on a microfluidic platform, reducing contamination risks and reagent consumption, and improving cell capture efficiency and uniformity.
Smart Images
Figure CN118146947B_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of cell culture chips, and more particularly to a device and preparation method for the combined regulation of light stimulation, electrical stimulation, and concentration gradient. Background Technology
[0002] Microfluidic chip technology originated from the micrototal analysis system proposed by Manz in his 1990 paper. Its aim was to maximize the transfer of analytical laboratory functions to portable analytical devices, even integrating them onto chips the size of centimeters, through miniaturization and integration of chemical analysis equipment. Microfluidic chips can be designed with multiple channels, using a network structure to distribute liquids to multiple units without interference between channels, allowing for simultaneous biochemical experiments in multiple units. The structure of microchips is controllable, enabling the control of microscopic objects (such as particles and cells) through microstructure design and fabrication, for example, in population cell studies. Two-dimensional cell culture within microfluidic chips has been widely used to study cell responses and cell viability. Cell culture systems based on microfluidic platforms provide a relatively stable microenvironment for two-dimensional cell culture under static and continuous perfusion conditions. Due to the advantages of microfluidic chips, such as requiring fewer reagents, high-throughput analysis, and automation, they are increasingly used in in vitro cell culture research.
[0003] Although microfluidic chip cell culture technology has been successfully applied in drug screening and pathological research, some problems still exist. Especially in the microfluidic in vitro culture of tumor cells, it is necessary to capture and culture cells locally, while simultaneously loading various physical and chemical influencing factors to monitor the growth and proliferation of target cells and perform subsequent metabolite and genomic analysis. Current microfluidic chip cell culture techniques cannot load multiple physical and chemical influencing factors during in vitro cell culture, thus hindering better research on cell differentiation and behavioral responses. Therefore, it is necessary to combine microfluidic chips with more efficient cell capture structures to achieve efficient and stable cell capture and localization culture, proposing a device and preparation method for the combined regulation of light stimulation, electrical stimulation, and concentration gradients. Summary of the Invention
[0004] To address the aforementioned shortcomings of existing technologies, this invention provides a device and preparation method for the combined regulation of light stimulation, electrical stimulation, and concentration gradient, thereby solving the problem that existing technologies based on microfluidic chips cannot load and regulate multiple factors during in vitro on-chip cell culture, thus hindering the study of cell differentiation and behavioral responses.
[0005] To achieve the above-mentioned objectives, the technical solution adopted by this invention is as follows:
[0006] A device for combined regulation of photostimulation, electrical stimulation, and concentration gradient is provided, comprising, from top to bottom, a photostimulation intensity gradient chip layer, a cell capture culture chip layer, and a microelectrode layer; a main sample injection section and a capture culture channel are connected in series on the cell capture culture chip layer, several main sample injection sections are connected in parallel, and several capture culture channels are connected in parallel; each capture culture channel is provided with a capture culture section, and several sets of capture culture chambers are connected in parallel on the capture culture section, each set of capture culture chambers includes two parallel capture culture chambers, and several capture structures are provided in each capture culture chamber; the photostimulation intensity gradient chip layer is connected in series on... The system is equipped with a main liquid inlet section and a light intensity regulating channel. Several main liquid inlet sections and several light intensity regulating channels are connected in parallel. Each light intensity regulating channel is equipped with a light intensity segment, which corresponds to the position of the capture culture chamber. Several first main electrodes and several second main electrodes are provided on the microelectrode layer. One end of each of the second main electrodes is connected in parallel. Several sets of first interdigital electrodes are provided on the first main electrodes, and several sets of second interdigital electrodes are provided on the second main electrodes. The several sets of first interdigital electrodes and several sets of second interdigital electrodes intersect to form several interdigital electrode regions, which correspond to the position of the capture culture chamber.
[0007] Furthermore, the capturing structure includes a concave body with two notches, which are symmetrically arranged with respect to the axis of symmetry of the concave body.
[0008] Furthermore, the main sample inlet section has two parallel inlet ports on its inlet side, and several capture culture channels have parallel outlet ports on their outlet sides. The inlet ports are located at the top of the cell capture culture chip layer, and the outlet ports are located at the bottom of the cell capture culture chip layer. The main liquid inlet section has two parallel liquid inlet ports on its inlet side, and the sub-liquid inlet section has parallel outlet ports on its outlet side.
[0009] Furthermore, the capture culture channel also includes a sample inlet section and a sample outlet section, which are connected sequentially; the light intensity modulation channel also includes a liquid inlet section, which is connected to the light intensity modulation section.
[0010] Furthermore, the sample inlet section, sample outlet section, and liquid inlet section are all serpentine microchannels.
[0011] Furthermore, both the main sample inlet section and the main liquid inlet section are serpentine microchannels.
[0012] Furthermore, the number of main sample inlet sections is less than the number of capture culture channels, and the number of main liquid inlet sections is less than the number of light intensity modulation channels.
[0013] Furthermore, the capture culture chamber is a rectangular chamber, with the inlet and outlet of the capture culture chamber located at diagonal positions. Several rows of capture structures are arranged along the width direction of the capture culture chamber, and several columns of capture structures are arranged along the length direction of the capture culture chamber. The capture structures located in adjacent columns are staggered.
[0014] A method for preparing a device for combined regulation of light stimulation, electrical stimulation, and concentration gradient includes the following steps:
[0015] S1: A photostimulated light intensity gradient chip layer was prepared by printing with a 3D printer;
[0016] S2. Prepare the cell capture culture chip layer;
[0017] S3. The microelectrode layer is prepared using a wet etching process;
[0018] S4: The light-stimulated light intensity gradient chip layer, microelectrode layer and cell capture culture chip layer are subjected to ultrasonic treatment and plasma cleaning. The plasma treatment time is 15-20s.
[0019] S5: First, bond the photostimulation intensity gradient chip layer to the cell capture culture chip layer at a bonding temperature of 100-120℃; then, place the cell capture culture chip layer upside down on the microelectrode layer and use a thermal bonding process to form a sealed cavity with the cell capture culture chip layer at a bonding temperature of 100-150℃.
[0020] Furthermore, the preparation of the cell capture culture chip layer in step S2 specifically includes:
[0021] S21. Select a 3-inch silicon wafer and spin-coat it with SU8-3025 photoresist.
[0022] S22. After spin coating and uniform coating, the silicon wafer is pre-baked and then exposed under a photolithography machine.
[0023] S23. After exposure, the chip is post-baked and hard-baked, then coated with a release agent;
[0024] S24. After preparing a certain proportion of PDMS, pour it into a silicon wafer mold and then place it in an oven at 60℃~100℃ for curing.
[0025] S25. Peel off the cured PDMS and cut it to fit the shape of the microelectrode layer.
[0026] Furthermore, the fabrication of the microelectrode layer in step S3 specifically includes:
[0027] S31. Quartz glass is selected as the substrate for the microelectrode layer.
[0028] S32. Spin-coat a 5μm layer of SU-83005 photoresist onto a quartz glass plate with 5-20nm Cr and 100-400nm Au sputtered by magnetron sputtering.
[0029] S33. The interdigitated electrode microarray structure is etched on the microelectrode layer by photolithography;
[0030] S34. The interdigitated electrode microarray structure is etched on the microelectrode layer by wet etching.
[0031] The beneficial effects of this invention are as follows:
[0032] The photostimulation, electrostimulation, and concentration gradient combined control device of the present invention can realize integrated multi-factor combined stimulation based on a microfluidic platform, parallel experiments, high integration, simultaneous operation of multiple units, low risk of contamination, and low reagent loss.
[0033] This invention enables the combined regulation of the cell proliferation and differentiation process under the stimulation of multiple factors such as light, electricity, and concentration gradient on a microfluidic platform.
[0034] In this invention, the cells to be captured enter the main injection section through the injection port. The cell movement trajectory is controlled by fluid resistance through the capture culture channel and flow control to the capture structure in the capture culture chamber. Then, pulse signals are applied for intermittent electrical stimulation. During this process, the double-slit notch of the capture structure can prevent the cells from being washed away by the fluid.
[0035] In this invention, after the cells to be cultured enter the capture culture chamber through the capture culture channel, an external electrical signal is introduced to the interdigital electrodes. An electric field of sufficient strength will be formed between adjacent microelectrodes to achieve intermittent electrical stimulation of the cells cultured on the sheet, thereby regulating the growth and differentiation process of the target cells. The capture structure can achieve a capture efficiency of more than 80% and a capture uniformity of more than 80%.
[0036] The top layer of this invention consists of a photostimulation intensity gradient chip layer with different concentration solutions controlled by absorbance. This chip layer is fabricated by a 3D printer. The upper end is composed of a Christmas tree-shaped serpentine microchannel consisting of a main liquid inlet section and sub-liquid inlet sections. After passive mixing, the solutions generate four concentration gradient solutions of 0, 1 / 3C, 2 / 3C, and C, which are then introduced into four photostimulation intensity segments. Based on the different absorbance of the solutions with different concentrations, photostimulation with different light intensities can be generated in the four photostimulation intensity segments.
[0037] The intermediate layer of this invention is composed of a cell capture culture chip layer. The concentration gradients of 0, 1 / 3C, 2 / 3C, and C are still generated by the Christmas tree model. This chip layer has a 4×4 array of capture culture chambers, and each capture culture chamber has two rows of single-cell capture structures. The intermediate chip layer can realize cell culture under different drug concentration gradients.
[0038] The lower electric field intensity gradient of the present invention is composed of a microelectrode layer, on which a plurality of interdigital electrode groups are provided. The interdigital electrode groups form an interdigital electrode region, corresponding to a 4×4 capture culture cavity in the cell capture culture chip layer. The amplitude and frequency of the electrical stimulation of the cells can be adjusted by a PCB circuit board with an AC sinusoidal electrical signal connected to the microelectrode layer.
[0039] The photostimulation, electrostimulation, and concentration gradient combined control device of the present invention can realize integrated multi-factor combined stimulation based on a microfluidic platform, parallel experiments, high integration, simultaneous operation of multiple units, low risk of contamination, and low reagent loss.
[0040] This invention enables the combined regulation of cell proliferation and differentiation processes in response to light stimulation, electrical stimulation, and concentration gradient stimulation. Attached Figure Description
[0041] Figure 1 This is a schematic diagram of the overall structure of the present invention;
[0042] Figure 2 A schematic diagram of the structure of a cell capture and culture chip layer;
[0043] Figure 3 This is a schematic diagram of the structure of a light-stimulated light intensity gradient chip layer.
[0044] Figure 4 This is a schematic diagram of the microelectrode layer structure;
[0045] Figure 5 for Figure 2 A magnified view of a portion of the image;
[0046] Figure 6 A schematic diagram of the structure for capturing the structure;
[0047] Figure 7 Physical experiments to capture the structure Figure 1 ;
[0048] Figure 8 Physical experiments to capture the structure Figure 2 ;
[0049] Figure 9 Experimental diagram of cell response when four signals of different frequencies are applied under the same stimulation amplitude of 2V in the electrical stimulation module;
[0050] Figure 10 This is a diagram of an experiment on light intensity gradient;
[0051] The symbols for the main components in the diagram are explained below:
[0052] 1. Cell capture culture chip layer; 11. Sample inlet; 12. Main sample inlet section; 13. Capture culture channel; 131. Sample inlet section; 132. Sample outlet section; 133. Capture culture section; 134. Capture culture chamber; 135. Capture structure; 1351. Concave main body; 1352. Notch; 14. Sample outlet;
[0053] 2. Photostimulation intensity gradient chip layer; 21. Liquid inlet; 22. Main liquid inlet section; 23. Light intensity modulation channel; 231. Sub-liquid inlet section; 232. Light intensity modulation section; 24. Liquid outlet;
[0054] 3. Microelectrode layer; 31. First main electrode; 32. First interdigitated electrode; 33. Second main electrode; 34. Second interdigitated electrode; 35. Interdigitated electrode region. Detailed Implementation
[0055] The specific embodiments of the present invention are described below to enable those skilled in the art to understand the present invention. However, it should be understood that the present invention is not limited to the scope of the specific embodiments. For those skilled in the art, various changes are obvious as long as they are within the spirit and scope of the present invention as defined and determined by the appended claims. All inventions utilizing the concept of the present invention are protected.
[0056] like Figure 1 As shown, a device and method for jointly regulating photostimulation, electrical stimulation, and concentration gradient includes, from top to bottom, a photostimulation intensity gradient chip layer 2, a cell capture and culture chip layer 1, and a microelectrode layer 3. Figure 2 Layered diagram of cell capture culture chip layer and light stimulation intensity gradient chip layer.
[0057] like Figure 2As shown, a main sample inlet section 12 and a capture culture channel 13 are connected in series on the cell capture culture chip layer 1. Several main sample inlet sections 12 and several capture culture channels 13 are connected in parallel. The number of main sample inlet sections 12 is less than the number of capture culture channels 13. In this embodiment, three main sample inlet sections 12 and four capture culture channels 13 are preferably provided, forming a Christmas tree shape. Each capture culture channel 13 is provided with a capture culture section 133, and several sets of capture culture chambers 134 are connected in parallel on the capture culture section 133. Each set of capture culture chambers 134 includes two parallel capture culture chambers 134, and several capture structures 135 are provided in each capture culture chamber 134. The capture culture channel 13 also includes a sample inlet section 131 and a sample outlet section 132, which are connected sequentially. The light intensity modulation channel 23 also includes a liquid inlet section 231, which is connected to the light intensity modulation section 232. The main sample inlet section 12, the liquid inlet section 131, and the sample outlet section 132 are all serpentine microchannels.
[0058] like Figure 3 As shown, a main liquid inlet section 22 and a light intensity regulating channel 23 are connected in series on the photostimulation intensity gradient chip layer 2. Several main liquid inlet sections 22 and several light intensity regulating channels 23 are connected in parallel. Each light intensity regulating channel 23 is provided with a light intensity regulating segment 232, which corresponds to the position of the capture culture chamber 134. The light intensity regulating segment 232 is a rectangular microchannel with a length of 12000 μm, a width of 1000 μm, and a depth of 500 μm. After passive mixing, the solution generates four concentration gradient solutions of 0, 1 / 3 C, 2 / 3 C, and C, which are introduced into the rectangular microchannels. Based on the different absorbance of the solutions of different concentrations, photostimulation of different intensities is generated in the four rectangular microchannels. Both the sub-liquid inlet section 231 and the main liquid inlet section 22 are serpentine microchannels. The main inlet section 22 has two inlets 21 connected in parallel on the inlet side, and the sub-inlet section 231 has an outlet 24 connected in parallel on the outlet side; the light intensity gradient experiment diagram is shown below. Figure 10 As shown. Figure 9 This is a cell response experiment diagram showing the application of four signals at different frequencies under the same amplitude condition after initial verification of the optimal stimulation amplitude of 2V in the electrical stimulation module.
[0059] In this embodiment, two inlets 11 are connected in parallel on the inlet side of the main sample inlet section 12, and outlets 14 are connected in parallel on the outlet side of several capture culture channels 13. The inlets 11 are located at the top of the cell capture culture chip layer 1, and the outlets 14 are located at the bottom of the cell capture culture chip layer 1. The two inlets 11 of the cell capture culture chip layer 1 are connected to the main sample inlet section 12. The channel width is 100-150 μm. The main sample inlet section 12 is a serpentine microchannel. The main sample inlet section 12 can achieve drug concentration gradients of 0, 1 / 3 C, 2 / 3 C, and C, and the meandering of the multiple serpentine channels can achieve better solution mixing. The diameters of the inlets 11 and outlets 14 are both 2-5 mm. The size of the inlets 11 and outlets 14 is mainly designed to match the external pressure pump pipeline required for the experiment. The number of main liquid inlet sections 22 is less than the number of light intensity regulating channels 23. Preferably, there are three main liquid inlet sections 22 and four light intensity regulating channels 23. The three main liquid inlet sections 22 and the four light intensity regulating channels 23 form a Christmas tree structure.
[0060] In this embodiment, the height of the cell capture culture chip layer 1 is 20-25 μm. Setting the height of the cell capture culture chip layer 1 to 20-25 μm can ensure smooth cell flow and prevent multiple cells from overlapping in the longitudinal direction, which would affect cell counting.
[0061] like Figure 4As shown, the microelectrode layer 3 is preferably made of gold, platinum, ITO, or similar materials. The microelectrode layer 3 is externally connected to the PCB board via conductive tape. An external electrical signal is introduced to the Au interdigitated electrodes, creating a sufficiently strong electric field between adjacent microelectrodes to achieve a gradient loading of the electric field intensity during on-chip culture. The main function of the cell capture culture chip layer 1 is to introduce cell buffer solution into the aforementioned structure. A serpentine channel is used to achieve a drug concentration gradient loading. After the solution enters the chamber, cells are captured, and excess cells are discharged from the outlet 14. The entire flow path control module includes the cell capture culture chip layer 1 and the conduit. The microelectrode layer 3 is provided with several first main electrodes 31 and several second main electrodes 33, with one end of each second main electrode 33 connected in parallel. The first main electrode 31 is provided with several sets of first interdigital electrodes 32, and the second main electrode 33 is provided with several sets of second interdigital electrodes 34. The sets of first interdigital electrodes 32 and the sets of second interdigital electrodes 34 intersect to form several interdigital electrode regions 35, which correspond to the positions of the capture culture chamber 134. The width of the interdigital electrodes is 100-200 μm, and the interdigital spacing is 200-400 μm. The amplitude and frequency of electrical stimulation of cells are adjustable by connecting the Au gold microelectrodes to an external AC sinusoidal electrical signal PCB circuit board. The comb tooth distance of the interdigital electrode array is controlled within the range of 100-200 μm to ensure good conductivity and reliability; the comb tooth width is within the range of 50-100 μm, which can be determined according to the density of the microelectrode array in actual use.
[0062] like Figure 5 , 6As shown in Figures 7 and 8, the capture culture chamber 134 is a rectangular chamber with its inlet and outlet located diagonally. Several capture culture chambers 134 are arranged in a 4×4 array. Each capture culture chamber 134 contains two rows of single-cell capture structures 135, with five columns in each row. Four rows of capture structures 135 are arranged along the width of the capture culture chamber 134, and four columns are arranged along its length. Capture structures 135 in adjacent columns are staggered by a displacement of 50–60 μm. To ensure a uniform flow rate of fluid reaching each column of chambers, the width of the capture culture channel 13 is preferably set to 100–150 μm, allowing cells to be evenly dispersed into the inlet of each capture culture chamber 134. The cell capture culture chip layer 1 enables in vitro cell culture under different drug concentration gradients. The capture structure 135 includes a concave body 1351 with two openings 1352 symmetrically arranged with respect to the axis of symmetry of the concave body 1351. The preferred tilt angle of the openings of the concave body 1351 is 55–65°. The tilt angle significantly affects cell capture efficiency; too large or too small an angle will reduce capture efficiency, leading to lower cell count uniformity within each chamber during culture. Furthermore, adjusting the tilt angle prevents cells from escaping the capture structure under the influence of the flow field, ensuring stable cell capture. The transverse spacing of the capture structures 135 is 40–50 μm, and the longitudinal spacing is 70–80 μm. Adjusting the row and column spacing of the capture structures 135 diverts the flow during capture, facilitating cell transport along the intended streamline to the corresponding capture structure. Two openings 1352 are provided in the concave body 1351. The width of the openings 1352 is 5-10 μm. By matching the fluid flow resistance through the double slits with a width of 5-10 μm, it can be ensured that the cells can smoothly enter the capture structure 135 and get stuck at the position of the opening 1352. Excess cells flow to the next empty capture structure 135.
[0063] In this embodiment, the capture culture chamber 134 is a diagonally rectangular geometric structure with a length of 1000-1200 μm and a width of 600-800 μm. The inlet and outlet of the capture culture chamber 134 are designed to enter and exit diagonally, providing a spatially distributed capture loading, which can achieve a more uniform spatial distribution of capture. It has been verified that the cells are more evenly distributed after capture, and have better capture uniformity.
[0064] The specific implementation principle of the combined control device of light stimulation, electrical stimulation and concentration gradient is as follows: The cells to be captured enter the channel through two inlet ports 11, pass through the main inlet section 12 and the capture culture channel 13, and enter the capture culture chamber 134. The cells are captured by fluid resistance. After the cell suspension is sampled, the cells are counted after capture. Then, culture medium is added to the cell capture culture chip layer 1 by continuous perfusion at a flow rate of 4 μl / min. At the same time, the bottom microelectrode layer 3 is connected to the electrical signal to intermittently electrically stimulate the cells. The cell capture culture chip layer 1 is placed in an incubator for culture.
[0065] A method for preparing a device for combined regulation of light stimulation, electrical stimulation, and concentration gradient includes the following steps:
[0066] S1: The photostimulated light intensity gradient chip layer 2 can be prepared by printing with a 3D printer or by photolithography.
[0067] S2. Prepare cell capture culture chip layer 1, as detailed below;
[0068] S21. Select a 3-inch silicon wafer and spin-coat it with SU8-3025 photoresist.
[0069] S22. After spin coating and uniform coating, the silicon wafer is pre-baked and then exposed under a photolithography machine.
[0070] S23. After exposure, the chip is post-baked and hard-baked, then coated with a release agent;
[0071] S24. After preparing a certain proportion of PDMS, pour it into a silicon wafer mold and then place it in an oven at 60℃~100℃ for curing. The oven temperature is preferably 60℃.
[0072] S25. Remove the cured PDMS and cut it to fit the shape of the microelectrode layer 3.
[0073] S3. Microelectrode layer 3 is prepared using a wet etching process, as detailed below;
[0074] S31. Quartz glass is selected as the substrate for microelectrode layer 3.
[0075] S32. Spin-coat a 5μm layer of SU-83005 photoresist onto a quartz glass plate with 5-20nm Cr and 100-400nm Au sputtered by magnetron sputtering. The thickness of Cr is preferably 7nm Cr and the thickness of Au is preferably 200nm Au.
[0076] S33. The interdigitated electrode microarray structure is etched on the microelectrode layer 3 by photolithography;
[0077] S34. An interdigitated electrode microarray structure is etched on the microelectrode layer by wet etching.
[0078] S4: The photostimulated light intensity gradient chip layer 2, microelectrode layer 3, and cell capture culture chip layer 1 are subjected to ultrasonic treatment and plasma cleaning. The plasma treatment time is 15-20 seconds. The plasma cleaning time mainly affects the bonding effect between cell capture culture chip layer 1 and microelectrode layer 3. If the cleaning time is too long, the bonding will be too tight, and the cell fluid will not be able to enter the structure. If the cleaning time is too short, the bonding will be loose and leakage will be easy. The purpose of ultrasonic treatment and plasma treatment of microelectrode layer 3 and cell capture culture chip layer 1 before bonding is to introduce hydrophilic -OH groups into cell capture culture chip layer 1 to make it hydrophilic, and at the same time change the chemical bonds of microelectrode layer 3 to make the two irreversible bonds. Ultrasonic treatment can clean the electrode and chip surface. The ultrasonic treatment conditions are to use triple-distilled water for ultrasonic treatment for 10-20 minutes, and then blow dry with nitrogen. The ultrasonic treatment time is the optimal time verified by practice. If the cleaning time is too short, the cleaning effect will be poor. In addition, since triple-distilled water is pure water, it can avoid interference from other ions and ensure the cleaning effect of the chip.
[0079] S5: First, the photostimulation intensity gradient chip layer 2 is bonded to the cell capture culture chip layer 1 at a bonding temperature of 100-120℃; then, the cell capture culture chip layer 1 is placed upside down on the microelectrode layer 3, and a thermal bonding process is used to form a closed cavity with the cell capture culture chip layer 1. The closed cavity allows the cell suspension to enter and exit only through the sample inlet 11 and the sample outlet 14 at a bonding temperature of 100-150℃. A bonding temperature of 100-150℃ is a suitable temperature to ensure that the bonding effect achieves the expected results.
Claims
1. A device for combined regulation of light stimulation, electrical stimulation, and concentration gradient, characterized in that, It includes a light-stimulated light intensity gradient chip layer (2), a cell capture and culture chip layer (1), and a microelectrode layer (3) arranged from top to bottom. The cell capture culture chip layer (1) is provided with a main injection section (12) and a capture culture channel (13) connected in series. Several main injection sections (12) are connected in parallel, and several capture culture channels (13) are connected in parallel. Each capture culture channel (13) is provided with a capture culture section (133). Several sets of capture culture chambers (134) are connected in parallel on the capture culture section (133). Each set of capture culture chambers (134) includes two parallel capture culture chambers (134). Several capture structures (135) are provided in each capture culture chamber (134). The photostimulated light intensity gradient chip layer (2) is provided with a main liquid inlet section (22) and a light intensity modulation channel (23) connected in series. Several main liquid inlet sections (22) are connected in parallel, and several light intensity modulation channels (23) are connected in parallel. Each light intensity modulation channel (23) is provided with a light intensity modulation segment (232), and the light intensity modulation segment (232) corresponds to the position of the capture culture chamber (134). The microelectrode layer (3) is provided with a plurality of first main electrodes (31) and a plurality of second main electrodes (33), with one end of the plurality of second main electrodes (33) connected in parallel; the first main electrodes (31) are provided with a plurality of first interdigital electrodes (32), and the second main electrodes (33) are provided with a plurality of second interdigital electrodes (34), with the plurality of first interdigital electrodes (32) and the plurality of second interdigital electrodes (34) intersecting to form a plurality of interdigital electrode regions (35), and the interdigital electrode regions (35) corresponding to the position of the capture culture chamber (134); The capture structure (135) includes a concave body (1351), on which two notches (1352) are provided, and the two notches (1352) are symmetrically arranged with respect to the axis of symmetry of the concave body (1351). The capture culture channel (13) further includes a sample injection section (131) and a sample discharge section (132), which are connected in sequence; the light intensity modulation channel (23) further includes a liquid injection section (231), which is connected to the light intensity modulation section (232); The capture culture chamber (134) is a rectangular chamber. The inlet and outlet of the capture culture chamber (134) are located at diagonal positions. The capture structure (135) is arranged in several rows along the width direction of the capture culture chamber (134). The capture structure (135) is arranged in several columns along the length direction of the capture culture chamber (134). The capture structures (135) located in adjacent columns are staggered.
2. The device for combined regulation of light stimulation, electrical stimulation, and concentration gradient according to claim 1, characterized in that, The main sample inlet section (12) has two inlet ports (11) connected in parallel on the inlet side, and the capture culture channels (13) have outlet ports (14) connected in parallel on the outlet side. The inlet ports (11) are located at the top of the cell capture culture chip layer (1), and the outlet ports (14) are located at the bottom of the cell capture culture chip layer (1). The main liquid inlet section (22) has two liquid inlet ports (21) connected in parallel on the inlet side, and the sub-liquid inlet section (231) has an outlet port (24) connected in parallel on the outlet side.
3. The device for combined regulation of light stimulation, electrical stimulation, and concentration gradient according to claim 1, characterized in that, The sample inlet section (131), sample outlet section (132), and liquid inlet section (231) are all serpentine microchannels.
4. The device for combined regulation of light stimulation, electrical stimulation, and concentration gradient according to claim 1, characterized in that, Both the main sample inlet section (12) and the main liquid inlet section (22) are serpentine microchannels.
5. A method for preparing a device for combined control of light stimulation, electrical stimulation, and concentration gradient according to any one of claims 1-4, characterized in that, Includes the following steps: S1: A photostimulated light intensity gradient chip layer was prepared by printing with a 3D printer (2). S2. Preparation of cell capture culture chip layer (1); S3. Microelectrode layer is prepared by wet etching process (3). S4: The photostimulation intensity gradient chip layer (2), microelectrode layer (3) and cell capture culture chip layer (1) are subjected to ultrasonic treatment and plasma cleaning. The plasma treatment time is 15-20s. S5: First, bond the light-stimulated light intensity gradient chip layer (2) to the cell capture culture chip layer (1) at a bonding temperature of 100-120℃; then, place the cell capture culture chip layer (1) inverted on the microelectrode layer (3) and use a thermal bonding process to form a closed cavity with the microelectrode layer (3) at a bonding temperature of 100-150℃.
6. The method for preparing the combined photostimulation, electrical stimulation, and concentration gradient control device according to claim 5, characterized in that, The preparation of the cell capture culture chip layer (1) in step S2 specifically includes: S21. Select a 3-inch silicon wafer and spin-coat it with SU8-3025 photoresist. S22. After spin coating and uniform coating, the silicon wafer is pre-baked and then exposed under a photolithography machine. S23. After exposure, the chip is post-baked and hard-baked, then coated with a release agent; S24. After preparing a certain proportion of PDMS, pour it into a silicon wafer mold and then place it in an oven at 60℃~100℃ for curing. S25. Remove the cured PDMS and cut it to fit the shape of the microelectrode layer (3). The preparation of the microelectrode layer (3) in step S3 specifically includes: S31. Quartz glass is selected as the substrate for the microelectrode layer (3); S32. Spin-coat a 5μm layer of SU-83005 photoresist onto a quartz glass plate with 5~20nm Cr and 100-400nm Au sputtered by magnetron sputtering. S33. The interdigitated electrode microarray structure is etched on the microelectrode layer (3) by photolithography; S34. The interdigitated electrode microarray structure is etched on the microelectrode layer by wet etching.