A device for combined regulation of gradient light stimulation and drug concentration gradient and its preparation method

Abstract

The present invention discloses a device for combined regulation of gradient light stimulation and drug concentration gradient and a preparation method, comprising a light stimulation light intensity gradient chip layer and a cell capture culture chip layer; the cell capture culture chip layer is provided with a main sample inlet section and a capture culture channel; the capture culture channel is provided with several groups of capture culture chambers in parallel, and several capture structures are provided in the capture culture chambers; the light stimulation light intensity gradient chip layer is provided with a main liquid inlet section and a light intensity regulation channel; and further preparation steps such as S1-S4 are included. The light stimulation light intensity gradient chip layer of the present invention generates different light intensity gradients through the different absorbances of channels with solutions of different concentrations; the cell capture culture chip layer is provided with several groups of capture culture chambers, and arrayed capture structures are provided in the capture culture chambers for capturing and culturing cells; the present invention can realize the differentiation process and behavior regulation of target cells cultured on a microfluidic chip under the multi-factor conditions of drug concentration gradient and gradient light intensity gradient.

Description

Technical Field

[0001] The present invention relates to the field of cell culture chips, and in particular to a device for combined regulation of gradient light stimulation and drug concentration gradient and a preparation method thereof. Background Art

[0002] Microfluidic chip technology originated from the micro-total analysis system proposed by Manz in his 1990 article. Its goal was to miniaturize and integrate chemical analysis equipment, thereby maximizing the transfer of analytical laboratory functions to portable analytical devices, even onto centimeter-sized chips. Microfluidic chips can be designed with multiple channels, using a network structure to distribute fluids to multiple cells without interfering with each other, allowing simultaneous biochemical experiments in multiple cells. The microchip's structure is controllable, enabling the manipulation of microscopic objects (e.g., particles and cells) through microstructural design and processing, such as in cell population studies. Two-dimensional cell culture within microfluidic chips has been widely used to study cell responses and cell activity. Cell culture systems based on microfluidic platforms provide a relatively stable microenvironment for two-dimensional cell culture in both static and continuous perfusion conditions. Microfluidic chips are increasingly being used in in vitro cell culture studies due to their advantages, such as low reagent requirements, high-throughput analysis, and automation.

[0003] Although microfluidic chip cell culture technology has been successfully applied in drug screening and pathological research, there are still some problems. Especially in the microfluidic in vitro culture of tumor cells, it is necessary to locate, capture and culture the cells, and at the same time load a variety of physical and chemical influencing factors to monitor the growth and proliferation of target cells and conduct subsequent metabolite and genomic analysis. When microfluidic chip cells are cultured in vitro on a chip in the existing technology, it is impossible to load a variety of physical and chemical influencing factors, making it impossible to better study the cell body differentiation and behavioral response. Therefore, it is necessary to combine microfluidic chips with more efficient cell capture structures to achieve efficient and stable cell capture and positioning culture, and propose a joint control device that loads a variety of physical and chemical influencing factors. Summary of the Invention

[0004] In response to the above-mentioned deficiencies in the prior art, the present invention provides a device and preparation method for combined regulation of gradient light stimulation and drug concentration gradient to solve the problem in the prior art that multiple factors cannot be loaded and regulated during on-chip culture of cells in vitro based on microfluidic chips, and that the study of cell differentiation and behavioral responses cannot be realized.

[0005] In order to achieve the above-mentioned object of the invention, the technical solution adopted by the present invention is:

[0006] Provided are a device for combined control of gradient light stimulation and drug concentration gradient and a preparation method, comprising a light stimulation light intensity gradient chip layer and a cell capture culture chip layer arranged in sequence from top to bottom; a main sample injection section and a capture culture channel are arranged in series on the cell capture culture chip layer, wherein several main sample injection sections are arranged in parallel, and several capture culture channels are arranged in parallel, and the number of main sample injection sections is smaller than the number of capture culture channels; a capture culture section is provided on each capture culture channel, and several groups of capture culture cavities are arranged in parallel on the capture culture section, each group of capture culture cavities includes two parallel capture culture cavities, and several capture structures are provided in each capture culture cavity; a main liquid inlet section and light intensity regulation channels are arranged in series on the light stimulation light intensity gradient chip layer, wherein several main liquid inlet sections are arranged in parallel, and several light intensity regulation channels are arranged in parallel, and the number of main liquid inlet sections is smaller than the number of light intensity regulation channels; a light emphasis segment is provided on each light intensity regulation channel, and the light emphasis segment corresponds to the position of the capture culture cavity.

[0007] Furthermore, the capture structure includes a concave main body, and two notches are provided on the concave main body, and the two notches are symmetrically arranged relative to the symmetry axis of the concave main body.

[0008] Furthermore, two sample inlets are arranged in parallel on the inlet side of the main sample inlet section, and sample outlets are arranged in parallel on the outlet sides of several capture culture channels. The sample inlet is located at the top of the cell capture culture chip layer, and the sample outlet is located at the bottom of the cell capture culture chip layer; two liquid inlets are arranged in parallel on the inlet side of the main liquid inlet section, and liquid outlets are arranged in parallel on the outlet side of the branch liquid inlet section.

[0009] Furthermore, the capture and culture channel also includes a sample injection section and a sample discharge section, which are connected in sequence; the light intensity regulation channel also includes a liquid injection section, which is connected to the light intensity regulation section.

[0010] Furthermore, the sample inlet section, the sample outlet section and the liquid inlet section are all serpentine microchannels.

[0011] Furthermore, the main sample inlet section and the main liquid inlet section are both serpentine microchannels.

[0012] Furthermore, the number of the main sample inlet sections is smaller than the number of the capture and culture channels, and the number of the main liquid inlet sections is smaller than the number of the light intensity regulating channels.

[0013] Furthermore, the capture culture chamber is a rectangular chamber, the inlet and outlet of the capture culture chamber are arranged at diagonal positions, the capture structure is arranged in several rows along the width direction of the capture culture chamber, and the capture structure is arranged in several columns along the length direction of the capture culture chamber, and the capture structures located in two adjacent columns are staggered.

[0014] A device for combined regulation of gradient light stimulation and drug concentration gradient and a preparation method thereof include the following steps:

[0015] S1: Preparation of the light intensity gradient chip layer for light stimulation:

[0016] Using a 3D printer to prepare and print a light stimulation intensity gradient chip layer;

[0017] S2: Preparation of cell capture culture chip layer;

[0018] The cell capture and culture chip layer was constructed using PDMS polymer through soft lithography and casting processes;

[0019] S3: Ultrasonic treatment and plasma cleaning are performed on the light stimulation intensity gradient chip layer and the cell capture culture chip layer. The plasma treatment time is 15-20 seconds.

[0020] S4: Bonding the light stimulation intensity gradient chip layer to the cell capture culture chip layer at a bonding temperature of 100-120°C.

[0021] The preparation of the cell capture culture chip layer in step S2 specifically includes:

[0022] The soft lithography process and the reverse molding process in step S2 specifically include the following steps:

[0023] S21: Using soft lithography, a cell capture culture chip layer mold with a structure height of 20-25 μm was fabricated.

[0024] S22: Fixing the cell capture culture chip layer mold on the acrylic mold;

[0025] S23: Pour in the PDMS mixed glue, let it stand and then vacuum;

[0026] S24: placing the acrylic mold in an oven for heating and curing, with the oven temperature being 60-80°C;

[0027] S25: Casting the mold to complete the preparation of the cell capture culture chip layer.

[0028] The beneficial effects of the present invention are:

[0029] In this technical solution, the cells to be captured enter the main injection section through the injection port, and 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, and then a pulse signal is applied for intermittent electrical stimulation; in this process, the double-slit notch setting of the capture structure can prevent the cells from being washed away by the fluid.

[0030] The top layer of the present invention is composed of a light stimulation intensity gradient chip layer based on absorbance regulation of solutions of different concentrations. This chip layer is prepared by a 3D printer. The upper end is composed of a Christmas tree model serpentine microchannel consisting of a main liquid inlet section and a branch liquid inlet section. After passive mixing, the solution generates four concentration gradient solutions of 0, 1 / 3C, 2 / 3C, and C, which are passed into four light emphasis segments. Based on the different absorbances of solutions of different concentrations, light stimulation of different light intensities can be generated in the four light emphasis segments.

[0031] The middle layer of the present invention is composed of a cell capture and culture chip layer. The concentration gradients of 0, 1 / 3C, 2 / 3C, and C are still generated by the Christmas tree model. This layer of chip is equipped with a 4×4 array chamber culture structure, and two rows of single-cell capture structures are arranged in a single culture chamber. The middle layer chip can realize in vitro cell culture under different drug concentration gradients.

[0032] The light stimulation intensity gradient chip layer of the present invention generates different light intensity gradients through the different absorbances of solution channels with different concentrations; the cell capture culture chip layer is provided with several groups of capture culture cavities, and the capture culture cavities are provided with arrayed capture structures for capturing cultured cells.

[0033] The present invention can realize the differentiation process and behavior regulation of target cells cultured on a microfluidic chip under the multi-factor conditions of drug concentration gradient and gradient light intensity gradient. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 This is a schematic diagram of the overall structure of the device for combined regulation of gradient light stimulation and drug concentration gradient;

[0035] Figure 2 This is a physical picture of the device for combined regulation of gradient light stimulation and drug concentration gradient;

[0036] Figure 3 Layered diagram of the cell capture culture chip layer and the light stimulation intensity gradient chip layer;

[0037] Figure 4 Schematic diagram of the structure of the cell capture culture chip layer;

[0038] Figure 5 This is a schematic diagram of the structure of the light intensity gradient chip layer for light stimulation;

[0039] Figure 6 for Figure 4 A partial enlarged view in FIG.

[0040] Figure 7 is a schematic diagram of the structure of the capture structure;

[0041] Figure 8 Experimental photos showing the generation of four-intensity gradients in the optical gradient chip layer for CMTPX dye verification;

[0042] Figure 9 Experimental data diagram for the generation of four light intensity gradients in the optical gradient chip layer for CMTPX dye verification;

[0043] Figure 10 Schematic diagram of the structure of the device for combined regulation of gradient light stimulation and drug concentration gradient in segments with different light intensity levels.

[0044] The main components in the figure are described as follows:

[0045] 1. Cell capture culture chip layer; 11. Sample inlet; 12. Main sample inlet section; 13. Capture culture channel; 131. Sub-sample inlet section; 132. Sub-sample outlet section; 133. Capture culture section; 134. Capture culture chamber; 135. Capture structure; 1351. Concave main body; 1352. Notch; 14. Sample outlet;

[0046] 2. Light stimulation intensity gradient chip layer; 21. Liquid inlet; 22. Main liquid inlet section; 23. Light intensity adjustment channel; 231. Sub-liquid inlet section; 232. Light intensity segment; 24. Liquid outlet. DETAILED DESCRIPTION

[0047] The following is further described in detail through specific embodiments, but the embodiments of the present invention are not limited thereto. Unless otherwise specified, the technical means used in the following embodiments are conventional means well known to those skilled in the art; the experimental methods used are all conventional methods; and the materials and reagents used are all commercially available.

[0048] like Figure 1 、 2 As shown in FIG3 , a device for combined control of gradient light stimulation and drug concentration gradient and a preparation method thereof include a light stimulation intensity gradient chip layer 2 and a cell capture and culture chip layer 1 arranged sequentially from top to bottom. Figure 2 Layer diagram of the cell capture culture chip layer and the light stimulation intensity gradient chip layer.

[0049] like Figure 4As shown, a main sample injection section 12 and a capture culture channel 13 are arranged in series on the cell capture culture chip layer 1, and several main sample injection sections 12 are arranged in parallel, and several capture culture channels 13 are arranged in parallel. The number of main sample injection sections 12 is less than the number of capture culture channels 13. In this embodiment, the main sample injection section 12 is preferably provided at three locations, and the capture culture channels 13 are preferably provided at four locations. The three main sample injection sections 12 and the four capture culture channels 13 are in the shape of a Christmas tree. A capture culture section 133 is provided on each capture culture channel 13, and several groups of capture culture chambers 134 are arranged in parallel on the capture culture section 133. Each group of capture culture chambers 134 includes two parallel capture culture chambers 134, and several capture structures 135 are arranged in each capture culture chamber 134. The capture culture channel 13 also includes a sub-sample injection section 131 and a sample outlet section 132. The sub-sample injection section 131, the capture culture section 133 and the sample outlet section 132 are connected in sequence. The light intensity regulation channel 23 further includes a liquid inlet section 231, which is connected to the light intensity regulation section 232. The main sample inlet section 12, the liquid inlet section 131 and the sample outlet section 132 are all serpentine microchannels.

[0050] like Figure 5 As shown, the light stimulation intensity gradient chip layer 2 is serially configured with a main liquid inlet section 22 and light intensity modulation channels 23. Several main liquid inlet sections 22 and light intensity modulation channels 23 are configured in parallel. Each light intensity modulation channel 23 is provided with a light enhancement segment 232, which corresponds to the position of the capture culture chamber 134. The light enhancement segment 232 is a rectangular microchannel with a length of 12,000 μm, a width of 1,000 μm, and a depth of 500 μm. After passive mixing, the solution generates four solutions with concentration gradients of 0, 1 / 3 C, 2 / 3 C, and C, which are then introduced into the rectangular microchannels. Based on the different absorbances of the solutions at different concentrations, different light intensities of light stimulation are generated in the four rectangular microchannels. Both the sub-liquid inlet section 231 and the main liquid inlet section 22 are serpentine microchannels. Two liquid inlets 21 are provided in parallel on the inlet side of the main liquid inlet section 22 , and a liquid outlet 24 is provided in parallel on the outlet side of the branch liquid inlet section 231 .

[0051] In this embodiment, two sample inlets 11 are provided in parallel on the inlet side of the main sample inlet section 12, and sample outlets 14 are provided in parallel on the outlet side of several capture culture channels 13. The sample inlets 11 are located at the top of the cell capture culture chip layer 1, and the sample outlets 14 are located at the bottom of the cell capture culture chip layer 1. The two sample inlets 11 of the cell capture culture chip layer 1 are connected to the main sample inlet section 12, and the channel width is 100 to 150 μm. The main sample inlet section 12 is a serpentine microchannel. The main sample inlet section 12 can achieve a drug concentration gradient of 0, 1 / 3C, 2 / 3C, and C, and the winding parts of the multiple serpentine channels can achieve a better solution mixing effect. The diameters of the sample inlet 11 and the sample outlet 14 are both 2 to 5 mm. The sizes of the sample inlet 11 and the sample outlet 14 are mainly designed to match the external pressure pump pipeline required for the experiment. The number of main liquid inlet sections 22 is smaller than the number of light intensity adjustment channels 23 . There are preferably three main liquid inlet sections 22 and four light intensity adjustment channels 23 . The three main liquid inlet sections 22 and the four light intensity adjustment channels 23 form a Christmas tree structure.

[0052] 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 the phenomenon of multiple cells overlapping in the longitudinal direction to affect counting.

[0053] like Figure 6 As shown, the capture culture chamber 134 is a rectangular chamber with the inlet and outlet positioned at diagonal locations. Several capture culture chambers 134 are arranged in a 4×4 array. Within each capture culture chamber 134 are two rows of single-cell capture structures 135, each row having five columns. Four rows of capture structures 135 are arranged along the width of the capture culture chamber 134, and four columns of capture structures 135 are arranged along the length of the capture culture chamber 134. The capture structures 135 in two adjacent columns are staggered, with a staggered displacement of 50 to 60 μm. To ensure the same flow rate for the fluid reaching each column, the width of the capture culture channel 13 is preferably set to 100 to 150 μm, allowing cells to be evenly distributed to the inlet of each capture culture chamber 134.

[0054] like Figure 7As shown, the cell capture culture chip layer 1 can achieve in vitro cell culture under different drug concentration gradients. The capture structure 135 includes a concave body 1351, which is provided with two notches 1352. The two notches 1352 are symmetrically arranged with respect to the symmetry axis of the concave body 1351. The opening inclination angle of the concave body 1351 is preferably 55-65 degrees. The inclination angle of the opening has a significant impact on the cell capture efficiency. An inclination angle that is too large or too small will lead to reduced cell capture efficiency, which in turn leads to a low consistency in the number of cells within each chamber during culture. In addition, by adjusting the opening inclination angle, cells can be prevented from escaping the capture structure under the action of the flow field, achieving stable cell capture. The lateral spacing of the capture structure 135 is 40-50 μm, and the longitudinal spacing is 70-80 μm. By adjusting the row and column spacing of the capture structure 135, a diversion effect is played during the capture process, facilitating the cells to shuttle along the expected streamline to the corresponding capture structure. Two notches 1352 are provided in the concave main body 1351, and the width of the notch 1352 is 5 to 10 μm. By matching the fluid flow resistance through the double-slit notch with a width of 5 to 10 μm, it can ensure that cells can smoothly enter the capture structure 135 and be stuck at the position of the notch 1352, and excess cells flow to the next empty capture structure 135.

[0055] In this embodiment, the capture culture chamber 134 is a diagonal rectangular geometric structure with a length of 1000 to 1200 μm and a width of 600 to 800 μm. The inlet and outlet of the capture culture chamber 134 are designed to enter and exit diagonally, providing a spatially distributed capture load, which can achieve a more uniform capture spatial distribution. It has been verified that the cells are more evenly distributed in space after capture, with better capture uniformity.

[0056] The specific implementation principle of the device for combined control of gradient light stimulation and drug concentration gradient is as follows: the cells to be captured enter the channel through two injection ports 11, pass through the main injection section 12 and the capture culture channel 13, and enter the capture culture chamber 134 to be captured by fluid resistance. When the cell suspension injection process is completed, the capture is completed and counted, and then the culture medium is added to the cell capture culture chip layer 1 by continuous perfusion, and the flow rate is controlled at 4μl / min. Figure 8 and 9 Shown are experimental photos and experimental data graphs for the generation of four light intensity gradients in the CMTPX dye-verified light gradient chip layer.

[0057] like Figure 10As shown, light stimuli of varying intensities are generated in the four rectangular microchannels based on the different absorbances of solutions of varying concentrations, using light-emphasis segments 232 at different heights. The Beer-Lambert law defines the relationship between light intensity and the concentration of a substance in a medium in optics. When monochromatic light passes through a homogeneous, non-luminescent solution or gas, the intensity of the light passing through the medium decreases as the concentration of the substance increases, and this change is proportional to the length of the medium. For a given incident light intensity of I0, the intensity of the light after passing through the medium of I, the concentration of the medium of C, and the length of the medium of l, the following equation holds: I = I0e(-εcb), where ε is the proportionality constant, also known as the absorption coefficient or molar absorptivity, and b and c represent the length and concentration of the medium, respectively. Absorbance (A) = ε × b × c, where A is absorbance, the logarithmic ratio of absorbed light intensity, typically measured by a spectrophotometer. ε is a constant representing the absorption coefficient of a substance. b is the optical path length, the actual distance light travels in the sample. c is the solution concentration, the molar concentration of the substance. A light gradient layer chip is fabricated by combining different concentration gradient absorbance solutions with channels of varying heights. Visible light is then applied to the chip. After passing through the light gradient layer, the varying absorbance of each channel generates four light spots with varying intensity gradients, which are then irradiated onto the underlying culture layer chip. This enables multi-gradient light stimulation to regulate the proliferation and differentiation of C2C12 cells. The heights of the four light-enhancing segments 232 are 2000-2500 μm, 1500-2000 μm, 1000-1500 μm, and 500-1000 μm, respectively. The different optical path lengths b of the four light-enhancing segments 232 produce different absorbance gradients.

[0058] A device for combined regulation of gradient light stimulation and drug concentration gradient and a preparation method thereof include the following steps:

[0059] S1: Preparation of light stimulation intensity gradient chip layer 2:

[0060] The printed light stimulation intensity gradient chip layer 2 is prepared by using a 3D printer; the printed light stimulation intensity gradient chip layer 2 can also be prepared by soft lithography, 3D printing and other technologies;

[0061] S2: Preparation of cell capture culture chip layer 1;

[0062] The cell capture culture chip layer 1 is constructed by using PDMS polymer through soft lithography and molding processes. Specifically;

[0063] S21: Using soft lithography, a cell capture culture chip layer mold with a structure height of 20-25 μm was fabricated.

[0064] S22: Fixing the cell capture culture chip layer mold on the acrylic mold;

[0065] S23: Pour in the PDMS mixed glue, let it stand and then vacuum;

[0066] S24: placing the acrylic mold in an oven for heating and curing, with the oven temperature being 60-80°C;

[0067] S25: Casting the mold to complete the preparation of the cell capture culture chip layer 1.

[0068] S3: Ultrasonic treatment and plasma cleaning are performed on the light stimulation intensity gradient chip layer 2 and the cell capture culture chip layer 1. The plasma treatment time is 15-20 seconds.

[0069] S4: Bonding the light stimulation intensity gradient chip layer 2 to the cell capture culture chip layer 1 at a bonding temperature of 100-120°C.

Claims

1. A device for combined regulation of gradient light stimulation and drug concentration gradient, characterized in that: It includes a light stimulation light intensity gradient chip layer (2) and a cell capture culture chip layer (1) arranged in sequence from top to bottom; The cell capture culture chip layer (1) is provided with a main sample injection section (12) and a capture culture channel (13) in series, a plurality of the main sample injection sections (12) are provided in parallel, and a plurality of the capture culture channels (13) are provided in parallel, and the number of the main sample injection sections (12) is less than the number of the capture culture channels (13); each capture culture channel (13) is provided with a capture culture section (133), and a plurality of groups of capture culture chambers (134) are provided in parallel on the capture culture section (133), each group of the capture culture chambers (134) includes two capture culture chambers (134) connected in parallel, and a plurality of capture structures (135) are provided in each capture culture chamber (134); The light stimulation light intensity gradient chip layer (2) is provided with a main liquid inlet section (22) and a light intensity regulation channel (23) in series, a plurality of the main liquid inlet sections (22) are provided in parallel, and a plurality of the light intensity regulation channels (23) are provided in parallel, and the number of the main liquid inlet sections (22) is less than the number of the light intensity regulation channels (23); a light emphasis segment (232) is provided on each of the light intensity regulation channels (23), and the light emphasis segment (232) corresponds to the position of the capture culture chamber (134); The capture and culture channel (13) further comprises a sample injection section (131) and a sample discharge section (132), wherein the sample injection section (131), the capture and culture section (133) and the sample discharge section (132) are connected in sequence; the light intensity regulation channel (23) further comprises a liquid injection section (231), wherein the liquid injection section (231) is connected to the light intensity regulation section (232).

2. The device for combined control of gradient light stimulation and drug concentration gradient according to claim 1, characterized in that: The capture structure (135) comprises a concave main body (1351), and two notches (1352) are provided on the concave main body (1351), and the two notches (1352) are symmetrically arranged relative to the symmetry axis of the concave main body (1351).

3. The device for combined control of gradient light stimulation and drug concentration gradient according to claim 1, characterized in that: The inlet side of the main sample inlet section (12) is provided with two sample inlets (11) in parallel, and the outlet side ports of the plurality of capture culture channels (13) are provided with sample outlets (14) in parallel, the sample inlet (11) is located at the top of the cell capture culture chip layer (1), and the sample outlet (14) is located at the bottom of the cell capture culture chip layer (1); the inlet side of the main liquid inlet section (22) is provided with two liquid inlets (21) in parallel, and the outlet side of the branch liquid inlet section (231) is provided with a liquid outlet (24) in parallel.

4. The device for combined control of gradient light stimulation and drug concentration gradient according to claim 1, characterized in that: The sample inlet section (131), the sample outlet section (132) and the liquid inlet section (231) are all serpentine microchannels.

5. The device for combined control of gradient light stimulation and drug concentration gradient according to claim 1, characterized in that: The main sample inlet section (12) and the main liquid inlet section (22) are both serpentine microchannels.

6. The device for combined control of gradient light stimulation and drug concentration gradient according to claim 1, characterized in that: The capture culture chamber (134) is a rectangular chamber, the inlet and outlet of the capture culture chamber (134) are arranged at diagonal positions, the capture structure (135) is arranged in a plurality of rows along the width direction of the capture culture chamber (134), and the capture structure (135) is arranged in a plurality of columns along the length direction of the capture culture chamber (134), and the capture structures (135) located in two adjacent columns are staggered.

7. A method for preparing the device for combined control of gradient light stimulation and drug concentration gradient according to any one of claims 1 to 6, characterized in that: The steps include: S1: Preparation of light stimulation intensity gradient chip layer (2): Using a 3D printer to prepare and print a light stimulation intensity gradient chip layer (2); S2: Preparation of cell capture culture chip layer (1); Using PDMS polymer through soft lithography and casting process to construct a cell capture culture chip layer (1); S3: Ultrasonic treatment and plasma cleaning are performed on the light stimulation intensity gradient chip layer (2) and the cell capture culture chip layer (1), and the plasma treatment time is 15-20 seconds; S4: Bonding the light stimulation intensity gradient chip layer (2) to the cell capture culture chip layer (1) at a bonding temperature of 100-120°C.

8. A method for preparing the device for combined control of gradient light stimulation and drug concentration gradient according to claim 7, characterized in that: The preparation of the cell capture culture chip layer (1) in step S2 specifically includes: The soft lithography process and the reverse molding process in step S2 specifically include the following steps: S21: Using soft lithography, a mold for the cell capture culture chip layer with a structure height of 20-25 μm was fabricated. S22: Fixing the cell capture culture chip layer mold on the acrylic mold; S23: Pour in the PDMS mixed glue, let it stand and then vacuum; S24: placing the acrylic mold in an oven for heating and curing, with the oven temperature being 60-80°C; S25: Casting the mold to complete the preparation of the cell capture culture chip layer (1).

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