Method for fabricating optically tunable organ-on-chip

Abstract

The application discloses a preparation and processing method of an optical tunable organ chip, and the method comprises the following steps: preparing soft channel A and B templates; pouring a processed polymer composite material into the prepared soft channel A template to obtain an initial organ chip channel to be processed; performing scanning and processing on the upper surface of the initial organ chip channel from bottom to top by using a femtosecond laser two-photon polymerization method to obtain a three-dimensional micro actuator; and performing post-processing on the initial organ chip with the three-dimensional micro actuator; and bonding the soft channel A and the soft channel B of the initial organ chip with the three-dimensional micro actuator to obtain the optical tunable organ chip.

Description

TECHNICAL FIELD

[0001] The present application relates to a preparation and processing method of an optically tunable organ chip, and belongs to the technical field of organ chip technology and laser micro-nano processing technology. BACKGROUND

[0002] An organ chip is a kind of chip system based on microfluidic technology, which is used to simulate the functions of different organs of the human body. This technology forms an in vitro model by implanting real human cells into a chip to simulate the complex microenvironment of the human body (such as pressure, shear stress, organ peristalsis, concentration gradient, etc.), so as to realize drug screening, drug safety detection, food safety detection and other tests closest to the real in vivo environment of the human body.

[0003] For example, related diseases in the intestinal tract such as intestinal inflammation, Crohn's disease, etc. are usually caused by the disorder of the intestinal flora, which induces a series of excessive inflammatory reactions, thereby damaging the intestinal barrier and mucus layer. At the same time, the intestinal flora can also affect brain health through the "brain-gut axis" signal transduction pathway. Therefore, it is particularly important to establish an in vitro coexistence model of intestinal cells and flora for specific disease research and related drug development. Cells are essentially sensors that receive signals from the microenvironment and change their own behavior and the surrounding environment. For intestinal cells, the mechanical force they feel can be roughly divided into two categories: one is the mechanical stimulation given by the peristalsis of the intestinal wall, and the rhythmic mechanical deformation brought by peristaltic movement can accelerate the functional differentiation of intestinal epithelial cells, so that the cells differentiate into crypts and villi structures, thereby forming a mucus layer; the second is the liquid shear stress generated when the liquid flows, and the liquid flow can remove excess microorganisms, thereby inhibiting the excessive growth of intestinal microorganisms and prolonging the coexistence time of microorganisms and the host. In summary, the liquid flow in the organ chip and the external force given to the cells are important parts to consider, so how to simulate the dynamic internal environment of the organ is the core problem of developing an organ chip. The establishment of traditional disease models mostly relies on in vitro two-dimensional static culture, which is simple and easy to implement but often faces problems such as long cell differentiation time and incomplete differentiation function, making it difficult to form structures such as crypts and villi. In order to form an organ surface with functional structures, researchers use hydrogels and extracellular matrix extract to construct scaffolds for three-dimensional culture, but this method cannot simulate the mechanical force stimulation on cells and microorganisms in a dynamic environment. Based on the above problems, researchers propose to construct an in vitro organ chip that can produce mechanical force stimulation on cells.

[0004] At present, research teams at home and abroad mainly rely on external hydraulic or pneumatic devices to simulate mechanical force stimulation in the intestinal chip. However, the above scheme still faces some problems. 1. If the low-speed flow of the liquid in the intestinal chip or the micro-scale peristalsis of the organ wall requires a high-precision external hydraulic or pneumatic device, the high-precision external device is expensive and bulky, which is contrary to the original design of the portable and movable intestinal chip, and reduces the functionality of the chip. 2. The chip integrated with multiple types of organs is the development trend, so the mechanical force stimulation inside the chip should pay more attention to accuracy and locality. However, under the regulation of the existing external device, the overall channel inside the chip is easily affected by the change of mechanical force. 3. In the face of some special application scenarios, such as the risk of exposure to infection, a remotely controllable mechanical force stimulation intestinal chip should be used for research. At present, the above problems restrict the development and application of the intestinal chip, and need to be solved urgently.

[0005] Femtosecond laser processing technology has outstanding advantages in three-dimensional precision manufacturing and micro-nano integration, and can prepare high-precision micro-nano three-dimensional devices on a three-dimensional substrate. This technology is based on the nonlinear interaction between the high-energy density light focus of ultrashort laser pulses (femtosecond level) and matter, such as material polymerization, reduction and phase change caused by multi-photon absorption. In recent years, femtosecond laser processing technology has made rapid progress, and its advantages in the preparation of three-dimensional micro-nano devices with different functions such as microelectronics, micro-optics, microfluidics and micromechanics have gradually emerged. Based on the above problems, it is urgent to develop an organ chip that can generate liquid flow and mechanical force on cells by external stimulation without external hydraulic or pneumatic devices. SUMMARY

[0006] The purpose of the present application is to provide a preparation and processing method of an optically tunable organ chip to solve the defect in the prior art that the low-speed flow of the liquid in the organ chip or the micro-scale peristalsis of the organ wall requires a high-precision external hydraulic or pneumatic device, and the high-precision external device is expensive and bulky, which is contrary to the original design of the portable and movable intestinal chip, and reduces the functionality of the chip.

[0007] The preparation and processing method of the optically tunable organ chip comprises:

[0008] Preparation of soft channel A and B templates;

[0009] Pour the processed polymer composite into the prepared soft channel A template to obtain a preliminary organ chip channel to be processed;

[0010] A three-dimensional micro actuator is processed on the upper surface of the preliminary organ chip channel from bottom to top by using femtosecond laser two-photon polymerization, and the preliminary organ chip with the three-dimensional micro actuator is post-processed;

[0011] The soft channel A and soft channel B of the initial organ chip with three-dimensional microactuators were bonded to obtain an optically tunable organ chip.

[0012] Furthermore, the method for preparing the soft channel A and B templates includes:

[0013] Use a carbon dioxide laser to carve the designed organ chip channels A and B on a machinable hard template.

[0014] The precursor of the soft re-moldable material and the curing agent are fully mixed and stirred, and the bubbles in the mixture are vacuum-extracted. The re-moldable material after the bubbles are removed is poured onto the hard channel templates A and B respectively, and placed in an oven to solidify and shape, completing the first re-mold of the channel;

[0015] The surfaces of the prepared soft channel A and B templates were fluorinated and re-molded. The re-molded soft channel A and B templates were placed in deionized water for ultrasonic cleaning, taken out, and then blown dry with dry nitrogen to finally obtain soft channel A and B template samples.

[0016] Furthermore, the processing method of the three-dimensional micro-actuator includes:

[0017] Fix the soft channel A template on the three-dimensional processing platform;

[0018] The processed polymer composite material is poured into the prepared soft channel A template, and a photoinitiator is added to the composite material to induce polymerization of the composite material;

[0019] The image sensor CCD imaging system is used to observe the spot shape and confirm the spot position, the femtosecond laser penetrates the polymer mixed material and focuses on the upper surface of the channel, and the pre-designed processing file is read into the processing program;

[0020] Femtosecond laser two-photon polymerization is used for processing, and scanning processing is performed from bottom to top to form a microactuator with a three-dimensional structure.

[0021] Furthermore, the three-dimensional processing platform is composed of a two-dimensional air-floating platform and a one-dimensional electric-controlled displacement platform; the two-dimensional air-floating platform is used to control the X-axis and the Y-axis; the one-dimensional electric-controlled displacement platform is used to control the Z-axis.

[0022] Furthermore, observing the light spot shape and confirming the light spot position by using an image sensor CCD imaging system includes:

[0023] Open the image sensor and the illumination mercury lamp light source, first adjust the one-dimensional electrically controlled displacement platform, raise the sample, find the mark line on the upper surface of the processing channel in the imaging system; open the processing control program, open the light shutter, control the movement of the two-dimensional air floating platform with the program, and adjust the Z-axis position until the clear mark line appears on the sample and the spot area is the smallest, and the spot position is confirmed.

[0024] Further, the movement range of the X-axis and the Y-axis of the two-dimensional air floating platform is 0-5 cm, and the accuracy is 50-100 nm, the movement range of the Z-axis of the one-dimensional electrically controlled displacement platform is 0-5 cm, and the accuracy is 10-20 nm, the X-axis and the Y-axis control the laser focus position by moving the sample with the air floating platform, the moving speed is 0-40 mm / s, and the moving accuracy is 50-100 nm.

[0025] Further, the post-processing of the initial organ chip with the three-dimensional micro actuator includes:

[0026] Developing the initial organ chip with the three-dimensional micro actuator by immersing it in a developing solution to remove the un-polymerized material;

[0027] Cleaning the surface and the inside of the channel with deionized water;

[0028] Then, the cleaned sample is first treated with acetone solution, then treated with ethanol solution, and finally treated with deionized water, and finally dried with a drying machine or dried with dry nitrogen flow.

[0029] Further, the method for bonding the soft channel A and the soft channel B of the initial organ chip with the three-dimensional micro actuator includes:

[0030] Cleaning the surface of the soft channel A and the soft channel B of the initial organ chip with the three-dimensional micro actuator together in the order of ethanol first and deionized water later.

[0031] Subsequently, the two samples are placed in an oxygen plasma cleaning machine for cleaning, and the surface is affected;

[0032] Subsequently, the cleaned soft channel A and the soft channel B of the initial organ chip with the three-dimensional micro actuator are bonded, and pressure is applied to integrate them together to form an optically tunable organ chip.

[0033] Further, the hard template material is polymethyl methacrylate, and the soft template material is polydimethylsiloxane.

[0034] Further, the three-dimensional micro actuator includes a Malan-goni micro actuator and a micro substrate actuator.

[0035] The Malan Goni micro-actuator is composed of a gear with asymmetric blades, the blades are composed of an arc-shaped side and a linear side, and a through hole is arranged in the center of the actuator, and the through hole is provided with a stand for limiting the movement of the actuator;

[0036] The three-dimensional micro-actuator is irradiated by an external light source, the heat generated by the absorption of light on both sides of the gear blade is accumulated differently, and an asymmetric light-heat field is generated on both sides of each blade of the gear, the existence of the asymmetric light-heat field induces the Malan Goni effect at the edge of the blade, so that the surface tension of two points located at the same diameter and close to the arc side and the linear side of the same blade respectively is different in size and opposite in direction, the surface tension of the point close to the arc side is greater than that of the point close to the linear side, so as to directionally drive the blade; when the directional driving force of each blade points to the same direction, the whole gear rotates directionally;

[0037] The micro-base actuator is designed based on the different thermal expansion capabilities of polymer materials with different cross-linking degrees, the polymer cross-linking network is divided into high network density C and low network density D according to the density, the high network density C and the low network density D are alternately connected and distributed on the X-axis and the Y-axis, and are also alternately connected and distributed in the Z-axis direction, which is realized by femtosecond laser double three-dimensional technology, the scanning path of the femtosecond laser is controlled by a program, the femtosecond laser performs photopolymerization cross-linking on the polymer material in the scanning path, and the control program can adjust the density of the polymerization path of the laser in a certain area, so as to generate areas with different cross-linking degrees in the same polymer, and the peripheral pattern of the laser polymerization path is designed in the program, so as to realize the overall three-dimensional structure of the actuator.

[0038] Compared with the prior art, the beneficial effects achieved by the present application are:

[0039] 1、The present application effectively improves the defects of inconvenience, inaccuracy and long distance caused by the mechanical force stimulation of traditional external pneumatic or hydraulic devices, so that the organ chip has remote light control, is more portable, and the mechanical force is more accurate;

[0040] 2、The present application uses femtosecond laser hetero-material conformal direct writing technology to prepare a micro Malan Goni actuator.

[0041] 3、Compared with the traditional organ chip which mostly uses external pneumatic devices to make the organ chip deform and simulate peristalsis, the present application uses femtosecond laser double three-dimensional direct writing technology to prepare a micro-base actuator, which simulates organ peristalsis under light tuning and produces local mechanical force stimulation to cells, so as to accelerate the functional differentiation of cells. BRIEF DESCRIPTION OF DRAWINGS

[0042] Figure 1 Preparation and operation flow chart of the present application;

[0043] Figure 2 Principle diagram of femtosecond laser double three-dimensional processing technology and optical driving of the present application;

[0044] Figure 3 Diagram of rotating the remote optical driving micro Malangoni actuator of the present application;

[0045] Figure 4 Shape of the micro substrate actuator in the present application and cycle deformation diagram of the optical driving micro substrate actuator;

[0046] Figure 5 Application of the optical tuner organ chip of the present application to an intestinal organ chip diagram. DETAILED DESCRIPTION

[0047] In order to make the technical means, creative features, purposes and effects of the present application easy to understand, the present application is further described below in combination with specific embodiments.

[0048] The preparation and processing method of the present application is femtosecond laser micro-nano processing. The micro Malangoni actuator and the micro substrate actuator are prepared by using composite materials, combining femtosecond laser hetero-material conformal processing technology and femtosecond laser double three-dimensional processing technology in the organ chip. Through the study of the optical modulation mechanism of the micro actuator, intelligent control of the micro actuator is realized. The micro Malangoni actuator and the micro substrate actuator in the organ chip simulate the flow of body fluid and the peristalsis of organs under the remote optical tuning without external liquid or pneumatic devices, thereby producing mechanical force stimulation on cells and microorganisms, promoting cell differentiation and prolonging the coexistence time of microorganisms and hosts. The specific method is as follows: EMBODIMENT

[0049] The present application discloses a preparation and processing method of an optical tunable organ chip, which comprises the following steps:

[0050] Step 1, preparing soft channel A and B templates;

[0051] Step 2, pouring the processed polymer composite material into the prepared soft channel A template to obtain a preliminary organ chip channel to be processed;

[0052] Step 3, using femtosecond laser double-photon polymerization to scan and process a three-dimensional micro actuator from bottom to top on the upper surface of the preliminary organ chip channel, and post-processing the preliminary organ chip with the three-dimensional micro actuator;

[0053] Step 4, bonding soft channel A and soft channel B of the initial organ chip with three-dimensional micro-actuator to obtain an organ chip capable of optical tuning.

[0054] For step 1, the method for preparing the soft channel A, B template comprises:

[0055] Using a carbon dioxide laser to engrave the designed organ chip channel A, B surface on the machinable hard template,

[0056] Mixing and stirring the precursor and curing agent of the soft moldable material, vacuuming the bubbles in the mixture, pouring the moldable material after the bubbles are removed on the hard channel template A, B respectively, and placing it in an oven to solidify and shape, completing the first mold turning of the channel;

[0057] The surface of the prepared soft channel A, B template is subjected to fluorination treatment, and the soft channel A, B template after mold turning is placed in deionized water for ultrasonic cleaning, then dried with dry nitrogen, and finally the soft channel A, B template sample is obtained.

[0058] For step 3, the processing method of the three-dimensional micro-actuator comprises:

[0059] Fixing the soft channel A template on the three-dimensional processing platform;

[0060] Pouring the processed polymer composite into the prepared soft channel A template, and adding a photoinitiator to the composite material for inducing polymerization of the composite material;

[0061] Using an image sensor CCD imaging system to observe the shape of the light spot and confirm the position of the light spot, focusing the femtosecond laser on the upper surface of the channel through the polymer mixed material, and reading the pre-designed processing file into the processing program;

[0062] Using the femtosecond laser two-photon polymerization method to process from bottom to top to form a micro-actuator with a three-dimensional structure.

[0063] The structure of the three-dimensional platform comprises:

[0064] The three-dimensional processing platform is composed of a two-dimensional air floating platform and a one-dimensional electrically controlled displacement platform; the two-dimensional air floating platform is used to control the X-axis and Y-axis; the one-dimensional electrically controlled displacement platform is used to control the Z-axis, the movement range of the X-axis and Y-axis of the two-dimensional air floating platform is 0-5cm, and the accuracy is 50-100nm, the movement range of the Z-axis of the one-dimensional electrically controlled displacement platform is 0-5cm, and the accuracy is 10-20nm, preferably the accuracy is 10nm, the X-axis and Y-axis are driven by the air floating platform to move the sample to control the position of the laser focal point, the moving speed is 0-40mm / s, the moving accuracy is 50-100nm, and preferably the moving accuracy is 50nm.

[0065] For confirming the position of the light spot, the observing the shape of the light spot by using the imaging system of the image sensor CCD comprises:

[0066] Turning on the image sensor and the illumination mercury lamp light source, first adjusting the one-dimensional electrically controlled displacement platform, raising the sample, finding the mark line on the upper surface of the processing channel in the imaging system; turning on the processing control program, opening the light shutter, moving the two-dimensional air floating platform under the control of the program, and adjusting the position of the Z axis until the clear mark line appears on the sample and the area of the light spot is the smallest, and the position is confirmed as the position of the light spot, wherein the processing file is written by 3Dmax software or Matlab software, the repetition frequency of the femtosecond laser is 80 MHz, the pulse width is 100 fs, the objective lens used is a 60 times lens with a numerical aperture of 1.4, the average power of the laser is about 30 mW, and the exposure time is 1 ms.

[0067] The post-processing of the initial organ chip with the three-dimensional micro actuator comprises:

[0068] The initial organ chip with the three-dimensional micro actuator is developed in a developing solution, developed for about 5 minutes, and the un-polymerized material is removed;

[0069] The surface and the inside of the channel are cleaned with deionized water;

[0070] Then, the cleaned sample is subjected to ultrasonic treatment in acetone solution, ethanol solution and deionized water in sequence, and the ultrasonic treatment lasts for 5 minutes, and finally the sample is dried by a drying machine or dried by a dry nitrogen flow.

[0071] For step 4, the method for bonding the soft channel A and the soft channel B of the initial organ chip with the three-dimensional micro actuator comprises:

[0072] The surface of the soft channel A and the soft channel B of the initial organ chip with the three-dimensional micro actuator are cleaned in sequence by ethanol and then deionized water.

[0073] Subsequently, the two samples are placed in an oxygen plasma cleaning machine for cleaning, and the surface of the samples is affected;

[0074] Subsequently, the cleaned soft channel A and the soft channel B of the initial organ chip with the three-dimensional micro actuator are bonded, and pressure is applied to the two samples to integrate them together to form an optically tunable organ chip.

[0075] In this embodiment, the hard template material is polymethyl methacrylate, and the soft template material is polydimethylsiloxane.

[0076] In the present embodiment, the three-dimensional micro-actuator comprises a Malan-goni micro-actuator and a micro-substrate actuator;

[0077] The Malan-goni micro-actuator comprises a gear composed of asymmetric blades, the blades are composed of an arc-shaped side and a linear side, and a through hole is arranged in the center of the actuator, the through hole is provided with a post for limiting the movement of the actuator;

[0078] Under the irradiation of external light source, the heat accumulated on the two sides of the gear blade is different, and an asymmetric light-heat field is generated on the two sides of each blade of the gear. The existence of the asymmetric light-heat field induces the Malan-goni effect at the edge of the blade, so that the surface tension of the two points at the same diameter of the same blade near the arc-shaped side and the linear side is different in size and direction. The surface tension of the point near the arc-shaped side is greater than that of the point near the linear side, thereby driving the blade in a directional manner. When the directional driving force of each blade points to the same direction, the entire gear rotates in a directional manner.

[0079] The micro-substrate actuator is designed based on the different thermal expansion capabilities of polymer materials with different cross-linking degrees. The polymer cross-linking network is divided into high network density C and low network density D according to the density, and the high network density C and the low network density D are alternately connected and distributed on the X-axis and the Y-axis, and also alternately connected and distributed in the Z-axis direction. The three-dimensional structure of the actuator is realized by femtosecond laser double three-dimensional technology. The scanning path of the femtosecond laser is controlled by a program, and the polymer material is subjected to photopolymerization cross-linking action on the scanning path traveled by the femtosecond laser. The control program can adjust the density of the polymerization path of the laser in a certain area, thereby generating areas with different cross-linking degrees in the same polymer. At the same time, the peripheral pattern of the laser polymerization path is designed in the program, thereby realizing the three-dimensional structure of the actuator.

[0080] Regarding the design of the Malan-goni micro-actuator, the actuator is a gear composed of asymmetric blades, the blades are composed of an arc-shaped side and a linear side, and the number of blades can be increased or decreased in the actuator as needed. A through hole is arranged in the center of the actuator, which functions to 1. reduce the weight of the actuator; 2. limit the movement of the actuator by preparing a post in the hole. Through the design of the asymmetric gear blades (see Figure 3 ), under the irradiation of external light source, the heat accumulated on the two sides of the gear blade is different, and an asymmetric light-heat field is generated on the two sides of each blade of the gear. The existence of the asymmetric light-heat field induces the Malan-goni effect at the edge of the blade, so that the surface tension of the two points at the same diameter of the same blade near the arc-shaped side and the linear side is different in size and direction. The surface tension of the point near the arc-shaped side is greater than that of the point near the linear side (F3 is greater than F2), thereby driving the blade in a directional manner. When the directional driving force of each blade points to the same direction, as shown in Figure 3The force F3 in the figure causes the entire gear to rotate in a certain direction.

[0081] Regarding the design of the micro-substrate actuator, the design of this type of actuator is based on the different thermal expansion capabilities of polymer materials with different cross-linking degrees. The polymer cross-linking network is divided into C (high network density) and D (low network density) according to the density, and C and D are alternately connected and distributed in the XY axis, and also alternately connected and distributed in the Z axis direction, as shown in the accompanying Figure 3 The number and size of each polymer block can be increased or decreased according to actual needs, and polymer blocks with new network densities different from the above two can also be added. Under the irradiation of external light sources, the actuator performs photo-thermal conversion. Due to the different network densities of different parts, different thermal expansions occur in different parts of the actuator at the same temperature. With the alternating distribution of C and D and the cyclic irradiation of the light source, the micro-substrate actuator produces high-low undulating motion, similar to peristaltic motion.

[0082] The preparation of this type of actuator is realized by femtosecond laser double three-dimensional technology. The scanning path of femtosecond laser is controlled by program, and the femtosecond laser can perform photopolymerization cross-linking on the polymer material in the scanned path. The control program can adjust the density of the polymerization path in a certain area, thereby generating areas with different cross-linking degrees in the same polymer, realizing the Figure 3 alternating connection of C and D in the accompanying

[0083] The micro-Malangoni actuator is prepared inside the organ chip by modulating the femtosecond laser composite material co-molding technology.

[0084] The femtosecond laser is a Mai Tai titanium sapphire femtosecond laser provided by Spectra-Physics Company; the wavelength of the femtosecond laser is 800 nm.

[0085] As Figure 1 shown, it is a flowchart of the design and processing method of an optical tuner organ chip of this embodiment, and the processing method comprises the following steps:

[0086] (1) Preparation of the sample to be processed;

[0087] ​Firstly, the carbon dioxide laser is used to engrave the designed organ chip channel A and B on the hard template. The precursor and curing agent of the soft moldable material are mixed thoroughly. The bubbles in the mixture are removed by vacuumizing. The moldable material after removing the bubbles is poured into the hard channel template A and B, and then placed in an oven for curing. The above steps complete the first film forming of the channel. The surface of the soft channel template prepared by the above steps is fluorinated and subjected to the second mold turning. The soft moldable material used in the second mold turning is the same as the above steps. The A and B soft channel templates are placed in deionized water for ultrasonic cleaning for 15 minutes, then dried with dry nitrogen. The preparation of the soft channel A and B templates is completed.

[0088] (2) Shape design and programming of three-dimensional micro Manganoni actuator;

[0089] According to the principle of Manganoni effect, an actuator that can generate an asymmetric light-thermal effect field needs to be designed, so the asymmetric structure and morphology of the actuator are designed. In this example, the asymmetric gear structure shown in FIG. 1 is used as the Manganoni actuator. The designed gear structure is drawn by software 3DMAX and converted and saved into a corresponding file for subsequent import into the machining program. Figure 3

[0090] (3) Femtosecond laser processing of three-dimensional micro Manganoni actuator by two-photon polymerization;

[0091] Firstly, the prepared processing sample is placed on the sample stage of the processing platform. Mark the position where the actuator needs to be processed in the channel. The side with the mark is placed upwards and the sample is fixed on the edge with 3M tape. Adjust the laser power and turn on the image sensor and illumination mercury lamp light source. Adjust the manual three-dimensional platform, first adjust the Z axis, slowly raise the sample, find the upper surface of the processing sample in the imaging system, adjust the X axis and Y axis, and find the mark line on the upper surface in the imaging system. Fill the composite material into the channel, turn on the processing control program, open the shutter, control the two-dimensional air floating platform with the computer program, and adjust the Z axis position until the laser exposure trace appears on the sample. At this time, the laser focus is focused on the upper surface of the sample. Then, close the shutter, import the pre-designed shape processing data of the Manganoni actuator, and start processing the micro Manganoni actuator in the channel.

[0092] The wavelength of femtosecond laser is 800 nm, the repetition frequency is 80 MHz, the objective lens is 60X lens, the numerical aperture is 1.4, the average power of laser is about 30 mW, and the exposure time is 1 ms.

[0093] (4) Sample etching;

[0094] ​First, the finished sample is immersed in a developing solution, i.e., thioxanthone dissolved in chloroform, to develop the directly-written device for about 5 minutes to remove the unpolymerized material. Subsequently, the surface and channel interior are cleaned with deionized water to clean the surface and channel interior of the remaining composite material after development; then the cleaned sample is subjected to ultrasonic treatment with acetone solution, then ethanol solution, and finally deionized water, each for 5 minutes, and finally dried with a dryer or dried with a dry nitrogen stream; the above steps can basically complete the preparation of the three-dimensional Malan-goni micro-actuator in the organ chip channel. Example

[0095] Preparation of a micro-base actuator in an organ chip channel by femtosecond laser double three-dimensional processing technology.

[0096] The processing flow is shown in Figure 1 , which is the same as Example 1.

[0097] The specific steps are as follows:

[0098] (1) Preparation of the sample to be processed;

[0099] The same as Example 2 will not be described in detail here.

[0100] (2) Shape design and programming of the three-dimensional micro-base actuator;

[0101] According to the principle that the thermal expansion effect is different depending on the polymer network density, an actuator that can produce an asymmetric thermal expansion effect needs to be designed, so according to the principle of femtosecond laser double three-dimensional processing, the internal area with different network densities can be prepared at the same time as the actuator shape structure is prepared. As shown in Figure 4 , this type of micro-base actuator is prepared, which can produce different thermal expansion effects under light due to different network densities in the interior, ultimately producing peristaltic motion. The designed micro-base structure is described by C language to control the laser path and converted and saved into the corresponding file for subsequent import into the processing program.

[0102] (3) Femtosecond laser processing double three-dimensional polymer processing three-dimensional micro-base actuator;

[0103] First, the prepared processing sample is placed on the sample stage of the processing platform, the side with the marks drawn is upward, and the sample is fixed at the edge of the sample with 3M tape; the laser power is adjusted and the image sensor and the illumination mercury lamp light source are turned on, the manual three-dimensional platform is adjusted, the Z axis is adjusted first, the sample is slowly raised, the upper surface of the processed sample is found in the imaging system, the X axis and the Y axis are adjusted, and the mark line of the upper surface is clearly found in the imaging system; at this time, the Z axis is adjusted to move the laser focal point 1 micrometer toward the inside of the channel, so as to ensure that the micro substrate is completely attached to the bottom of the channel. The processing data of the micro substrate actuator designed in advance is imported, and the processing and preparation of the micro substrate actuator in the channel are started, which is the same as in Example 1.

[0104] (4) sample corrosion;

[0105] The same as in Example 2, which will not be repeated here.

[0106] The internal liquid flow and mechanical force stimulation to cells of the traditional organ chip are both dependent on external liquid and pneumatic equipment. The design and invention of the present application is to integrate and prepare a micro optical actuator in the channel of the organ chip by means of femtosecond laser processing, to drive the micro actuator to move by using external light irradiation, so as to push the liquid flow and generate mechanical force stimulation to cells in the organ chip.

[0107] The above is only the preferred embodiment of the present application, and it should be pointed out that for ordinary skilled persons in the art, a number of improvements and modifications can be made without departing from the technical principles of the present application, and these improvements and modifications should also be considered as the protection scope of the present application.

Claims

1. A method for preparing and processing a light-tunable organ chip, characterized in that: The method comprises: Prepare soft channel A and B templates; The polymer composite material to be processed is poured into the prepared soft channel A template to obtain the initial organ chip channel to be processed; Using femtosecond laser two-photon polymerization, three-dimensional micro-actuators are scanned from bottom to top on the upper surface of the early organ chip channel, and the early organ chip with the three-dimensional micro-actuators is post-processed; Bonding soft channel A and soft channel B of the initial organ chip with three-dimensional microactuators to obtain an optically tunable organ chip; The three-dimensional microactuator includes a Marangoni microactuator and a micro-substrate actuator; The Marangoni microactuator is a gear consisting of asymmetric blades, each blade having an arc-shaped side and a straight side. There is a through hole in the center of the actuator, and the through hole is provided with a column to limit the movement of the actuator. When the three-dimensional micro-actuator is illuminated by an external light source, the heat converted into by absorbing light on both sides of the gear blade accumulates differently, generating an asymmetric photothermal field on both sides of each blade of the gear. The presence of the asymmetric photothermal field induces a Marangoni effect at the blade edge, causing the surface tensions of two points located at the same diameter of the same blade, respectively close to the arc edge and the straight edge, to be different in magnitude and opposite in direction. The surface tension of the arc edge point is greater than that of the straight edge point, thereby directionally driving the blade. When the directional driving force of each blade points in the same direction, the entire gear undergoes directionally rotation. The micro-substrate actuator is designed based on the principle that polymer materials with different degrees of cross-linking have different thermal expansion capabilities. The polymer cross-linking network is divided into high network density C and low network density D according to the density. The high network density C and low network density D are alternately connected and distributed on the X-axis and Y-axis, and are also alternately connected and distributed in the Z-axis direction. It is realized by femtosecond laser dual three-dimensional technology. The scanning path of the femtosecond laser is controlled by a program. The femtosecond laser performs photopolymerization and cross-linking on the polymer material along the scanning path. The control program can adjust the density of the laser polymerization path in a certain area, thereby generating areas with different degrees of cross-linking in the same polymer. At the same time, the peripheral graphics of the laser polymerization path are designed in the program to realize the overall three-dimensional structure of the actuator.

2. The method for preparing a light-tunable organ chip according to claim 1, characterized in that: The method for preparing the soft channel A and B templates comprises: Use a carbon dioxide laser to carve the designed organ chip channels A and B on a machinable hard template. The precursor of the soft re-moldable material and the curing agent are fully mixed and stirred, and the bubbles in the mixture are vacuum-extracted. The re-moldable material after the bubbles are removed is poured onto the hard channel templates A and B respectively, and placed in an oven to solidify and shape, completing the first re-mold of the channel; The surfaces of the prepared soft channel A and B templates were fluorinated and re-molded. The re-molded soft channel A and B templates were placed in deionized water for ultrasonic cleaning, taken out, and then blown dry with dry nitrogen to finally obtain soft channel A and B template samples.

3. The method for preparing a light-tunable organ chip according to claim 1, characterized in that: The processing method of the three-dimensional micro actuator includes: Fix the soft channel A template on the three-dimensional processing platform; The processed polymer composite material is poured into the prepared soft channel A template, and a photoinitiator is added to the composite material to induce polymerization of the composite material; The image sensor CCD imaging system is used to observe the spot shape and confirm the spot position, the femtosecond laser penetrates the polymer mixed material and focuses on the upper surface of the channel, and the pre-designed processing file is read into the processing program; Femtosecond laser two-photon polymerization is used for processing, and scanning processing is performed from bottom to top to form a microactuator with a three-dimensional structure.

4. The method for preparing a light-tunable organ chip according to claim 3, characterized in that: The three-dimensional processing platform consists of a two-dimensional air-floating platform and a one-dimensional electric-controlled displacement platform; the two-dimensional air-floating platform is used to control the X-axis and the Y-axis; the one-dimensional electric-controlled displacement platform is used to control the Z-axis.

5. The method for preparing a light-tunable organ chip according to claim 4, characterized in that: The method of observing the light spot shape and confirming the light spot position by using an image sensor CCD imaging system includes: Turn on the image sensor and the mercury lamp light source. First, adjust the one-dimensional electric displacement platform, raise the sample, and find the mark line on the upper surface of the processing channel in the imaging system. Open the processing control program, open the light gate, and use the program to control the movement of the two-dimensional air flotation platform. At the same time, adjust the Z-axis position until a clear mark line appears on the sample and the spot area is minimized. This is confirmed as the spot position.

6. The method for preparing and processing a light-tunable organ chip according to claim 4, characterized in that: The moving range of the X-axis and Y-axis of the two-dimensional air flotation platform is 0-5cm, with an accuracy of 50-100nm. The moving range of the Z-axis of the one-dimensional electric displacement platform is 0-5cm, with an accuracy of 10-20nm. The X-axis and Y-axis are controlled by the air flotation platform to move the sample to control the laser focus position. The moving speed is 0-40mm / s and the moving accuracy is 50-100nm.

7. The method for preparing a light-tunable organ chip according to claim 1, characterized in that: Post-processing of early Organ-on-Chips with 3D microactuators includes: Immersing the early organ chip with the three-dimensional microactuator in a developing solution for development to remove unpolymerized materials; Use deionized water to clean the surface and inside the channel; The cleaned sample is then ultrasonically treated with an acetone solution, an ethanol solution, and finally deionized water, and finally dried in a dryer or blown dry with a dry nitrogen flow.

8. The method for preparing a light-tunable organ chip according to claim 1, characterized in that: The method for bonding the soft channel A and the soft channel B of the early organ chip with the three-dimensional microactuator includes: Clean the soft channels A and B of the early organ chip with three-dimensional microactuators using ethanol first and then deionized water. Then the two samples were placed in an oxygen plasma cleaning machine for cleaning to act on their surfaces; The two samples of soft channel A and soft channel B of the initial organ chip with three-dimensional microactuators were then bonded after cleaning, and pressure was applied to them to integrate them together to form an optically tunable organ chip.

9. The method for preparing a light-tunable organ chip according to claim 2, characterized in that: The hard channel template material is polymethyl methacrylate, and the soft channel template material is polydimethylsiloxane.

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