Preparation method of a self-filling microfluidic chip based on joint driving and application thereof

Abstract

The application discloses a kind of based on joint drive's bionic self perfusion microfluidic chip preparation method and its application, belong to microfluidic chip field, the preparation method includes: preparation with the reserved groove for accommodating hydrophilic porous material flow channel layer;A hydrophilic porous material is embedded in the reserved groove of the flow channel layer, to form capillary flow starting area;Provide a substrate layer, and a hydrophobic porous evaporation membrane is arranged in the predetermined evaporation area position of substrate layer surface;And with hydrophilic porous material the flow channel layer with hydrophobic porous evaporation membrane of the substrate layer is aligned bonding, so that the flow channel area for evaporation on the flow channel layer corresponds with the position of the hydrophobic porous evaporation membrane, to obtain the microfluidic chip.The preparation method of the application should be simple and low cost.

Description

Technical Field

[0001] This invention relates to the field of microfluidic chips, and in particular to a method for fabricating a biomimetic self-perfusion microfluidic chip based on joint drive and its application. Background Technology

[0002] Microfluidic chip technology, also known as lab-on-a-chip, holds immense potential in fields such as biology, chemistry, and medicine. However, traditional microfluidic systems heavily rely on expensive and bulky external pumps to drive the fluid, resulting in problems such as low system integration, high power consumption, high cost, difficulty in achieving high-throughput parallel experiments, and lack of portability.

[0003] In existing technologies, some methods have been developed to achieve "self-driven" fluid flow, such as capillary actuation, which utilizes the hydrophilicity of the flow channel surface to generate capillary action. However, this method has limited driving force, the flow rate is difficult to maintain, and it is easily interrupted by liquid evaporation. Furthermore, this approach is complex to construct, and maintaining the solution concentration is challenging. Therefore, there is an urgent need in the field for a self-perfusion driving technology that can generate stable, long-term, and externally-force-free flow. Summary of the Invention

[0004] One of the objectives of this invention is to provide a biomimetic self-perfusion microfluidic chip fabrication method based on joint drive, so as to solve the problem that maintaining solution concentration is difficult in the prior art.

[0005] This invention is achieved through the following technical solution: a method for fabricating a biomimetic self-perfusion microfluidic chip based on joint driving, comprising the following steps:

[0006] A flow channel layer with reserved grooves for accommodating a hydrophilic porous material is prepared; a hydrophilic porous material is embedded in the reserved grooves of the flow channel layer to form a capillary initiation region; a substrate layer is provided, and a hydrophobic porous evaporation film is disposed at a predetermined evaporation region position on the surface of the substrate layer; and the flow channel layer with the hydrophilic porous material and the substrate layer with the hydrophobic porous evaporation film are aligned and bonded such that the flow channel region on the flow channel layer for evaporation corresponds to the position of the hydrophobic porous evaporation film, thereby obtaining the microfluidic chip.

[0007] Furthermore, the flow channel layer is made of polydimethylsiloxane.

[0008] Furthermore, polydimethylsiloxane is prepared by mixing polydimethylsiloxane prepolymer and curing agent at a weight ratio of 10:1 and then heat curing at 80°C for 2 hours.

[0009] Furthermore, the hydrophilic porous material is chromatography filter paper; and / or, the hydrophobic porous evaporation membrane is a polytetrafluoroethylene membrane.

[0010] Furthermore, the average pore size of the polytetrafluoroethylene membrane is 0.45 μm.

[0011] Furthermore, the alignment bonding includes: performing plasma treatment on the surface of the channel layer and the surface of the substrate layer to be bonded, so as to activate the surfaces.

[0012] Furthermore, the plasma treatment conditions were as follows: treatment for 45 seconds at a power of 50W and an oxygen pressure of 100 mTorr.

[0013] Furthermore, the preparation method also includes a step of heating the bonded integral chip at 80°C for 30 minutes to enhance the bonding strength.

[0014] Furthermore, when the hydrophilic porous material is anisotropic bacterial cellulose / polyvinyl alcohol aerogel, its preparation steps include: composite slurry preparation: mixing bacterial cellulose nanofiber suspension with polyvinyl alcohol solution, adding surfactant, and centrifuging to remove bubbles to obtain a uniform slurry; directional cryocasting: placing the slurry in a mold, and controlling the temperature gradient to generate directionally growing ice crystals in the slurry; post-treatment: freeze-drying the frozen sample to remove ice crystals to form directional channels, and performing chemical cross-linking to enhance structural stability.

[0015] Furthermore, liquid nitrogen was used as a cold source to control the vertical upward growth of ice crystals; glutaraldehyde vapor was used for fumigation and cross-linking.

[0016] Furthermore, when embedding the hydrophilic porous material into the reserved groove, the direction of the microchannels inside the hydrophilic porous material is parallel to the fluid flow direction of the flow channel layer.

[0017] Another aspect of the present invention provides an application of a biomimetic self-perfusion microfluidic chip based on joint drive. The biomimetic self-perfusion microfluidic chip prepared according to the above steps has the following applications: (1) application in advanced cell and tissue culture; (2) application in drug development and toxicology screening.

[0018] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0019] 1. This invention constructs a combined drive engine of capillary initiation and evaporation maintenance by ingeniously integrating hydrophilic porous materials and hydrophobic porous evaporation membranes on a chip. This engine can generate stable and continuous fluid perfusion without any external pump or power supply, greatly improving the chip's integration and portability.

[0020] 2. This invention ensures that the liquid can quickly and bubble-free fill the entire flow channel network and start the flow through capillary action; while the subsequent evaporation pump effect takes over the drive, and pulls the liquid through continuous and stable evaporation loss, forming a stable micro flow rate (such as 2.5 μL / h) for several days, which solves the problem of rapid flow rate decay and unsustainability of traditional capillary drive, and achieves true self-driven flow.

[0021] 3. The materials used in this invention (such as PDMS, filter paper, PTFE membrane, and glass slide) are inexpensive and readily available. Furthermore, the preparation process is mainly based on mature soft lithography and plasma bonding technologies, which are simple to operate, have good repeatability, and are easy to mass-produce. They have the advantages of simple preparation process and low cost. Attached Figure Description

[0022] The accompanying drawings, which are included to provide a further understanding of embodiments of the invention and form part of this application, do not constitute a limitation thereof. In the drawings:

[0023] Figure 1 This is an overall structural diagram provided for Embodiment 1 of the present invention.

[0024] Figure 2 This is a scanning electron microscope image of the hydrophilic porous material provided in Embodiment 2 of the present invention. Detailed Implementation

[0025] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are some embodiments of the present invention, but not all embodiments.

[0026] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated herein by reference to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail. The terms “comprising,” “including,” “having,” “containing,” etc., as used herein are open-ended terms, meaning that they include but are not limited to. Unless the context clearly indicates otherwise, the expressions “a” and “an” as used herein include plural references. It should be noted that “first,” “second,” etc., are used merely for convenience of description and distinction and should not be construed as indicating or implying relative importance. The term “about” as used herein indicates a range of ±20% of the following numerical value. In some embodiments, the term “about” indicates a range of ±10% of the following numerical value. In some embodiments, the term “about” indicates a range of ±5% of the following numerical value.

[0027] Example 1

[0028] This embodiment provides a self-perfusion cell culture microfluidic chip based on an evaporation pump and its usage method, specifically including the following steps:

[0029] Step 1: Chip manufacturing.

[0030] Preparation of the flow channel layer and the top cover layer:

[0031] Using polydimethylsiloxane as raw material, PDMS prepolymer and curing agent were accurately weighed at a ratio of 10:1 (w / w), thoroughly mixed in a vacuum stirrer and vacuum defoamed for 30 minutes to prepare a uniformly mixed PDMS mixture.

[0032] The PDMS mixture is then poured onto a silicon-based mold prefabricated by photolithography. The mold has a raised structure opposite to the flow channel network, with a height of 100 μm.

[0033] The cast mold is placed in a constant temperature oven at 80℃ for heat curing for 2 hours. After curing, the flow channel layer with a recessed flow channel structure is peeled off from the mold.

[0034] Using the same method, a 2mm thick PDMS flat film was poured into a clean petri dish, cured, and used as the top cover layer. A 2mm diameter punch was used to punch through the corresponding position of the top cover layer 1 to form the sample inlet and outlet.

[0035] Step 2: Preparation of the substrate layer and integration of functional materials:

[0036] In this embodiment, the substrate layer is a 1 mm thick glass slide (25 mm x 75 mm in area). The hydrophilic porous material is chromatography filter paper, which is cut into small circular pieces using a 1.5 mm diameter punch. Before subsequent bonding, the circular filter paper pieces are precisely pressed into and embedded in the pre-reserved grooves in the capillary initiation region of the flow channel layer.

[0037] The porous evaporation membrane used was a hydrophobic polytetrafluoroethylene (PTFE) membrane from Pall Corporation of the United States, with an average pore size of 0.45 μm and a thickness of 150 μm; it was cut into 5 mm x 5 mm squares for later use.

[0038] Step 3: Overall bonding:

[0039] The flow channel layer with embedded filter paper, the flat top cover layer, and the glass slide serving as the substrate are ultrasonically cleaned with deionized water and ethanol and then dried. The porous evaporation membrane is then placed on the substrate layer at the corresponding evaporation zone position.

[0040] Subsequently, all components (the flow channel surface of the flow channel layer, the flat surface of the top cover layer, and the base layer with the evaporation film) are placed together in a plasma cleaner and treated for 45 seconds at a power of 50W and an oxygen pressure of 100 mTorr to activate their surfaces and generate hydrophilic groups.

[0041] After processing, the porous evaporation membrane is first permanently bonded to the corresponding position of the substrate layer.

[0042] Then, under a microscope, the channel layer and the substrate layer are precisely aligned, so that the evaporation channels of the channel layer are aligned with the evaporation film on the substrate layer, forming a preliminary bond. Finally, the top cap layer is aligned and bonded to the channel layer. The bonded chip is then placed in an 80°C oven and heated for 30 minutes to enhance the bonding strength, ultimately forming a complete and irreversibly bonded microfluidic chip.

[0043] The specific dimensions of the chip prepared in this embodiment are as follows:

[0044] The liquid storage area is a circular region with a diameter of 5 mm;

[0045] The main irrigation channel is 300 μm wide and 100 μm high;

[0046] The biomimetic microcavity is a circular cavity with a diameter of 1 mm and a height of 100 μm;

[0047] The capillary initiation zone has a width of 1.5 mm; the evaporation zone channel 23 is a 5 mm x 5 mm wide area. Figure 1 The overall structure diagram of the chip prepared in this embodiment is disclosed.

[0048] Step 4: After the chip is fabricated, its performance is verified.

[0049] 1) Cell seeding and initiation of autoperfusion:

[0050] This embodiment uses human liver cancer cells (HepG2) for culture experiments.

[0051] First, the chip was sterilized by UV irradiation and 75% ethanol solution. Pre-cultured HepG2 cells were digested, centrifuged, and resuspended in DMEM high-glucose medium containing 10% fetal bovine serum (FBS), adjusting the cell density to 2 × 10⁻⁶ cells / day. 6 per mL.

[0052] Using a pipette, 50 μL of cell suspension was slowly injected into the reservoir through the inlet. During injection, the liquid first came into contact with the hydrophilic porous material in the capillary initiation zone. Due to the capillary force of the material, the liquid was rapidly and bubble-free drawn in and filled the entire flow channel network, and the cells entered and settled uniformly at the bottom of the biomimetic microchamber.

[0053] The self-perfusion process automatically starts when the liquid front reaches the evaporation zone channel and completely wets the porous evaporation membrane below.

[0054] 2) Cell culture and efficacy verification:

[0055] The cell-inoculated chip was placed in a cell culture incubator at 37°C and 95% relative humidity. The evaporation pump started working: because the porous evaporation membrane 6 is hydrophobic, liquid water cannot pass through, but water vapor can pass through freely; there is a water vapor pressure difference between the unsaturated humidity environment in the incubator and the 100% humidity liquid inside the chip, which drives the liquid to evaporate from the membrane in the form of water vapor.

[0056] This continuous liquid loss causes the liquid in the storage zone 12 to flow slowly and continuously toward the evaporation zone under the combined action of capillary force and water pressure gradient, thus forming a stable self-flow.

[0057] By mixing polystyrene microspheres with a diameter of 1 μm into the culture medium and taking long-term sequential photographs using an inverted microscope, the average flow rate inside the biomimetic microchamber 4 was measured to be stable at approximately 2.5 μL / h using particle image velocimetry (PIV).

[0058] Example 2

[0059] The overall steps in this embodiment are the same as in Embodiment 1. The difference is that the hydrophilic porous material used in this embodiment is a self-made hydrophilic porous material.

[0060] This embodiment discloses anisotropic BC / PVA hydrophilic porous material based on liquid nitrogen directional freezing. The aim of this embodiment is to prepare a hydrophilic material with highly oriented microchannels to achieve rapid startup and low flow resistance transport in microfluidic chips. The preparation method of this material includes the following steps:

[0061] 1. Preparation of composite slurry:

[0062] (1) Substrate treatment: Bacterial cellulose (BC) wet film was pulped and subjected to high pressure homogenization (800 bar, 5 cycles) to prepare BC nanofiber suspension with a solid content of 0.5 wt%.

[0063] (2) Mixing ratio: Dissolve polyvinyl alcohol (PVA-1799) in deionized water to prepare a 10 wt% solution. Mix the BC suspension and PVA solution at a mass ratio of 7:3 (dry weight ratio).

[0064] (3) Additives: Add 0.1% by volume of Triton X-100 surfactant to the mixture to reduce surface tension.

[0065] (4) Defoaming: The mixture is centrifuged at 2000 rpm for 5 minutes to remove air bubbles and obtain a uniform slurry.

[0066] 2. Directional cryogenic casting:

[0067] (1) Use a heat-insulating mold with a copper plate bottom and polytetrafluoroethylene (PTFE) around the edges (20mm inner diameter, 10mm height).

[0068] (2) Pour the slurry into the mold and immerse the bottom of the mold directly into liquid nitrogen (-196℃).

[0069] (3) Temperature gradient control: Using the extreme supercooling provided by liquid nitrogen, ice crystals are controlled to grow vertically upward at a speed of about 15-20 μm / s, forcing BC / PVA segments to be arranged in the gaps between ice crystals. The freezing process lasts for 10 minutes until completely frozen.

[0070] 3. Post-treatment and cross-linking:

[0071] (1) Freeze-drying: Place the frozen sample in a freeze dryer and dry it for 48 hours at -50℃ and 10 Pa vacuum to remove ice crystals and form oriented channels.

[0072] (2) Chemical crosslinking: The dried aerogel was placed in a desiccator containing 25% glutaraldehyde solution and crosslinked by fumigation in a 60°C oven for 2 hours using glutaraldehyde vapor to enhance its structural stability in an aqueous environment.

[0073] Figure 2The image shows a scanning electron microscope (SEM) image of the hydrophilic porous material prepared in this embodiment. The image reveals parallel-arranged honeycomb-like through-channels within the material. The prepared hydrophilic porous material was cut into 1.5 mm diameter cylinders and embedded into the chip channels of Example 1 (channel direction parallel to fluid direction). 50 μL of cell culture medium was added, and the liquid rapidly filled the 2 cm long channel within 2 seconds without generating bubbles, demonstrating that the hydrophilic porous material in this embodiment has excellent instantaneous start-up capability.

[0074] The specific embodiments described above further illustrate the purpose, technical solution, and beneficial effects of the present invention. It should be understood that the above description is only a specific embodiment of the present invention and is not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for fabricating a biomimetic self-perfusion microfluidic chip based on joint driving, characterized in that, The preparation method includes: Prepare a flow channel layer with reserved grooves for accommodating hydrophilic porous materials; A hydrophilic porous material is embedded in the reserved groove of the flow channel layer to form a capillary initiation region; A substrate layer is provided, and a hydrophobic porous evaporation membrane is disposed at a predetermined evaporation zone location on the surface of the substrate layer; and The flow channel layer with hydrophilic porous material is aligned and bonded to the base layer with hydrophobic porous evaporation membrane, so that the flow channel region on the flow channel layer for evaporation corresponds to the position of the hydrophobic porous evaporation membrane, thereby obtaining the microfluidic chip. The hydrophilic porous material is chromatography filter paper or anisotropic bacterial cellulose / polyvinyl alcohol aerogel; and / or, The hydrophobic porous evaporation membrane is a polytetrafluoroethylene membrane; When the hydrophilic porous material is anisotropic bacterial cellulose / polyvinyl alcohol aerogel, its preparation steps include: Preparation of composite slurry: Bacterial cellulose nanofiber suspension was mixed with polyvinyl alcohol solution, and surfactant was added. The mixture was then centrifuged to remove bubbles and obtain a homogeneous slurry. Directional cryogenic casting: The slurry is placed in a mold, and the directional growth of ice crystals is caused by controlling the temperature gradient. Post-processing: The frozen sample was freeze-dried to remove ice crystals and form oriented channels, and chemical cross-linking was performed to enhance structural stability; Liquid nitrogen was used as a cold source to control the vertical upward growth of ice crystals; glutaraldehyde vapor was used for fumigation and cross-linking. When embedding the hydrophilic porous material into the reserved groove, the direction of the microchannels inside the hydrophilic porous material is parallel to the fluid flow direction of the flow channel layer.

2. The method for fabricating a biomimetic self-perfusion microfluidic chip based on joint driving according to claim 1, characterized in that, The flow channel layer is made of polydimethylsiloxane.

3. The method for fabricating a biomimetic self-perfusion microfluidic chip based on joint driving according to claim 2, characterized in that, The polydimethylsiloxane is prepared by mixing polydimethylsiloxane prepolymer and curing agent at a weight ratio of 10:1 and then heat-curing at 80°C for 2 hours.

4. The method for fabricating a biomimetic self-perfusion microfluidic chip based on joint driving according to claim 1, characterized in that, The polytetrafluoroethylene membrane has an average pore size of 0.45 μm.

5. The method for fabricating a biomimetic self-perfusion microfluidic chip based on joint driving according to claim 1, characterized in that, The alignment bonding includes: performing plasma treatment on the surface of the channel layer and the surface of the substrate layer to be bonded, so as to activate the surfaces.

6. The method for fabricating a biomimetic self-perfusion microfluidic chip based on joint drive according to claim 5, characterized in that, The plasma treatment conditions are: 45 seconds of treatment at a power of 50W and an oxygen pressure of 100 mTorr.

7. The method for fabricating a biomimetic self-perfusion microfluidic chip based on joint driving according to claim 1, characterized in that, The preparation method further includes: The bonded chip is heated at 80°C for 30 minutes to enhance the bonding strength.

8. An application of a biomimetic self-perfusion microfluidic chip based on joint drive, characterized in that, The microfluidic chip prepared according to the preparation method according to any one of claims 1 to 7 has the following characteristics: (1) Applications in advanced cell and tissue culture; (2) Application in drug development and toxicology screening.

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