A microfluidic cell culture chip and pollutant toxicity monitoring platform

Abstract

The present invention provides a microfluidic cell culture chip and a pollutant toxicity monitoring platform. The microfluidic cell culture chip includes a sealed housing, wherein a plurality of cell culture chambers are provided within the sealed housing. A gas exchange chamber is provided on the periphery of the cell culture chamber, one end of the cell culture chamber is connected to a liquid inlet, and the other end is connected to a liquid outlet, and the gas exchange chamber is connected to an air inlet and an air outlet, respectively. The gap between the cell culture chamber and the gas exchange chamber is filled with a PDMS layer, wherein the PDMS layer has micropores for gas to pass through, and the surface of the PDMS layer is modified with any one of polylysine, human recombinant fibronectin, and fish collagen. The pollutant toxicity monitoring platform constructed by coupling the microfluidic cell culture chip with cell electrochemical technology can realize online monitoring of pollutant toxicity, has the advantages of high sensitivity and low cost, and provides a new technical platform for environmental pollutant toxicity detection.

Description

Technical Field

[0001] The present invention belongs to the technical field of pollutant toxicity monitoring, and in particular relates to a microfluidic cell culture chip and a pollutant toxicity monitoring platform. Background Art

[0002] In recent years, the environmental pollution and ecotoxic effects of emerging pollutants have become a major environmental issue facing my country and the world. Emerging pollutants have a wide range of sources and complex toxic mechanisms. Currently, domestic research on emerging pollutants in areas such as hazard identification, exposure prediction, environmental risk assessment, and green alternatives is weak, and a systematic evaluation and monitoring system is lacking. This severely restricts the environmental risk assessment and management of emerging pollutants.

[0003] With the advent of the international "3R" principle and the development and maturity of cell culture technology, in vitro cultured cells have become an important tool for toxicity assessment of environmental pollutants. Cell viability assays reflect the toxic effects of exogenous substances (such as poisons or drugs) on cells by evaluating indicators such as cell proliferation, survival rate, and cell death rate. They are key assays for determining the growth status of in vitro cultured cells under specific treatment conditions (such as poison exposure, drug intervention, or changes in the culture environment). However, traditional in vitro toxicity assessment methods, such as the methylthiazolium tetrazolium (MTT) colorimetric assay and staining counting assay, typically indirectly assess cell viability based on differences in the effects of dyes on cells. These methods have disadvantages such as the need for labeling, complex and time-consuming procedures, and the toxicity of dyes.

[0004] The recently established cellular electrochemical sensing technology based on nucleotide metabolism has provided a new technical approach for environmental toxicological assessment. This technology primarily analyzes cellular electrochemical signals by combining electrochemistry with cellular and molecular biology techniques, revealing the effects of exogenous molecules on their activity and function. With its advantages of simplicity, rapidity, high sensitivity, non-toxicity, and low cost, it has become an important tool for pollutant toxicity research. However, to achieve efficient early warning and toxicity assessment of a large number of new pollutants, cellular electrochemical sensors still need to be improved in terms of microquantification, high throughput, and online detection. Therefore, it is necessary to combine them with other technical approaches.

[0005] As a cutting-edge technology for testing in the biological and pharmaceutical fields, microfluidic chip technology has become a research hotspot in recent years. Also known as a micro-total analysis system, microfluidic chips integrate essential functions required for chemical, physical, and biological experiments onto a chip measuring a few square centimeters, enabling microscale control of fluids. This technology achieves highly optimized reaction conditions and automated experimental procedures by precisely controlling liquid flows at the micrometer scale. Its advantages include low sample and reagent consumption, enabling rapid reactions within microscale reaction chambers; a high level of integration enabling high-throughput, multi-channel parallel testing; miniaturization and portability; and a wide range of chip designs, allowing for the design and fabrication of chips with diverse structures tailored to specific functional needs. By designing complex microchannel networks, microfluidic chips can be used for sample separation, mixing, reactions, and detection. Furthermore, the integration of microfluidic chip technology with optical (fluorescence detectors), electrochemical (conductivity meters, ammeters, and voltmeters), mass spectrometry, and biodetection techniques has further expanded its application. Microfluidics has become a versatile platform capable of analyzing a wide range of substances, showing promising applications in environmental testing, food hygiene, and biomedicine. Coupling microfluidics with cell-based electrochemical sensors could provide new approaches for studying pollutant toxicity. Summary of the Invention

[0006] In order to solve the problems of high cost and inability to achieve online monitoring in existing pollutant toxicity monitoring systems, the present invention provides a microfluidic cell culture chip, which can be coupled with cell electrochemical technology to construct a pollutant toxicity monitoring platform. The constructed pollutant toxicity monitoring platform can achieve online monitoring of pollutant toxicity, has the advantages of high sensitivity and low cost, and provides a new technical platform for environmental pollutant toxicity detection.

[0007] The present invention is achieved through the following technical solutions:

[0008] The present invention provides a microfluidic cell culture chip, comprising a sealed housing, wherein a plurality of cell culture chambers are provided in the sealed housing, a gas exchange chamber is provided on the periphery of the cell culture chamber, one end of the cell culture chamber is connected to a liquid inlet, and the other end is connected to a liquid outlet, and the gas exchange chamber is connected to an air inlet and an air outlet, respectively;

[0009] The gap between the cell culture chamber and the gas exchange chamber is filled with a PDMS layer, the PDMS layer has micropores for gas to pass through, and the surface of the PDMS layer is modified with any one of polylysine, human recombinant fibronectin and fish collagen.

[0010] Furthermore, the cell culture chamber has a length of 48±0.5 mm, a width of 2±0.05 mm, and a height of 2±0.05 mm;

[0011] The number of the cell culture chambers is 9.

[0012] Furthermore, the preparation method of the PDMS layer (8) is as follows:

[0013] The vinyl-terminated polydimethylsiloxane and the cross-linking agent are uniformly mixed in a mass ratio of 10:1, and then subjected to vacuum degassing treatment to obtain a mixture;

[0014] The mixture was injected into a mold, and then kept at 80±2°C for 2±0.1 h to obtain a cured PDMS;

[0015] The surface of the solidified PDMS in contact with the cell culture chamber (2) is brought into contact with a coating solution to perform surface modification, followed by drying to obtain the PDMS layer (8);

[0016] Wherein, the coating solution comprises any one of a polylysine solution, a human recombinant fibronectin solution and a fish collagen solution;

[0017] The concentrations of the polylysine solution, the human recombinant fibronectin solution, and the fish collagen solution are 100 μg / mL, 10 μg / mL, and 10 μg / mL, respectively.

[0018] Furthermore, the sealed housing includes, from top to bottom, a catheter support layer, an upper sealing layer, a cell culture layer and a lower sealing layer;

[0019] The cell culture layer includes a rectangular frame and several PDMS layers, wherein the several PDMS layers are arranged horizontally and parallel to each other within the rectangular frame. A cavity penetrating the upper and lower surfaces of the PDMS layer is provided within the PDMS layer, which is the cell culture chamber. The space between the several PDMS layers and between the PDMS layer and the rectangular frame is the gas exchange chamber.

[0020] The rectangular frame and the PDMS layer have the same thickness, and the upper sealing layer and the lower sealing layer are respectively connected to the upper surface and the lower surface of the cell culture layer to seal the cell culture chamber and the gas exchange chamber;

[0021] The conduit support layer is connected to the upper surface of the upper sealing layer, and the lower surface of the lower sealing layer is provided with a support sheet.

[0022] The air inlet, the air outlet, the liquid inlet and the liquid outlet all pass through the upper sealing layer and the conduit supporting layer.

[0023] Furthermore, the upper sealing layer is a PDMS film with a thickness of 0.2±0.01 mm, and the lower sealing layer is a PDMS film with a thickness of 0.1±0.005 mm;

[0024] The conduit support layer includes two conduit support bodies with a thickness of 4±0.1 mm, one of which is provided with the air inlet and several liquid inlet holes, and the other is provided with the air outlet and several liquid outlet holes;

[0025] The catheter support body and the rectangular frame are both made of PDMS material, and the catheter support layer, the upper sealing layer, the cell culture layer and the lower sealing layer are all tightly connected by PDMS-PDMS bonding;

[0026] The surface of the PDMS membrane is modified with any one of polylysine, human recombinant fibronectin and fish collagen.

[0027] The supporting sheet is a glass sheet.

[0028] Based on the same inventive concept, the present invention provides a pollutant toxicity monitoring platform based on microfluidic chip coupled with cell electrochemical technology, the pollutant toxicity monitoring platform comprising a split syringe pump, a cell culture chamber, an electrochemical workstation and the above-mentioned microfluidic cell culture chip;

[0029] The microfluidic cell culture chip is used for cell culture, the split injection pump provides air and nutrients for cell circulation in the microfluidic cell culture chip through the air inlet, the air outlet, the liquid inlet and the liquid outlet, the microfluidic cell culture chip is placed in the cell culture incubator for culture, and the electrochemical workstation is used to detect cell electroactive substances in cell metabolic products.

[0030] Furthermore, the working electrode of the electrochemical workstation is prepared by the following method:

[0031] Graphitized carboxyl multi-walled carbon nanotubes (G-MWCNT-COOH), ionic liquid (BMIM PF6 － ,IL) (1-butyl-3-methylimidazolium hexafluorophosphate, purity: 97%) and copper oxide (CuO) were mixed and coated on the surface of the screen-printed electrode to obtain the G-MWCNT-COOH / IL / CuO / SPE electrode;

[0032] The mass ratio of the multi-walled carbon nanotubes to copper oxide is 1:2.

[0033] Furthermore, the electrochemical workstation further comprises a reference electrode and a counter electrode, wherein the reference electrode and the counter electrode are made of silver / silver chloride material and carbon material, respectively.

[0034] Furthermore, the cell electroactive substance includes xanthine and / or guanine.

[0035] One or more technical solutions in the embodiments of the present invention have at least the following technical effects or advantages:

[0036] 1. The present invention discloses a microfluidic cell culture chip, which is applied to cell culture and pollutant toxicity monitoring, which can greatly reduce the amount of cells used and achieve micro-quantification detection. The single channel area of ​​the chip is only about 1 cm 2 , which is much smaller than the area of ​​conventional cell culture dishes (27 cm 2 ), each channel in the microfluidic chip only requires 200 μL of cell sample, which is less than 7% of the amount used in conventional cell culture dishes.

[0037] 2. The present invention provides a pollutant toxicity monitoring platform based on a microfluidic chip coupled with cell electrochemical sensing technology. The platform uses a microfluidic cell culture chip as a cell culture container and couples it with cell electrochemical sensing technology to achieve high-throughput analysis of samples. The microfluidic chip can culture and contaminate cells in 9n (n is the number of chips actually used) channels in a single experiment, achieving high-throughput toxicity testing and significantly reducing testing costs. The microfluidic chip uses a small amount of cells and can be reused after cleaning, reducing the cost of toxicity research.

[0038] 3. The present invention provides a pollutant toxicity monitoring platform based on microfluidic chip coupled cell electrochemical sensing technology. Compared with existing pollutant toxicity detection methods, the present invention can realize online monitoring of pollutant toxicity, has the advantages of high sensitivity and low cost, and provides a new technical platform for environmental pollutant toxicity detection, solving the problems of existing in vitro pollutant toxicity detection methods such as the need for labeling, complex operation, time-consuming, and difficulty in achieving micro-quantification and online monitoring. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following is a brief introduction to the drawings required for use in the description of the embodiments. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0040] Figure 1 Schematic diagram of the pollutant toxicity monitoring platform based on microfluidic chip coupled with cell electrochemical technology of the present invention.

[0041] Figure 2 Schematic diagram of the structure of the microfluidic cell culture chip of the present invention: (A) is the main view; (B) is the left view; (C) is the top view; (D) is the 3D exploded view.

[0042] Figure 3 Schematic diagram of the cell culture layer structure in the microfluidic cell culture chip of the present invention.

[0043] Figure 4 This is a morphological diagram of HepG2 cells cultured on the microfluidic cell culture chip of the present invention.

[0044] Figure 5 This is a top view of each layer of the microfluidic cell culture chip of the present invention: (A) is the conduit support layer; (B) is the upper sealing layer; (C) is the cell culture layer; and (D) is the lower sealing layer.

[0045] Figure 6 Figure 3: The morphology of HepG2 cells after culturing in microfluidic chips treated with different coatings for 48 h: (A) uncoated; (B) coated with poly-lysine; (C) coated with human recombinant fibronectin; (D) coated with fish collagen.

[0046] Figure 7 Figure 3: The morphology of HepG2 cells after 48 h of culture on a microfluidic chip at different seeding densities: (A) Seeding density 7.5 × 10 5 cells / mL; (B) inoculation density 5.0 × 10 5 cells / mL; (C) seeding density 3.75 × 10 5 pieces / mL.

[0047] Figure 8 Figure 3: Morphological images of HepG2 cells cultured in a microfluidic chip for 48 h at different serum concentrations: (A) 5% serum concentration; (B) 10% serum concentration; (C) 20% serum concentration.

[0048] Figure 9 The surface morphology of the microfluidic chip treated in different ways: (A) unwashed; (B) trypsin digestion; (C) DMSO immersion and ultrasonication; (D) rinsing with anhydrous ethanol and double distilled water.

[0049] Figure 10 The toxicity of different concentrations of drinking water disinfection by-products to HepG2 cells.

[0050] Figure 11 Morphological images of HepG2 cells after (A) blank control and (B) treatment with 125.89 µM, (C) 251.19 µM, (D) 501.19 µM, (E) 1000.00 µM, and (F) 1995.26 µM 2-CPAN for 36 h.

[0051] Figure 12Morphological images of HepG2 cells after (A) blank control and (B) treatment with 125.89 µM, (C) 251.19 µM, (D) 501.19 µM, (E) 1000.00 µM, and (F) 1995.26 µM 3-CPAN for 36 h.

[0052] Figure 13 Morphological images of HepG2 cells after (A) blank control and (B) treatment with 125.89 µM, (C) 251.19 µM, (D) 501.19 µM, (E) 1000.00 µM, and (F) 1995.26 µM 4-CPAN for 36 h.

[0053] The component names corresponding to the reference numerals are as follows:

[0054] 1-sealed shell, 11-catheter support layer, 111-catheter support body, 12-upper sealing layer, 13-cell culture layer, 131-rectangular frame, 14-lower sealing layer, 2-cell culture chamber, 3-gas exchange chamber, 4-liquid inlet, 5-liquid outlet, 6-air inlet, 7-air outlet, 8-PDMS layer. DETAILED DESCRIPTION

[0055] The present invention will be described in detail below in conjunction with specific embodiments and examples, and the advantages and various effects of the present invention will be more clearly presented. It should be understood by those skilled in the art that these specific embodiments and examples are for illustrating the present invention, rather than for limiting the present invention.

[0056] Throughout this specification, unless otherwise specified, the terms used herein should be understood as having the same meaning as commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In the event of any conflict, the present specification shall take precedence.

[0057] Unless otherwise specified, various raw materials, reagents, instruments and equipment used in the present invention can be purchased from the market or prepared by existing methods.

[0058] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0059] Current microfluidic technology still has some limitations in pollutant toxicity detection. However, combining it with electrochemical sensing technology can effectively address these technical bottlenecks. Specifically, traditional microfluidic chips primarily rely on optical labels (such as fluorescent dyes) for detection. This approach not only increases the complexity of experimental operations but can also significantly interfere with test results due to the biotoxicity of the labeling reagents themselves. In contrast, electrochemical sensors can directly detect cellular metabolic activity in a label-free manner, simplifying the experimental process and significantly reducing the impact of reagent interference on test results. Furthermore, microfluidic chips generally face the technical challenge of insufficient signal strength in trace pollutant detection. Electrochemical sensors, however, offer high signal strength and sensitivity, enabling sensitive detection of low-concentration pollutants. Furthermore, traditional microfluidic systems have significant limitations in dynamic multi-parameter monitoring. Electrochemical sensors, by integrating multi-channel electrode arrays, can simultaneously monitor multiple toxicity indicators, including cellular respiration metabolism, membrane potential changes, and ion channel activity, in real time. In summary, by coupling electrochemical sensing technology with the microfluidic platform, not only can the advantages of microfluidic technology such as high throughput, automation, and low sample consumption be fully utilized, but it can also effectively make up for its shortcomings in detection sensitivity, labeling dependence, and multi-parameter simultaneous monitoring capabilities, providing technical support for the construction of a new pollutant toxicity detection platform.

[0060] Based on this, the present invention provides a microfluidic cell culture chip and a pollutant toxicity monitoring platform.

[0061] The microfluidic cell culture chip and pollutant toxicity monitoring platform of the present invention will be described in detail below with reference to the examples and experimental data.

[0062] Example 1

[0063] This embodiment provides a microfluidic cell culture chip.

[0064] This embodiment provides a microfluidic cell culture chip, comprising a sealed housing 1, wherein a plurality of cell culture chambers 2 are provided within the sealed housing 1, and a gas exchange chamber 3 is provided on the periphery of the cell culture chamber 2. One end of the cell culture chamber 2 is connected to a liquid inlet 4, and the other end is connected to a liquid outlet 5. The gas exchange chamber 3 is connected to an air inlet 6 and an air outlet 7, respectively.

[0065] The gap between the cell culture chamber 2 and the gas exchange chamber 3 is filled with a PDMS layer 8 having micro-channels for gas to pass through. The surface of the PDMS layer 8 is modified with any one of polylysine, human recombinant fibronectin and fish collagen.

[0066] Preferably, the cell culture chamber 2 has a length of 48 mm, a width of 2 mm, and a height of 2 mm;

[0067] The number of the cell culture chambers 2 is 9.

[0068] Furthermore, the sealed housing 1 includes, from top to bottom, a catheter support layer 11, an upper sealing layer 12, a cell culture layer 13 and a lower sealing layer 14;

[0069] The cell culture layer 13 includes a rectangular frame 131 and several PDMS layers 8. The several PDMS layers 8 are arranged horizontally and parallel to each other within the rectangular frame 131. A cavity is provided in the PDMS layer 8 that passes through the upper and lower surfaces of the PDMS layer 8, which is the cell culture chamber 2. The space between the several PDMS layers 8 and between the PDMS layers 8 and the rectangular frame 131 is the gas exchange chamber 3.

[0070] The rectangular frame 131 and the PDMS layer 8 have the same thickness. The upper sealing layer 12 and the lower sealing layer 14 are respectively connected to the upper surface and the lower surface of the cell culture layer 13 to seal the cell culture chamber 2 and the gas exchange chamber 3.

[0071] The conduit support layer 11 is connected to the upper surface of the upper sealing layer 12, and a support sheet is provided on the lower surface of the lower sealing layer 14. The air inlet 6, the air outlet 7, the liquid inlet 4 and the liquid outlet 5 all pass through the upper sealing layer 12 and the conduit support layer 11.

[0072] Preferably, the upper sealing layer 12 is a PDMS film with a thickness of 0.2 mm, and the lower sealing layer 14 is a PDMS film with a thickness of 0.1 mm;

[0073] The conduit support layer 11 includes two conduit support bodies 111 with a thickness of 4 mm, one of which is provided with the air inlet 6 and the plurality of liquid inlet holes 4, and the other conduit support body 111 is provided with the air outlet 7 and the plurality of liquid outlet holes 5;

[0074] The catheter support body 111 and the rectangular frame 131 are both made of PDMS, and the catheter support layer 11, the upper sealing layer 12, the cell culture layer 13 and the lower sealing layer 14 are all tightly connected by PDMS-PDMS bonding;

[0075] The surface of the PDMS membrane is modified with any one of polylysine, human recombinant fibronectin and fish collagen;

[0076] The supporting sheet is a glass sheet.

[0077] This embodiment also provides a pollutant toxicity monitoring platform based on microfluidic chip coupled with cell electrochemical technology, the pollutant toxicity monitoring platform comprising a split syringe pump, a cell culture chamber, an electrochemical workstation and the above-mentioned microfluidic cell culture chip;

[0078] The microfluidic cell culture chip is used for cell culture, and the split injection pump provides nutrients and air for the cell circulation in the microfluidic cell culture chip through the air inlet 6, the air outlet 7, the liquid inlet 4 and the liquid outlet 5. The microfluidic cell culture chip is placed in the cell culture incubator for culture, and the electrochemical workstation is used to detect cell electroactive substances in cell metabolic products.

[0079] Example 2

[0080] This embodiment provides a method for preparing a microfluidic cell culture chip, which is as follows:

[0081] 1. Design of Microfluidic Cell Culture Chip

[0082] Use CAD drawing software to design the chip, the top view of each layer of the chip is as follows Figure 5 As shown. The microfluidic cell culture chip is designed as a rectangle of 85 × 75 mm. The first layer is a rectangular tube support 111 of 85 × 20 × 4 mm for stabilizing the inlet and outlet tubes, and a circular hole with a diameter of 2 mm is opened at the corresponding inlet and outlet ports ( Figure 5 A). The second layer ( Figure 5 B) is a PDMS film of 85 × 75 × 0.2 mm used to cover the cell culture layer 13, with circular holes opened at the corresponding liquid inlets and outlets. Figure 5 C) is the cell culture layer 13, which belongs to the cell culture area and contains 9 50×2×2 mm rectangular hollow channels for cell culture, named cell culture chamber 2. The PDMS layer 8 on the left and right sides of the channel has a wall thickness of 2 mm. The two ends of the cell culture chamber 2 are circular inlet and outlet ports. In addition, Figure 5 The area outside the cell culture area within the 74 × 64.5 mm marked in C is the entire through-hole gas exchange chamber 3. The fourth layer is a PMDS film measuring 85 × 75 × 0.1 mm for cell attachment, which serves as the bottom of the third layer and is bonded to it (the third layer's channel is a hollow, bottomless structure, so the fourth layer is required as the bottom), and is bonded to a 100 × 100 × 1 mm glass film to provide support ( Figure 5 D).

[0083] 2. Fabrication of Microfluidic Cell Culture Chips

[0084] The chip is manufactured using soft lithography. The specific steps are as follows:

[0085] (1) Mask production

[0086] The designed chip structure (i.e. Figure 5 The 4-layer structure designed in the figure is transferred to a photomask, a chrome-plated glass plate is selected as the photomask, and printed with UV opaque ink.

[0087] (2) Mold preparation

[0088] A uniform, 2 mm thick layer of SU-8 photosensitive resin was applied to a silicon wafer. UV light was then irradiated through a photomask, curing only the exposed areas while leaving the remaining areas unexposed. After a development process, the unexposed areas were removed, leaving the hardened resin structure as part of the mold. To ensure smooth removal of the PDMS device from the mold, the mold surface was treated with trimethylchlorosilane to make it hydrophobic.

[0089] (3) PDMS mixing and degassing

[0090] PDMS prepolymer (an uncured liquid base material, chemically a linear polysiloxane chain containing reactive (ethyl) end groups, typically described as vinyl-terminated polydimethylsiloxane) and its crosslinker (methylhydrogensiloxane) were weighed in a 10:1 mass ratio and thoroughly stirred to ensure complete mixing. Because bubbles inevitably form during mixing, the mixed PDMS was degassed in a vacuum environment until all visible bubbles disappeared.

[0091] (4) Pouring and curing

[0092] Pour the degassed PDMS solution into the prepared mold, taking care to avoid introducing new bubbles. Place the entire device in an oven and heat at 80°C for approximately 2 hours to solidify the PDMS. (The PDMS material prepared in this example has good gas permeability, and the gas exchange rate is slow, but sufficient for cell culture.)

[0093] (5) Stripping and post-processing

[0094] After curing, the PDMS is allowed to cool to room temperature and then gently peeled off from the mold. This leaves a PDMS replica with a microchannel structure. Based on the designed chip structure, a punch is used to create inlet and outlet holes at the corresponding locations to facilitate connection to external fluidic systems.

[0095] (6) Surface treatment and bonding

[0096] To seal the microchannels and ensure a good seal, the PDMS surface is plasma treated to increase adhesion between the PDMS and glass or another PDMS sheet, creating a tight and durable bond. Immediately after the plasma treatment, the treated PDMS is placed in contact with another flat sheet of PDMS or glass, applying gentle pressure to achieve a tight bond.

[0097] The plasma treatment method is as follows:

[0098] First, gently press the surfaces of the two prepared PDMS sheets with dust-free tape to remove debris. Then, ultrasonically clean them with isopropyl alcohol and deionized water for 5 minutes each, and blow dry them with nitrogen.

[0099] The PDMS surface was treated using a Harrick Plasma Cleaner at a power of 30 W and an air flow rate of 10–30 sccm for 60 seconds. The vacuum pressure was maintained at 0.2–0.3 mbar during the entire treatment process.

[0100] Processing steps: Place the PDMS sheet (with the bonding surface facing up) into the reaction chamber to avoid stacking; start the vacuum pump and start the plasma after the chamber pressure stabilizes; after the treatment is completed, slowly release the vacuum to avoid airflow disturbance and contamination of the surface.

[0101] Align the activated surfaces of the two PDMS sheets and bond them together (operate under a microscope and use a fixture to assist alignment), gently press to remove bubbles; then place them in an 80°C oven and heat for 40 minutes to enhance the bonding strength.

[0102] (7) Cleaning and inspection

[0103] The finished chip is thoroughly cleaned to remove residual material, and the quality of the microchannels is inspected microscopically to ensure that they meet the expected design specifications.

[0104] Example 3

[0105] Optimization of cell culture conditions in microfluidic chips.

[0106] 1. Coating optimization

[0107] PDMS is inherently hydrophobic, requiring hydrophilic modification before use in cell culture. Currently, the method of modifying the PDMS surface with extracellular matrix proteins is widely used. Extracellular matrix proteins include fibronectin and collagen. These polar substances can adsorb to the material surface through hydrogen bonding and other interactions, thereby improving the hydrophilicity of PDMS and promoting cell adhesion.

[0108] The dried microfluidic chip was placed in a sterile operating table, irradiated with UV light for 30 minutes, and washed three times with 75% alcohol to fully disinfect it. The culture chip was coated with 100 μg / mL polylysine, 10 μg / mL human recombinant fibronectin, and 10 μg / mL fish collagen overnight. After coating, the coating was aspirated and dried at room temperature. 5 HepG2 cells at a concentration of 100 cells / mL were inoculated into the microfluidic chip and cultured in a cell incubator for 48 h. The microfluidic chip was then removed and the cells were observed under an inverted microscope. Figure 6 (A-D) show HepG2 cells cultured on microfluidic chips after 48 hours of culture, either uncoated or coated with polylysine, fibronectin, or fish collagen, respectively. The results showed that all three coatings improved the hydrophobicity of the microfluidic chip. After 48 hours of culture on the uncoated microfluidic chip, most cells were round and agglomerated. After rinsing with phosphate-buffered saline (PBS), a large number of cells shed. In contrast, after 48 hours of culture on the coated microfluidic chip, most cells were spindle-shaped and showed no detachment after rinsing with PBS. Furthermore, cells cultured on microfluidic chips coated with polylysine, fibronectin, and fish collagen all grew well, with similar oxidation peak currents. However, fibronectin is expensive (approximately 1,000 times and 1,000,000 times the price of polylysine and fish collagen, respectively), and fish collagen is complex to prepare and exhibits significant batch-to-batch variability. Therefore, polylysine was chosen as the coating for cell culture on microfluidic chips in this study.

[0109] 2. Optimization of seeding density

[0110] Cell seeding density is a crucial parameter in cell culture. It not only influences the initial distribution of cells but also determines the subsequent intercellular contact and resulting colony effect. If the density is too low, cells may lack sufficient signaling; conversely, if the density is too high, it can easily lead to problems such as localized hypoxia. Therefore, optimizing the cell seeding density is crucial.

[0111] The microfluidic chip was coated with poly-lysine at a concentration of 100 μg / mL, and the cell seeding density (7.5× 10 5 cells / mL, 5.0 × 10 5 cells / mL and 3.75 × 10 5 The results are as follows: Figure 7 As shown in Figure 2, it can be seen that when the initial cell seeding density is 7.5 × 10 5 / mL ( Figure 7A) After 48 h of culture, the cells grew too densely, and cell stacking appeared in some areas, preventing the dye from fully contacting the cells. When the initial cell inoculation concentration was 3.75 × 10 5 / mL( Figure 7 C), the cell confluence did not reach 70%-80% after 48 h of culture. In contrast, the initial cell seeding concentration in the flow control chip was 5.0 × 10 5 / mL( Figure 7 B) is more suitable. Therefore, 5.0 × 10 5 The cell seeding density was 100 cells / mL for subsequent experiments.

[0112] 3. Serum concentration optimization

[0113] In a microfluidic chip with polylysine as coating material and a cell seeding density of 5 × 10 5 The effects of different serum ratios (5%, 10% and 20%) on cells were investigated under the condition of 400 μg / mL. The results are as follows Figure 8 As shown in the figure, cells in the microfluidic chip grow best when the serum ratio is 10%, with the highest oxidation peak current, outperforming the serum ratios of 5% and 20%. This may be because when the serum ratio is too low, it cannot provide sufficient nutrients and growth factors to the cells, leading to cell growth stagnation or even death. When the serum ratio is too high, the concentration of certain growth factors or hormones in the serum is too high, which has an inhibitory effect on cells. Therefore, a serum ratio of 10% was selected for cell culture in the microfluidic chip.

[0114] Example 4

[0115] This example conducts an experiment to detect the toxicity of new pollutants using microfluidic chip coupled cell electrochemical sensing technology.

[0116] 1. Cell Adherence Culture

[0117] After sterilizing the clean microfluidic chip with UV light for 30 minutes, rinse three times with 75% alcohol to remove impurities. Polylysine (PDMS) at a concentration of 100 µg / mL was added to the culture area and coated overnight to improve the hydrophilicity of the PDMS, making it suitable for cell adhesion. After coating, the polylysine solution was removed from the microfluidic chip and set aside for future use. Cells reaching approximately 80% confluence were dissociated with trypsin, and the cell suspension was centrifuged at 1000 rpm for 5 minutes. The supernatant was discarded, and the cells were diluted with culture medium to a density of 5 × 10 cells / mL. 5 The cells were inoculated into the culture area and cultured in a cell culture incubator at 37°C and 5% CO2.

[0118] 2. Cell Infection

[0119] Based on the growth curve of HepG2 cells on the chip, exposure experiments were performed 12 hours after the cells had fully adhered. First, a Langer TS-1B / 4*W0109-1B four-channel push-pull syringe pump was used to aspirate the cell culture wastewater from the culture area at a flow rate of 0.8 mm / s. Then, pre-prepared culture media containing varying concentrations of contaminants were injected into the cell culture area of ​​the chip at the same flow rate. Finally, the microfluidic chip was returned to the incubator and cultured for an additional 36 hours at 37°C and 5% CO2.

[0120] 3. Collection of Cell Secretions

[0121] After the incubation period, remove the wastewater and rinse the cells two to three times with PBS (pH 7.4) to remove cellular metabolites and residual contaminants, minimizing interference with subsequent testing. Add 200 µL of PBS to the cell culture area and incubate in an incubator for 1.5 hours to allow for secretion. After secretion is complete, collect the secretion fluid for testing.

[0122] 4. Toxicity testing and evaluation

[0123] 5 mg of graphitized carboxyl multi-walled carbon nanotubes (G-MWCNT-COOH), 150 µL of ionic liquid (IL), and 10 mg of copper oxide (CuO) were mixed and then blade-coated onto the surface of a screen-printed electrode (SPE) to create the G-MWCNT-COOH / IL / CuO / SPE. Detection was performed using a Chenhua CHI 760E electrochemical workstation using the G-MWCNT-COOH / IL / CuO composite, silver / silver chloride, and carbon materials as the working, reference, and counter electrodes, respectively. 100 µL of cell secretion fluid was pipetted onto the G-MWCNT-COOH / IL / CuO / SPE surface. Amperometric it curves were then used to analyze the concentration of the sample prior to detection. Immediately thereafter, DPV (Differential Pulse Voltammetry) was used to measure the oxidation peak current. The G-MWCNT-COOH / IL / CuO / SPE surface sample was then removed by aspiration and placed in 0.2 M PBS for electrode regeneration using CV (Cyclic Voltammetry). This procedure was repeated until all samples were analyzed. The relevant parameters for the electrochemical method are shown in Table 1.

[0124] The object of detection is the electroactive substances (xanthine / guanine) in cells. Xanthine / guanine are both products of cellular nucleotide metabolism. The better the cell activity and the more vigorous the metabolism, the more xanthine / guanine is produced. Therefore, the electrochemical signal of xanthine / guanine is positively correlated with cell activity. The more toxic the pollutant is to the cell, the greater the decrease in the electrical signal. The goal of the test is to reflect cell activity by measuring the magnitude of the oxidation peak current of xanthine / guanine, thereby conducting a toxicity assessment. The toxicity assessment formula is shown in Formula (1).

[0125] Table 1 Electrochemical detection methods and parameters

[0126] ;

[0127] Note: “-” means this method does not involve the adjustment of this parameter.

[0128] Cytotoxicity was calculated according to formula (1), and a linear correlation equation between the logarithm of pollutant concentration and cytotoxicity was established to calculate the half-maximal inhibitory concentration (IC 50 ) value to evaluate the toxicity of pollutants.

[0129] Cytotoxicity = [( I pa,1 – I pa,2 ) / I pa,1 ] × 100% (1)

[0130] in, I pa,1 and I pa,2 is the oxidation peak current value of the cells in the control group and the experimental group.

[0131] Example 5

[0132] 1. Cell Adherence Culture

[0133] The culture steps are the same as in Example 4

[0134] 2. Cell Infection

[0135] Based on the growth curve of HepG2 cells on the chip, exposure experiments were performed 12 hours after cells had fully adhered. First, a Langer TS-1B / 4*W0109-1B four-channel push-pull syringe pump was used to aspirate the cell culture wastewater from the culture area at a flow rate of 0.8 mm / s. Then, pre-prepared culture solutions containing varying concentrations of 2-chlorophenylacetonitrile (2-CPAN), 3-chlorophenylacetonitrile (3-CPAN), and 4-chlorophenylacetonitrile (4-CPAN) (125.89, 251.19, 501.19, 1000.00, and 1995.26 µM, respectively) were injected into the cell culture area of ​​the chip at the same flow rate. Finally, the microfluidic chip was returned to a constant temperature incubator and incubated at 37°C, 5% CO₂ for 36 hours.

[0136] 3. Collection of Cell Secretions

[0137] The steps for collecting secretion fluid are the same as those in Example 4.

[0138] 4. Toxicity testing and evaluation

[0139] Toxicity detection and evaluation steps are the same as in Example 4

[0140] 5. Experimental Results

[0141] The toxicity of chlorobenzeneacetonitrile was detected using the constructed cell electrochemical sensing platform based on microfluidic chip. The results are as follows: Figure 10 As shown. It can be seen that the toxicity of the three chlorobenzene acetonitrile to HepG2 cells increases with the increase of their concentration. Cytotoxicity was calculated according to formula (1) in Example 4, and a linear correlation equation of the logarithm of the pollutant concentration-cytotoxicity was established to calculate the half-maximal inhibitory concentration (IC 50 ) values ​​were used to evaluate the toxicity of pollutants. The results are shown in Table 2.

[0142] Table 2 Logarithm of chlorophenylacetonitrile concentration ( X ) and cytotoxicity to HepG2 cells ( Y ) linear equation and IC 50 value

[0143] ;

[0144] IC values ​​of 2-CPAN, 3-CPAN and 4-CPAN on HepG2 cells at 36 h 50The values ​​were 710.59, 968.43, and 892.15 µM, respectively. The order of toxicity was 2-CPAN > 4-CPAN > 3-CPAN. This is consistent with the order of toxicity of 2-CPAN, 3-CPAN, and 4-CPAN against human liver cancer cells tested in the paper "A Low-Cost Electrochemical Cell Sensor Based on MWCNT-COOH / α-Fe2O3 for Toxicity Detection of Drinking Water Disinfection Byproducts."

[0145] 6. Regeneration of Microfluidic Chip

[0146] After the test is completed, the waste liquid in the microfluidic chip is removed for cleaning and regeneration. First, trypsin is added to the culture area for digestion for 5 minutes, and then the chip is placed in an ultrasonic cleaning tank for 15 minutes to remove all cells ( Figure 9 B). After ultrasonic cleaning, add dimethyl sulfoxide (DMSO) to the culture area and soak for 5 minutes, then place it in an ultrasonic cleaning tank for 5 minutes to remove the residue on the surface of the PDMS chip ( Figure 9 C). Finally, rinse the culture area three times with anhydrous ethanol and double distilled water, respectively, and then place it in a constant temperature drying oven to dry for later use ( Figure 9 D). Therefore, compared with the uncleaned chip ( Figure 9 A) Compared with the above treatment, the above treatment can effectively remove the cells and impurities remaining on the surface of the microfluidic chip.

[0147] The effects of different concentrations of 2-CPAN, 3-CPAN, and 4-CPAN on the morphology of HepG2 cells are shown in Figure 2. Figures 11 to 13 As shown. Figure 11-13 A are blank control groups, Figure 11-13 The BF concentrations were 125.89, 251.19, 501.19, 1000.00, and 1995.26 µM, respectively. The results showed that the HepG2 cell density in the control group was high, and the cells were mostly irregular in shape. However, after 36 hours of treatment with 2-CPAN, 3-CPAN, and 4-CPAN, the HepG2 cell number decreased, the cells became smaller and rounder, and a large number of shrunken and dead cells appeared.

[0148] Finally, it should be noted that the terms "comprises," "includes," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that includes a list of elements includes not only those elements, but also other elements not expressly listed, or elements inherent to such process, method, article, or apparatus.

[0149] Although the preferred embodiments of the present invention have been described, those skilled in the art may make additional changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0150] Obviously, those skilled in the art may make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if such changes and modifications fall within the scope of the claims and their equivalents, the present invention is intended to include such changes and modifications.

Claims

1. A microfluidic cell culture chip, characterized in that: The microfluidic cell culture chip comprises a sealed housing (1), wherein a plurality of cell culture chambers (2) are provided in the sealed housing (1), a gas exchange chamber (3) is provided on the periphery of the cell culture chamber (2), one end of the cell culture chamber (2) is connected to a liquid inlet (4), and the other end is connected to a liquid outlet (5), and the gas exchange chamber (3) is respectively connected to an air inlet (6) and an air outlet (7); The gap between the cell culture chamber (2) and the gas exchange chamber (3) is filled with a PDMS layer (8), the PDMS layer (8) has micro-channels for gas to pass through, and the surface of the PDMS layer (8) is modified with any one of polylysine, human recombinant fibronectin and fish collagen; The sealed housing (1) comprises, from top to bottom, a catheter support layer (11), an upper sealing layer (12), a cell culture layer (13) and a lower sealing layer (14); The upper sealing layer (12) is a PDMS film with a thickness of 0.2±0.01 mm, and the lower sealing layer (14) is a PDMS film with a thickness of 0.1±0.005 mm; The cell culture chamber (2) has a length of 48±0.5 mm, a width of 2±0.05 mm, and a height of 2±0.05 mm; and the number of the cell culture chambers (2) is 9.

2. A microfluidic cell culture chip according to claim 1, characterized in that: The preparation method of the PDMS layer (8) is as follows: The vinyl-terminated polydimethylsiloxane and the cross-linking agent are uniformly mixed in a mass ratio of 10:1, and then subjected to vacuum degassing treatment to obtain a mixture; The mixture was injected into a mold, and then kept at 80±2°C for 2±0.1h to obtain a cured PDMS; The surface of the solidified PDMS in contact with the cell culture chamber (2) is brought into contact with a coating solution to perform surface modification, followed by drying to obtain the PDMS layer (8); Wherein, the coating solution comprises any one of a polylysine solution, a human recombinant fibronectin solution and a fish collagen solution; The concentrations of the polylysine solution, the human recombinant fibronectin solution, and the fish collagen solution are 100 μg / mL, 10 μg / mL, and 10 μg / mL, respectively.

3. The microfluidic cell culture chip according to claim 1, characterized in that: The cell culture layer (13) comprises a rectangular frame (131) and a plurality of PDMS layers (8), wherein the plurality of PDMS layers (8) are arranged horizontally and parallel to each other within the rectangular frame (131), a cavity penetrating the upper and lower surfaces of the PDMS layer (8) is provided within the PDMS layer (8), namely, the cell culture chamber (2), and the space between the plurality of PDMS layers (8) and between the PDMS layer (8) and the rectangular frame (131) is the gas exchange chamber (3); The rectangular frame (131) and the PDMS layer (8) have the same thickness, and the upper sealing layer (12) and the lower sealing layer (14) are respectively connected to the upper surface and the lower surface of the cell culture layer (13) to seal the cell culture chamber (2) and the gas exchange chamber (3); The conduit support layer (11) is connected to the upper surface of the upper sealing layer (12), a support sheet is provided on the lower surface of the lower sealing layer (14), and the air inlet (6), the air outlet (7), the liquid inlet (4) and the liquid outlet (5) all penetrate the upper sealing layer (12) and the conduit support layer (11).

4. The microfluidic cell culture chip according to claim 3, characterized in that: The conduit support layer (11) comprises two conduit support bodies (111) with a thickness of 4±0.1 mm, wherein one conduit support body (111) is provided with the air inlet (6) and a plurality of the liquid inlet holes (4), and the other conduit support body (111) is provided with the air outlet (7) and a plurality of the liquid outlet holes (5); The catheter support body (111) and the rectangular frame (131) are both made of PDMS material, and the catheter support layer (11), the upper sealing layer (12), the cell culture layer (13) and the lower sealing layer (14) are all tightly connected through PDMS-PDMS bonding to achieve close connection between the layers; The surface of the PDMS membrane is modified with any one of polylysine, human recombinant fibronectin and fish collagen.

5. The microfluidic cell culture chip according to claim 4, characterized in that: The supporting sheet is a glass sheet.

6. A pollutant toxicity monitoring platform based on microfluidic chip coupled with cell electrochemical technology, characterized in that: The pollutant toxicity monitoring platform comprises a split syringe pump, a cell culture chamber, an electrochemical workstation and a microfluidic cell culture chip according to any one of claims 1 to 3; The microfluidic cell culture chip is used for cell culture, the split injection pump provides nutrients and air for cell circulation in the microfluidic cell culture chip through the air inlet (6), the air outlet (7), the liquid inlet (4) and the liquid outlet (5), the microfluidic cell culture chip is placed in the cell culture box for culture, and the electrochemical workstation is used for detecting cell electroactive substances in cell metabolic products.

7. The pollutant toxicity monitoring platform based on microfluidic chip coupled with cell electrochemical technology according to claim 6, characterized in that: The working electrode of the electrochemical workstation was prepared by the following method: Graphitized carboxyl multi-walled carbon nanotubes, ionic liquid (IL) and copper oxide were mixed and coated on the surface of the screen-printed electrode to obtain G-MWCNT-COOH / IL / CuO / SPE electrode; The mass ratio of the multi-walled carbon nanotubes to copper oxide is 1:

2.

8. The pollutant toxicity monitoring platform based on microfluidic chip coupled with cell electrochemical technology according to claim 6, characterized in that: The electrochemical workstation further comprises a reference electrode and a counter electrode, wherein the reference electrode and the counter electrode are made of silver / silver chloride material and carbon material respectively.

9. The pollutant toxicity monitoring platform based on microfluidic chip coupled with cell electrochemical technology according to claim 6, characterized in that: The cell electroactive substances include xanthine and / or guanine.

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