Microfluidic single-cell chip and gas-liquid exposure particulate matter toxicity analysis method

Abstract

The present application relates to the technical field of single cell analysis, and particularly relates to a microfluidic single cell chip and a gas-liquid exposure particulate matter toxicity analysis method, the chip comprising a liquid inlet, a gas inlet, a plurality of parallel single cell capture arrays, the plurality of single cell capture arrays sharing an outlet, the single cell capture array comprising a serpentine main channel, a plurality of capture trap-cavity units being arranged between two adjacent rows of segments, the capture trap-cavity unit comprising a cell capture trap, a front limiting channel and a cell culture cavity in communication, the inlet of the cell capture trap being in communication with the last row of segments, the middle of the cell culture cavity being in communication with the rear limiting channel, and the rear limiting channel being in communication with the next row of segments. The present application adopts the method of parallel capture trap-cavity units to increase the number of capture-cavity units per unit area, ensuring that the chip has a high enough cell capture amount; and ensuring that the cells are still immersed in the culture solution during the gas-liquid exposure process, meeting the application of particulate matter toxicity analysis.

Description

Technical Field

[0001] This invention relates to the field of single-cell analysis technology, specifically to a microfluidic single-cell chip and a method for analyzing the toxicity of gas-liquid exposed particulate matter. Background Technology

[0002] In vitro cell experiments are an important tool for studying the toxicity of atmospheric particulate matter. Traditional in vitro cell experiments often use multi-well plate cell culture to detect the average response of cell populations induced by atmospheric particulate matter to assess its toxicity, but they cannot analyze single-cell responses. However, cellular heterogeneity is a ubiquitous biological phenomenon, and the most significantly varied individual or group of cells often reveal important biological effects. Therefore, studying the heterogeneity of single cells induced by atmospheric particulate matter at multiple levels, including genotype and phenotype, is crucial for understanding the toxicity and biological effects of atmospheric particulate matter.

[0003] Flow cytometry can analyze single-cell signals but cannot provide the dynamic response of a specific single cell. The development of microfluidics has provided an excellent technical means to capture single cells and track their time-varying dynamic responses. Microfluidics is a technique for manipulating and controlling microfluidics in micrometer-scale channels, offering advantages such as small volume, high efficiency, high throughput, and automation. Simultaneously, microfabrication techniques provide methods for manufacturing microstructures similar in size to single cells, enabling accurate capture and identification of single cells.

[0004] Patent CN105441307A describes a single-cell capture chip comprising a fluid layer, an elastic membrane layer, and a drive structure. It achieves high-efficiency single-cell capture through pre-placed cell cavities, and the captured cells are not easily detached from the capture site. However, this chip requires the configuration of an elastic membrane and a drive structure, resulting in complex microfabrication processes and high costs. Patent CN110004043B describes a single-cell capture microfluidic chip with only one layer and simple fabrication. By setting up reasonable dispersion columns, buffer columns, and capture traps, it can achieve uniform injection of the cell carrier fluid. Cells can remain stable in the capture traps under fluid shear forces, making them difficult to flow out. However, the number of capture units that can be accommodated per unit area of ​​the chip is limited, resulting in a limited cell capture throughput. Related literature describes a single-cell capture array chip device (Environmental Science & Technology, 2020, 54, 13121-13130), which has multiple units containing capture traps and chambers, each unit consisting of a front capture trap and a rear chamber. This chip utilizes fluid dynamics principles to trap cells in a capture trap. By applying pressure at the inlet, the cells are fixed in the rear chamber, preventing cell loss during subsequent stimulation with particulate solutions. However, during the process of pushing cells from the capture trap into the chamber, cells can easily escape through the flow channels at the rear of the chamber, resulting in low single-cell capture efficiency for this chip device.

[0005] Furthermore, none of the currently disclosed technologies involve methods for analyzing the toxicity of particulate matter exposed to gas and liquid. The single-cell capture microstructure design of the above methods cannot ensure that the cells remain immersed in the cell culture medium during gas-liquid exposure. This technical limitation can easily lead to significant deviations in the toxicity analysis results. Summary of the Invention

[0006] To address the problems existing in the prior art, this invention provides a microfluidic single-cell chip and a method for analyzing the toxicity of particulate matter exposed to gas and liquid.

[0007] This invention is achieved through the following technical solution:

[0008] A microfluidic single-cell chip includes a liquid inlet, a gas inlet, and multiple parallel single-cell capture arrays. The multiple single-cell capture arrays share a single outlet. The single-cell capture array includes a serpentine main channel. Multiple capture trap-chamber units are provided between adjacent rows of segments. Each capture trap-chamber unit includes a connected cell capture trap, a front confinement channel, and a cell culture chamber. The inlet of the cell capture trap is connected to the previous row of segments. The middle of the cell culture chamber is connected to a rear confinement channel, which is connected to the next row of segments.

[0009] Preferably, there are two rear restriction channels.

[0010] Preferably, the two rear limiting channels are symmetrically arranged.

[0011] Preferably, the entrance to the rear restricted passage is located at the same horizontal level.

[0012] Preferably, the cell trap has a lateral length of H1, a longitudinal length of H2, and a depth of H3, with the following dimensions: the depth H3 of the cell trap is greater than the lateral length H1 of the cell trap, and both the lateral length H1 and the depth H3 are greater than the diameter of a single cell, and the longitudinal length H2 is greater than or equal to the lateral length H1.

[0013] Preferably, the lateral length H4 of the anterior limiting channel is less than the lateral length H1 of the cell trap, the longitudinal length H5 of the anterior limiting channel is less than the longitudinal length H2 of the cell trap, the depth H6 of the anterior limiting channel is less than the depth H3 of the cell trap, and the depth H6 of the anterior limiting channel is less than the diameter of a single cell.

[0014] Preferably, the transverse length H7 of the cell culture chamber is greater than the transverse length H1 of the cell trap, the longitudinal length H8 of the cell culture chamber is greater than the longitudinal length H2 of the cell trap, and the depth H9 of the cell culture chamber is greater than the diameter of a single cell.

[0015] Preferably, the lateral length H10 of the rear confinement channel is less than the lateral length H1 of the cell trap, and the longitudinal length H11 of the rear confinement channel satisfies that H11 is less than the longitudinal length H2 of the cell trap; the depth H12 of the rear confinement channel satisfies that H12 is less than the depth H3 of the cell trap and is less than the diameter of a single cell.

[0016] Application of a microfluidic single-cell chip in the toxicity analysis of particulate matter exposed to gas and liquid.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] This invention discloses a microfluidic single-cell chip. The microfluidic single-cell chip adopts a parallel trap-chamber unit method, which increases the number of trap-chamber units that can be accommodated per unit area, thereby ensuring that the chip has a sufficiently high cell capture capacity.

[0019] The microfluidic single-cell chip features two flow path inlets: one for gas and one for liquid, preventing unnecessary errors such as inlet rupture during flow path switching. Simultaneously, based on fluid dynamics principles, the chip utilizes shear force to capture single cells, resulting in simple operation, low cost, and good functionality.

[0020] The present invention provides a microfluidic single-cell chip that allows cells to remain immersed in the culture medium even during gas-liquid exposure by setting the cell culture chamber so that the culture medium flows out from both sides of the middle of the chamber.

[0021] This invention discloses a microfluidic single-cell chip that improves cell capture efficiency by allowing the cell culture medium to flow out from both sides of the center of the cell chamber. This design ensures that cells do not escape from the chamber during the process of pushing them into the chamber from the capture trap. Simultaneously, this design also ensures that during the introduction of particulate matter through gas-liquid exposure, cells remain in contact with the particulate matter while simultaneously being immersed in the cell culture medium. This avoids deviations in toxicity response results caused by a sub-optimal state of cell health due to the absence of culture medium. Attached Figure Description

[0022] Figure 1 This is a schematic diagram of a microfluidic single-cell chip provided by the present invention;

[0023] Figure 2 This is a top view of a trap-chamber unit in a microfluidic single-cell chip provided by the present invention.

[0024] Figure 3 This is a three-dimensional structural diagram of a trap-chamber unit in a microfluidic single-cell chip provided by the present invention.

[0025] In the diagram, 11 is the liquid inlet; 12 is the gas inlet; 13 is the single-cell trap array; 14 is the outlet; 111 is the trap trap; 112 is the front confinement channel; 113 is the cell culture chamber; and 114 is the side confinement channel. Detailed Implementation

[0026] The present invention will be further described in detail below with reference to specific embodiments. These descriptions are for explanation purposes only and are not intended to limit the scope of the invention.

[0027] This invention discloses a microfluidic single-cell chip, referring to... Figure 1 It includes a liquid inlet, a gas inlet, and multiple parallel single-cell capture arrays, all sharing a single outlet. Each single-cell capture array includes a main channel arranged in a serpentine pattern with multiple rows of segments. Between adjacent rows of segments are multiple capture trap-chamber units. Each capture trap-chamber unit includes a connected cell capture trap, a front confinement channel, and a cell culture chamber. The inlet of the cell capture trap communicates with the previous row of segments, and the middle of the cell culture chamber is connected to a rear confinement channel, which communicates with the next row of segments. There are two rear confinement channels, symmetrically arranged, with their inlets at the same horizontal level.

[0028] Assume there are n rows of segments. The inlet of the liquid is set as the head of the segment according to the direction of liquid flow, and the outlet of the liquid is set as the tail of the segment. The tail of the first row of segments is connected to the head of the second row of segments, the tail of the second row of segments is connected to the head of the third row of segments, and so on.

[0029] Reference Figure 2 , 3 Let the transverse length of the cell trap be H1, the longitudinal length be H2, and the depth be H3. The dimensions satisfy the following: the depth H3 of the cell trap is greater than the transverse length H1 of the cell trap, and both the transverse length H1 and the depth H3 are greater than the diameter of a single cell. The longitudinal length H2 is greater than or equal to the transverse length H1.

[0030] The lateral length H4 of the anterior confinement channel is less than the lateral length H1 of the cell trap, the longitudinal length H5 of the anterior confinement channel is less than the longitudinal length H2 of the cell trap, the depth H6 of the anterior confinement channel is less than the depth H3 of the cell trap, and the depth H6 of the anterior confinement channel is less than the diameter of a single cell.

[0031] The transverse length H7 of the cell culture chamber is greater than the transverse length H1 of the cell trap, the longitudinal length H8 of the cell culture chamber is greater than the longitudinal length H2 of the cell trap, and the depth H9 of the cell culture chamber is greater than the diameter of a single cell.

[0032] The lateral length H10 of the rear confinement channel is less than the lateral length H1 of the cell trap, and the longitudinal length H11 of the rear confinement channel satisfies the condition that H11 is less than the longitudinal length H2 of the cell trap; the depth H12 of the rear confinement channel satisfies the condition that H12 is less than the depth H3 of the cell trap and is less than the diameter of a single cell.

[0033] An application of a microfluidic single-cell chip in the toxicity analysis of particulate matter exposed to gas and liquid, characterized by comprising the following steps:

[0034] S1, inject cell suspension into microfluidic chip through liquid inlet; the height of liquid inlet of microfluidic chip is higher than the height of outlet, and the flow rate of cell suspension is 5~10 µl / min.

[0035] S2, the cell carrier fluid enters the single-cell capture array through the liquid inlet. The cell capture trap retains the target cells, and the waste liquid is discharged from the microfluidic chip through the outlet. The specific operation is as follows: A 200µl pipette tip is inserted into the liquid inlet, and a polyethylene tube is connected to the outlet. By placing the end of the polyethylene tube at a lower position, a height difference is created between the inlet and outlet, forming a gravity-driven flow that loads the cell suspension from the pipette tip into the microfluidic chip. The cell carrier fluid enters the single-cell capture array through the cell suspension inlet, causing the cells to flow along the main channel. Under shear force, the cells are trapped by the capture trap, and the remaining waste liquid is discharged from the microfluidic chip through the outlet.

[0036] S3. After the cell trap captures the cells, the cell suspension at the liquid inlet is removed and cell culture medium is added. Then, the first syringe is connected to the liquid inlet, the first syringe is pushed in, and the cells in the microfluidic chip are observed under a microscope to see if they enter the cell culture chamber from the cell trap through the pre-restriction channel. When the cells enter the cell culture chamber, the first syringe is disconnected and the chip is cultured to obtain the culture chip.

[0037] During cultivation, the chip was placed in a saturated humidity, 37 ℃, 5% CO2 incubator for 24 hours, and the waste liquid outlet was placed at the same height as the chip.

[0038] S4. A gas-liquid exposure particulate toxicity assessment test is conducted using a culture chip. A second syringe is connected to the gas inlet, and gas carrying the test particles is introduced into the culture chip. The second syringe is placed in the injection pump, ensuring that the syringe is filled with the gas carrying the test particles. Within a gas injection rate range of 20–40 µl / min and for a gas-liquid exposure experiment lasting up to 4 hours, the cell culture chamber designed in this invention can still ensure that the cells are immersed in the cell culture medium.

[0039] In the gas-liquid exposure test, after 1-4 hours of clean air introduction, the cell survival rate was not significantly different from that of the negative control group without air introduction, indicating that the chip of the present invention is fully applicable to gas-liquid exposure particulate matter toxicity assessment experiments. The fluorescent indicator used to assess particulate matter toxicity can be injected into the chip before or after the gas-liquid exposure test to stain the cells, depending on the experimental needs.

[0040] The fabrication process of the microfluidic single-cell chip in this invention is as follows:

[0041] (1) Pretreatment of silicon wafers: The surface of the silicon wafers is cleaned by degreasing, polishing, isopropanol washing and water washing, and then placed on a 150℃ hot plate for 20 minutes and dried.

[0042] (2) Coating: A uniform layer of photoresist is coated on the surface of the treated silicon wafer. The spin-coating speed can be set according to the required pattern depth. In this embodiment of the invention, the spin-coating speed is set to 3000 rpm and run for 30 seconds.

[0043] (3) Pre-baking: The solvent in the photoresist evaporates at a certain temperature. The pre-baking temperature and time are set according to the properties of the photoresist used. In this embodiment of the invention, the silicon wafer is placed on a hot plate at 80 °C for min.

[0044] (4) Exposure: A photomask is placed on the silicon wafer, and ultraviolet light is used to irradiate the photoresist through the photomask. The exposure time is set according to the power of the exposure machine, the thickness of the photoresist film, the thickness of the photomask film, and the distance between the light source and the silicon wafer. In this embodiment of the invention, the exposure time is 60s.

[0045] It should be noted that the mold is manufactured using a three-step photolithography process. The first two steps manufacture the cell culture chamber and the two limiting channels on both sides of the middle of the chamber. The third step manufactures the cell trap and the main channel.

[0046] (5) Post-baking: After exposure, the template is removed and subjected to a short post-baking process. In this embodiment of the invention, the temperature for post-baking is set to 100 ℃ and the time is 2 min.

[0047] (6) Development: The silicon wafer is placed in propylene glycol monomethyl ether acetate developer to remove the unexposed photoresist. The development time depends on the type of developer and the development temperature, and the process is repeated 2 to 3 times. In this embodiment of the invention, the development temperature is set to 25°C and the development time is 60 seconds.

[0048] (7) Hardening: After the developed substrate is cleaned, it is baked at 100 °C to completely remove the solvent or moisture remaining in the photoresist film after development, so that the photoresist film adheres tightly to the substrate and prevents the photoresist layer from peeling off. The temperature of the hardening process is set according to the thickness of the photoresist. In the embodiment of the present invention, the hardening temperature is 100 °C.

[0049] (8) Development and inspection: Check the difference between the developed photoresist pattern and the standard pattern. At this point, the mold processing is complete.

[0050] (9) Surface coupling: The coupling agent is evaporated onto the surface of the silicon wafer in a closed vacuum space to form a coupling layer. The coupling time is at least 60 minutes. The coupling agent can be tridecafluoro-1,1,2,2-tetrahydrooctyl-1-trichlorosilane or (3-acryloyloxypropyl)-trichlorosilane.

[0051] (10) PDMS adhesive preparation: The PDMS matrix and curing agent are mixed in a certain ratio, and then degassed in a closed vacuum space. The ratio of matrix to curing agent is configured according to the required hardness of the PDMS material. In this embodiment of the invention, the ratio of matrix to curing agent is 15:1.

[0052] (11) Pouring the adhesive: Fix the silicon wafer in the culture dish, pour the prepared PDMS adhesive evenly into the dish, and ensure that there are no air bubbles in the adhesive.

[0053] (12) Heat treatment: Place the PDMS adhesive in a 75℃ constant temperature drying oven for at least 2 hours.

[0054] (13) Peeling: After cooling, use a scalpel to peel the cured PDMS off the silicon wafer.

[0055] (14) Cutting: Cut along the outer frame of the chip with a scalpel. Wipe the side of the chip with the graphic with Scotch invisible tape and then stick the invisible tape on.

[0056] (15) Drilling: Drill holes at the chip inlet and outlet using a hole punch. The diameter of the hole punch is selected based on the diameter of the conduit connecting the chip inlet and outlet. In this embodiment of the invention, the diameter of the hole punch is 1.5 mm.

[0057] (16) Bonding: Wipe the PDMS substrate and glass slide clean with disinfectant alcohol, use a plasma machine or corona discharge instrument to modify the bonding surface of the PDMS substrate and the glass slide, and then bond and compact the two treated surfaces.

[0058] (17) Baking: Place the chip on a 110℃ hot plate and bake for 10 minutes.

[0059] The method for preparing the cell suspension in this invention is as follows:

[0060] (1) Cell culture: Alveolar macrophages were grown in cell culture medium and placed in a saturated humidity, 37 ℃, 5% CO2 incubator. The cell culture medium consisted of RPMI-1640 medium, 10% fetal bovine serum and 1% penicillin-streptomycin bispecific antibiotics.

[0061] (2) Cell extraction: When the cells have grown to 80%~90% confluence, remove the cells from the carbon dioxide incubator, add trypsin to digest the cells, separate the adherent cells, use 3ml of trypsin at a time, centrifuge the separated mixture at 1000r / min for 10min, remove the supernatant, add 5ml of culture medium and mix thoroughly to obtain a cell suspension.

[0062] Example 1

[0063] Microfluidic single-cell chips and cell suspensions were prepared using the above method. The dimensions of the microfluidic single-cell chip were as follows: H1=15 µm, H2=12 µm, H3=20 µm, H4=10 µm, H5=10 µm, H6=10 µm, H7=20 µm, H8=36 µm, H9=20 µm, H10=8 µm, H11=8 µm, H12=6 µm. The chip was cleaned by sequentially introducing deionized water and phosphate buffer solution using a syringe. The cell suspension was introduced into the chip through the liquid inlet, and the height difference between the liquid inlet and waste outlet was adjusted to control the inflow rate of the cell suspension at 10 µl / min. The capture of single cells in the capture traps was observed under a microscope. After the cells were captured in the cell capture traps in front of each capture trap-chamber unit, the cell suspension at the liquid inlet was removed and cell culture medium was added. A syringe was then connected to the liquid inlet to push the cells into the cell culture chamber. After culturing the cells in a CO2 incubator for 24 hours, a gas-liquid exposure experiment was performed. Air was introduced into the chip gas inlet for 1 hour, with the gas injection rate controlled at 40 µl / min.

[0064] In Example 1, the capture efficiency of single cells in the trap reached 82%, and the cell survival rate was 96% after 1 hour of gas-liquid exposure experiment, which was not significantly different from the negative control group that did not introduce air.

[0065] Example 2

[0066] Microfluidic single-cell chips and cell suspensions were prepared using the above method. The dimensions of the microfluidic single-cell chip were as follows: H1=15 µm, H2=12 µm, H3=20 µm, H4=10 µm, H5=10 µm, H6=10 µm, H7=20 µm, H8=36 µm, H9=20 µm, H10=8 µm, H11=8 µm, H12=6 µm. The chip was cleaned by sequentially introducing deionized water and phosphate buffer solution using a syringe. The cell suspension was introduced into the chip through the liquid inlet, and the height difference between the liquid inlet and waste outlet was adjusted to control the inflow rate of the cell suspension at 5 µl / min. The capture of single cells in the capture traps was observed under a microscope. After the cells were captured in the cell capture traps in front of each capture trap-chamber unit, the cell suspension at the liquid inlet was removed and cell culture medium was added. A syringe was connected to the liquid inlet to push the cells into the cell culture chamber. After culturing the cells in a CO2 incubator for 24 hours, a gas-liquid exposure experiment was performed. Air was introduced into the chip gas inlet for 2 hours, with the gas injection rate controlled at 30 µl / min.

[0067] In Example 2, the capture efficiency of single cells in the trap reached 88%, and after 2 hours of gas-liquid exposure, the cell survival rate was 93%, which was not significantly different from the negative control group that did not introduce air.

[0068] Example 3

[0069] Microfluidic single-cell chips and cell suspensions were prepared using the above method. The dimensions of the microfluidic single-cell chip were as follows: H1=15 µm, H2=12 µm, H3=20 µm, H4=10 µm, H5=10 µm, H6=10 µm, H7=20 µm, H8=36 µm, H9=20 µm, H10=8 µm, H11=8 µm, H12=6 µm. The chip was cleaned by sequentially introducing deionized water and phosphate buffer solution using a syringe. The cell suspension was introduced into the chip through the liquid inlet, and the height difference between the liquid inlet and waste outlet was adjusted to control the inflow rate of the cell suspension at 8 µl / min. The capture of single cells in the capture traps was observed under a microscope. After the cells were captured in the cell capture traps in front of each capture trap-chamber unit, the cell suspension at the liquid inlet was removed and cell culture medium was added. A syringe was connected to the liquid inlet to push the cells into the cell culture chamber. After culturing the cells in a CO2 incubator for 24 hours, a gas-liquid exposure experiment was performed. Air was introduced into the chip gas inlet for 4 hours, with the gas injection rate controlled at 20 µl / min.

[0070] In Example 3, the capture efficiency of single cells in the trap reached 85%. After 4 hours of gas-liquid exposure, the cell survival rate was 92%, which was not significantly different from the negative control group that did not introduce air.

[0071] The above description is merely a preferred embodiment of the present invention and is not intended to limit the technical solution of the present invention in any way. Those skilled in the art should understand that, without departing from the spirit and principles of the present invention, the technical solution can be modified and replaced in several simple ways, and these modifications and replacements are all within the scope of protection covered by the claims.

Claims

1. An application of a microfluidic single-cell chip in the toxicity analysis of particulate matter exposed to gas and liquid, characterized in that, This microfluidic single-cell chip includes a liquid inlet, a gas inlet, and multiple parallel single-cell capture arrays. The multiple single-cell capture arrays share a single outlet. The single-cell capture array includes a main channel arranged in a serpentine pattern with multiple segments. Multiple capture trap-chamber units are provided between two adjacent rows of segments. Each capture trap-chamber unit includes a connected cell capture trap, a front confinement channel, and a cell culture chamber. The inlet of the cell capture trap is connected to the previous row of segments, and the middle of the cell culture chamber is connected to a rear confinement channel, which is connected to the next row of segments. There are two rear restriction channels; the two rear restriction channels are symmetrically arranged; the entrances of the rear restriction channels are located at the same horizontal level; The dimensions of each channel in the microfluidic single-cell chip meet the following requirements: H1=15 µm, H2=12 µm, H3=20 µm, H4=10 µm, H5=10 µm, H6=10 µm, H7=20 µm, H8=36 µm, H9=20 µm, H10=8 µm, H11=8 µm, H12=6 µm; where H1 is the lateral length of the cell trap, H2 is the longitudinal length of the cell trap, H3 is the depth of the cell trap, H4 is the lateral length of the front confinement channel, H5 is the longitudinal length of the front confinement channel, H6 is the depth of the front confinement channel, H7 is the lateral length of the cell culture chamber, H8 is the longitudinal length of the cell culture chamber, H9 is the depth of the cell culture chamber, H10 is the lateral length of the rear confinement channel, H11 is the longitudinal length of the rear confinement channel, and H12 is the depth of the rear confinement channel. Gas-liquid exposure experiments were conducted using the aforementioned microfluidic single-cell chip. Cell suspension was introduced into the chip through the liquid inlet, and the height difference between the liquid inlet and waste outlet was adjusted to control the inflow rate of the cell suspension at 10 µl / min. The capture of single cells in the capture traps was observed under a microscope. After the cells were captured in the cell capture traps in front of each capture trap-chamber unit, the cell suspension at the liquid inlet was removed and cell culture medium was added. A syringe was connected to the liquid inlet to push the cells into the cell culture chamber. After culturing the cells in a CO2 incubator for 24 hours, gas-liquid exposure experiments were performed. Air was introduced through the chip's gas inlet for 1 hour, with the gas injection rate controlled at 40 µl / min. The single-cell capture efficiency in the trap reached 82%, and the cell survival rate was 96% after 1 hour of gas-liquid exposure experiment.

2. An application of a microfluidic single-cell chip in the toxicity analysis of particulate matter exposed to gas and liquid, characterized in that, This microfluidic single-cell chip includes a liquid inlet, a gas inlet, and multiple parallel single-cell capture arrays. The multiple single-cell capture arrays share a single outlet. The single-cell capture array includes a main channel arranged in a serpentine pattern with multiple segments. Multiple capture trap-chamber units are provided between two adjacent rows of segments. Each capture trap-chamber unit includes a connected cell capture trap, a front confinement channel, and a cell culture chamber. The inlet of the cell capture trap is connected to the previous row of segments, and the middle of the cell culture chamber is connected to a rear confinement channel, which is connected to the next row of segments. There are two rear restriction channels; the two rear restriction channels are symmetrically arranged; the entrances of the rear restriction channels are located at the same horizontal level; The dimensions of each channel in the microfluidic single-cell chip meet the following requirements: H1=15 µm, H2=12 µm, H3=20 µm, H4=10 µm, H5=10 µm, H6=10 µm, H7=20 µm, H8=36 µm, H9=20 µm, H10=8 µm, H11=8 µm, H12=6 µm; where H1 is the lateral length of the cell trap, H2 is the longitudinal length of the cell trap, H3 is the depth of the cell trap, H4 is the lateral length of the front confinement channel, H5 is the longitudinal length of the front confinement channel, H6 is the depth of the front confinement channel, H7 is the lateral length of the cell culture chamber, H8 is the longitudinal length of the cell culture chamber, H9 is the depth of the cell culture chamber, H10 is the lateral length of the rear confinement channel, H11 is the longitudinal length of the rear confinement channel, and H12 is the depth of the rear confinement channel. Gas-liquid exposure experiments were conducted using the aforementioned microfluidic single-cell chip. Cell suspension was introduced into the chip through the liquid inlet, and the height difference between the liquid inlet and waste outlet pipes was adjusted to control the inflow rate of the cell suspension at 5 µl / min. The capture of single cells in the capture traps was observed under a microscope. After the cells were captured in the cell capture traps in front of each capture trap-chamber unit, the cell suspension at the liquid inlet was removed and cell culture medium was added. A syringe was connected to the liquid inlet to push the cells into the cell culture chamber. After culturing the cells in a CO2 incubator for 24 hours, gas-liquid exposure experiments were performed. Air was introduced through the chip's gas inlet for 2 hours, with the gas injection rate controlled at 30 µl / min. The single-cell capture efficiency in the trap reached 88%, and the cell survival rate was 93% after 2 hours of gas-liquid exposure experiment.

3. An application of a microfluidic single-cell chip in the toxicity analysis of particulate matter exposed to gas and liquid, characterized in that, This microfluidic single-cell chip includes a liquid inlet, a gas inlet, and multiple parallel single-cell capture arrays. The multiple single-cell capture arrays share a single outlet. The single-cell capture array includes a main channel arranged in a serpentine pattern with multiple segments. Multiple capture trap-chamber units are provided between two adjacent rows of segments. Each capture trap-chamber unit includes a connected cell capture trap, a front confinement channel, and a cell culture chamber. The inlet of the cell capture trap is connected to the previous row of segments, and the middle of the cell culture chamber is connected to a rear confinement channel, which is connected to the next row of segments. There are two rear restriction channels; the two rear restriction channels are symmetrically arranged; the entrances of the rear restriction channels are located at the same horizontal level; The dimensions of each channel in the microfluidic single-cell chip meet the following requirements: H1=15 µm, H2=12 µm, H3=20 µm, H4=10 µm, H5=10 µm, H6=10 µm, H7=20 µm, H8=36 µm, H9=20 µm, H10=8 µm, H11=8 µm, H12=6 µm; where H1 is the lateral length of the cell trap, H2 is the longitudinal length of the cell trap, H3 is the depth of the cell trap, H4 is the lateral length of the front confinement channel, H5 is the longitudinal length of the front confinement channel, H6 is the depth of the front confinement channel, H7 is the lateral length of the cell culture chamber, H8 is the longitudinal length of the cell culture chamber, H9 is the depth of the cell culture chamber, H10 is the lateral length of the rear confinement channel, H11 is the longitudinal length of the rear confinement channel, and H12 is the depth of the rear confinement channel. Gas-liquid exposure experiments were conducted using the aforementioned microfluidic single-cell chip. Cell suspension was introduced into the chip through the liquid inlet, and the height difference between the liquid inlet and waste outlet was adjusted to control the inflow rate of the cell suspension at 8 µl / min. The capture of single cells in the capture traps was observed under a microscope. After the cells were captured in the cell capture traps in front of each capture trap-chamber unit, the cell suspension at the liquid inlet was removed and cell culture medium was added. A syringe was connected to the liquid inlet to push the cells into the cell culture chamber. After culturing the cells in a CO2 incubator for 24 hours, gas-liquid exposure experiments were performed. Air was introduced through the chip's gas inlet for 4 hours, with the gas injection rate controlled at 20 µl / min. The single-cell capture efficiency in the trap reached 85%, and the cell survival rate was 92% after 4 hours of gas-liquid exposure experiment.

Images