A single-cell capture microfluidic chip device, its fabrication method, and related components

Abstract

This invention discloses a single-cell capture microfluidic chip device, its fabrication method, and related components. The single-cell capture microfluidic chip device includes a glass substrate and a silicon-based cap. Grooves are formed in the glass substrate to create a main channel and side channels. Inlet and outlet are provided in the grooves. The depth of the grooves, the width of the main channel, and the width of the side channels are all greater than the diameter of the cell. The main channel and side channels are respectively located between the inlet and outlet. The main channel is a straight channel, while the side channels are non-linear channels. A capture unit protrudes from the main channel for capturing cells. The silicon-based cap is fixedly connected to the glass substrate at the opening end of the groove, and a sensing window is formed on the silicon-based cap, located above the main channel and side channels. This invention can isolate a vacuum environment, maintain cell viability, and facilitate observation and detection.

Description

Technical Field

[0001] This invention relates to the field of microfluidic device technology, and in particular to a single-cell capture microfluidic chip device, its fabrication method, and related components. Background Technology

[0002] Cancerous cells can be distinguished from dying cells by changes in their surface shape and stiffness. Furthermore, observing changes in cancerous cells over several minutes at nanometer resolution can advance the elucidation of cell death mechanisms. Previous studies have used scanning electron microscopy (SEM) to observe cells in liquids by separating the liquid from a vacuum using a silicon nitride (Si3N4) film. Atmospheric scanning electron microscopy (ASEM), utilizing a 30nm thick Si3N4 film window, has achieved resolutions as high as 8nm. However, commercially available SEMs are quite difficult to use. With ASEM, because the sample is placed on a thin-film dish, the instrument needs to be inverted compared to traditional SEM. Therefore, ASEM requires a specialized setup. This undoubtedly adds considerable trouble for cell researchers. Moreover, once the sample is placed on the stage and observation begins, it inevitably takes place in a sealed environment, making it difficult for cells to remain viable during observation.

[0003] The above background information is provided only to aid in understanding the concept and technical solution of this invention. It does not necessarily belong to the prior art of this patent application. In the absence of clear evidence that the above information was disclosed on the filing date of this patent application, the above background information should not be used to evaluate the novelty and inventiveness of this application. Summary of the Invention

[0004] To address the aforementioned technical problems, this invention proposes a single-cell capture microfluidic chip device, its fabrication method, and related components, which can isolate a vacuum environment, maintain cell activity, and facilitate observation and detection.

[0005] To achieve the above objectives, the present invention adopts the following technical solution:

[0006] In a first aspect, the present invention discloses a single-cell capture microfluidic chip device, comprising a glass substrate and a silicon-based cap. A groove is formed on the glass substrate to create a main flow channel and side flow channels. An inlet and an outlet are provided on the groove. The depth of the groove, the width of the main flow channel, and the width of the side flow channels are all greater than the diameter of a cell. The main flow channel and the side flow channels are respectively disposed between the inlet and the outlet. The main flow channel is a straight flow channel, and the side flow channels are non-straight flow channels. A capture unit protrudes from the main flow channel for capturing cells. The silicon-based cap is fixedly connected to the glass substrate at the opening end of the groove, and a sensing window is formed on the silicon-based cap, located above the main flow channel and the side flow channels.

[0007] Preferably, the silicon-based cap is made of an SOI wafer comprising a substrate layer, an oxide layer, and a device layer, and the sensing window is a thin silicon film window formed by removing the substrate layer and the oxide layer at a position on the silicon-based cap above the main flow channel and the side flow channel.

[0008] Preferably, the thickness of the silicon-based cover at the sensing window is 0.5–2 μm.

[0009] Preferably, the length of the sensing window is 200-250 μm and the width is 100-150 μm.

[0010] Preferably, the depth of the groove, the width of the main flow channel, and the width of the side flow channel are all 20-25 μm.

[0011] Preferably, the side channel is an annular channel.

[0012] Preferably, the capture unit includes two symmetrically arranged arc-shaped cylinders to form a trap structure at the middle position of the main channel for capturing cells, and the gap between the two arc-shaped cylinders and the sidewall of the main channel is 2 to 5 μm.

[0013] Preferably, the capturing unit comprises at least one cylinder or arc-shaped cylinder.

[0014] In a second aspect, the present invention discloses a method for fabricating the single-cell capture microfluidic chip device described in the first aspect, comprising the following steps:

[0015] S1: Pattern the photoresist on the substrate side of the SOI wafer, and use deep silicon etching process to etch a portion of the substrate layer at the location where the sensing window is to be opened on the SOI wafer.

[0016] S2: Sputtered Cr and Au are used as seed layers for Ni electroplating on the glass substrate to form a mask layer. The seed layer is etched by Xe ion ball milling, and then the groove is etched by reactive ion etching to form the main channel, the side channel and the trapping unit.

[0017] S3: Using dry molding compound, the underside of the glass substrate is sandblasted to form through holes;

[0018] S4: The SOI wafer from step S1 is bonded to the glass substrate by anodic bonding.

[0019] S5: Deep silicon etching is performed on the substrate layer of the SOI wafer until the oxide layer is exposed at the sensing window location, and wet etching is performed on the oxide layer at the sensing window location of the SOI wafer to form the silicon-based cap having the sensing window.

[0020] Thirdly, the present invention discloses a cell-capturing microfluidic chip assembly, comprising a plurality of single-cell-capturing microfluidic chip devices as described in the first aspect, wherein the outlet of the preceding single-cell-capturing microfluidic chip device is connected to the inlet of the following single-cell-capturing microfluidic chip device, such that the single-cell-capturing microfluidic chip devices are connected in series sequentially.

[0021] Compared with existing technologies, the advantages of this invention are as follows: The single-cell capture microfluidic chip device, its fabrication method, and related components proposed in this invention employ a silicon thin-film window to fabricate a main channel and side channel structure on a glass substrate. The capture unit in the main channel is used to capture single cells flowing into the microfluidic suspension, while the side channels facilitate the inflow of remaining cells. Simultaneously, the captured cells remain in the center of the main channel, facilitating precise cell positioning. Finally, an SOI wafer with a thin-film window is anoly bonded to the glass substrate to complete the encapsulation. This achieves the capture of suspended cells in liquid samples under vacuum conditions. The cells can remain active in the flowing suspension for a relatively long time, while avoiding background noise in ESR-MRFM measurements caused by window crystal defects. This enables long-term observation of living cells in liquids under vacuum conditions. Attached Figure Description

[0022] Figure 1a and Figure 1b This is a schematic diagram of the structure of the single-cell capture microfluidic chip device disclosed in a preferred embodiment of the present invention;

[0023] Figures 2a to 2h This is a flowchart illustrating the fabrication process of the single-cell capture microfluidic chip device disclosed in a preferred embodiment of the present invention;

[0024] Figure 3This is a schematic diagram of a magnetic resonance force microscopy imaging system based on a single-cell capture microfluidic chip device in the atmospheric environment disclosed in this invention. Detailed Implementation

[0025] The embodiments of the present invention will be described in detail below. It should be emphasized that the following description is merely exemplary and not intended to limit the scope and application of the present invention.

[0026] It should be noted that when a component is referred to as "fixed to" or "set on" another component, it can be directly on or indirectly on that other component. When a component is referred to as "connected to" another component, it can be directly connected to or indirectly connected to that other component. Furthermore, a connection can be used for both fixing and circuit / signal connectivity.

[0027] It should be understood that the terms "length", "width", "up", "down", "front", "back", "left", "right", "vertical", "horizontal", "top", "bottom", "inner", and "outer" indicate the orientation or positional relationship based on the orientation or positional relationship shown in the accompanying drawings. They are only for the convenience of describing the embodiments of the present invention and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation. Therefore, they should not be construed as limitations on the present invention.

[0028] Furthermore, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. In the description of embodiments of the present invention, "a plurality of" means two or more, unless otherwise explicitly specified.

[0029] like Figure 1a and Figure 1bAs shown, a preferred embodiment of the present invention discloses a single-cell capture microfluidic chip device 100, including a glass substrate 10 and a silicon-based cap 20. A groove 11 is formed on the glass substrate 10 to form a main channel 111 and a side channel 112. The groove 11 is provided with an inlet 113 and an outlet 114. The depth of the groove 11, the width of the main channel 112, and the width of the side channel 113 are all greater than the diameter of the cell 200. The main channel 111 and the side channel 112 are respectively disposed between the inlet 113 and the outlet 114. The main channel 111 is a straight channel, and the side channel 112 is a non-straight channel. A capture unit 12 is protruding on the main channel 111 for capturing cells. The silicon-based cap 20 is fixedly connected to the glass substrate 10 at the opening end of the groove 11, and a sensing window 24 is formed on the silicon-based cap 20. The sensing window 24 is located above the main channel 111 and the side channel 112.

[0030] The silicon cap 20 is fabricated using an SOI wafer (SOI: silicon on insulator) comprising a substrate layer 21, an oxide layer 22, and a device layer 23. The sensing window 24 is a thin silicon film window formed by removing the substrate layer 21 and the oxide layer 22 at a position above the main flow channel 111 and the side flow channel 112 of the silicon cap 20; that is, there is only one silicon film (device layer 23) at the sensing window 24. The thickness of the silicon cap 20 at the sensing window 24 is 0.5–2 μm, the length of the sensing window 24 is 200–250 μm, and the width is 100–150 μm.

[0031] In this embodiment, the depth of the groove 11 (the depth of the main channel 111 and the depth of the side channel 112 are both equal to the depth of the groove 11), the width of the main channel 111, and the width of the side channel 112 are all 20-25 μm. The side channel 112 is an annular channel.

[0032] In this embodiment, the capture unit 12 includes two symmetrically arranged arcuate cylinders to form a trap structure at the middle position of the main channel 111 for capturing cells 200. The gap between the two arcuate cylinders and the sidewall of the main channel 111 is 2-5 μm. In other embodiments, the capture unit 12 may also include at least one or more cylinders or arcuate cylinders, such as at least one cylinder or arcuate cylinder protruding at the middle position of the main channel 111, as long as the purpose of cell capture is achieved.

[0033] In a preferred embodiment of the present invention, etching a silicon thin film window on the SOI wafer can isolate the vacuum environment, maintain cell viability, and the thin observation window facilitates observation and detection. The trapping structure enables cells to be trapped in a liquid environment, maintaining cell viability during long-term observation. Furthermore, when cells are trapped in the trapping structure, the remaining cells will flow out with the liquid from the side channel, ensuring the capture of a single cell.

[0034] Specifically, Figure 1a and Figure 1b The diagram shows a schematic of a single-cell capture microfluidic chip with a silicon thin-film window. Silicon-based thin-film window structure (length h) w 200–250 μm; width w w 100–150 μm; thickness d w A single-crystal silicon thin film window (500 nm to 2 μm) is bonded to a glass substrate 10 with a structure having a main flow channel 111, a side flow channel 112, and a cell-capturing microfluidic channel 12 structure via an anodic bonding process. Using the single-crystal silicon thin film window fabricated on an SOI wafer, the single-cell capture process and results can be observed via SEM or optical microscopy, while ensuring high sensitivity of the vacuum-encapsulated magnetic cantilever beam sensor to the free radical signals within the captured cells. The thickness (d) of the silicon thin film window (sensing window 24) is... w Size (h) w w w ) and the thickness around the window (d) s This also determines the observation and capture state, signal sensing capability, and processing difficulty. In specific applications, the allowable stress of the silicon thin film sensing window can be calculated based on factors such as material, structure, and pressure difference between internal and external environments. Finite element simulation analysis can be used to verify that the stress in the silicon thin film window does not exceed the allowable stress. Then, the optimal thickness scheme for processing the silicon thin film window can be established through experimental means.

[0035] Two symmetrically placed arc-shaped pillars (i.e., capture units 12) are designed in the main channel 111 to form a trap structure at the center of the main channel 111, ensuring that single-cell capture can be completed in a specific area of ​​the main channel 111. The symmetrical arc-shaped pillars form a trap structure at the center of the main channel 111, so that the captured cell is located in the center of the main channel 111, which facilitates the positioning of the target during observation. A gap of 2-5 μm is maintained between the capture unit 12 and the side wall of the main channel 111 to ensure that when a cell is captured, the main channel can still pass through a small flow of liquid without being completely blocked. The diameter of human cells is generally between 7 and 20 μm. In order to ensure that cells can smoothly enter the flow channel, the depth (d) of the microchannel (including the main channel 111 and the side channel 112) is... c ) and width (w c The diameters are 20μm–25μm and 20μm–25μm, respectively. Compared to the straight structure of the main channel 111, the side channel 112 adopts a ring structure, which facilitates the passage of other cells in the suspension through the side channel 112 in the case of single-cell capture. The inner walls of the channels are rounded, which makes the flow of liquid in the channels more stable on the one hand, and avoids the cells from colliding with sharp corners and being damaged, thus affecting the observation of cell structure.

[0036] Based on microchannel size (d) c w c Parameters such as the capture unit structure and cell suspension concentration are used to establish a fluid-structure interaction simulation model to ensure that the flow rate of the main channel dominates in the initial stage, which is conducive to transporting single cells to the capture area. After single cell capture, the flow rate of the main channel 111 should be less than the flow rate of the side channel 112, so that subsequent cells can flow out through the side channel 112.

[0037] Furthermore, this microfluidic chip device can be designed in series on a substrate material to achieve batch single-cell capture. That is, another preferred embodiment of the present invention discloses a cell capture microfluidic chip assembly, including multiple of the above-mentioned single-cell capture microfluidic chip devices, wherein the outlet of one single-cell capture microfluidic chip device is connected to the inlet of the next single-cell capture microfluidic chip device, so that the single-cell capture microfluidic chip devices are connected in series sequentially.

[0038] like Figures 2a to 2h As shown, a preferred embodiment of the present invention discloses a method for fabricating the above-mentioned single-cell capture microfluidic chip device 100, the specific micro-nano fabrication process of which is as follows:

[0039] S1: Pattern the photoresist on the substrate side of the SOI wafer, and use deep silicon etching process to etch a portion of the substrate layer at the location where the sensing window is opened on the SOI wafer.

[0040] like Figure 2a The silicon thin film window structure is made of the device layer 23 of the SOI wafer, and the thickness of the device layer 23 is 500nm to 2μm.

[0041] like Figure 2b After the photoresist is patterned on the substrate side, a 25-35 μm thick Si layer (substrate layer 21) is etched using a deep silicon etching process.

[0042] S2: Sputtered Cr and Au are used as seed layers for electroplating Ni on a glass substrate to form a mask layer. The seed layer is etched by Xe ion ball milling, and then the grooves are etched by reactive ion etching to form the main channel, side channel and trapping unit.

[0043] like Figure 2c The glass substrate 10 is cut to a size of 10mm×10mm (±0.5mm) with a thickness of 285~315μm;

[0044] like Figure 2dThe glass substrate 10 was etched using reactive ion etching (RIE) with SF6 gas, an operating power of 90–110 W, and a process pressure of 0.3–0.5 Pa. A patterned Ni layer was used as a mask layer. To form the mask pattern for the Ni layer, sputtered Cr (20 nm (±1 nm) thick) and Au (180 nm (±5 nm) thick) were used as seed layers for Ni electroplating; the Ni layer thickness was 3.5–4.5 μm. After electrodepositing the Ni layer and removing the photoresist, the CrAu seed layer was etched by Xe ion ball milling at an operating power of 50–70 W and a process pressure of 0.3–0.5 Pa. Recesses 11 were formed within the channels by reactive ion etching to obtain microchannels and trapping units 12, including a main channel 111 and side channels 112.

[0045] S3: Dry molding compound is used to sandblast the underside of the glass substrate to form through holes;

[0046] like Figure 2e Using dry film adhesive, the rear end of the glass substrate 10 is sandblasted to form a through hole, through which liquid can be input and output from the outside.

[0047] S4: Bond the SOI wafer from step S1 onto the glass substrate using anodic bonding.

[0048] like Figure 2f The prepared glass substrate 10 and the processed SOI wafer are bonded under atmospheric conditions using an anodic bonding method.

[0049] S5: Deep silicon etching is performed on the substrate layer of the SOI wafer until the oxide layer is exposed at the sensing window location, and wet etching is performed on the oxide layer at the sensing window location of the SOI wafer to form a silicon-based cap with a sensing window.

[0050] like Figure 2g The entire surface of the substrate layer 21 of the SOI wafer is etched using a deep silicon etching process;

[0051] like Figure 2h After being etched by steam HF, the SiO2 insulating layer (oxide layer 22) is safely removed without surface tension during the wet etching process.

[0052] The single-cell capture microfluidic chip device provided in the preferred embodiment of this invention features a specially designed capture structure that enables the capture of single cells in a suspension while maintaining cell viability, effectively solving the problem of long-term observation of live cells in suspension. Furthermore, the preferred embodiment of this invention uses an SOI wafer to etch a silicon thin film window (observation window), which not only isolates the liquid environment from the external environment, meeting the vacuum environment requirements for SEM or MRFM observation, but also allows electrons to penetrate the extremely thin silicon thin film window at relatively low accelerating voltages, reducing the difficulty of observation and detection. Additionally, in the main channel with the trap structure, when a cell is captured, the main channel becomes blocked, and cells remaining in the liquid sample flow to the next trap structure through side channels. Accordingly, a series of continuous cell capture chip arrays can be formed, achieving the goal of simultaneously capturing and observing multiple single cells.

[0053] Magnetic resonance force microscopy (MRFM) is an imaging technique based on highly sensitive mechanical cantilever beam sensing, offering nanometer-level resolution. Electron spin resonance (ESR)-MRFM has been developed for three-dimensional imaging of biological samples. However, MRFM observation of biological samples requires prolonged sample capture at a specific location and operation under high vacuum. When using ESR-MRFM to image free radical-based biological samples, Si3N4 is unsuitable as a window in the system due to background noise caused by crystal defects in Si3N4.

[0054] Due to the small size of cells, failure to accurately locate their position significantly increases the difficulty of observation. Furthermore, the accumulation of multiple cells can cause compression, collisions, and overlaps, which can substantially impact observation. Therefore, precise capture and accurate localization of individual cells are necessary.

[0055] The single-cell capture microfluidic chip device and its components proposed in this invention can be used for the detection of intracellular free radicals. By combining the single-cell capture microfluidic chip device with a vacuum-packaged magnetic cantilever beam sensor magnetic resonance force microscopy system, a novel magnetic resonance force microscopy system is constructed under standard atmospheric pressure. The specific layout is as follows... Figure 3As shown, in the vacuum-encapsulated magnetic cantilever beam sensor 400, the small magnet 420 at the tip of the cantilever beam 410 applies a certain gradient magnetic field in the vertical direction to magnetize the cells within the single-cell capture microfluidic chip device 100; the radio frequency coil 500 applies a certain radio frequency field in the horizontal direction to excite the electron spin resonance of free radicals inside the cells; simultaneously, the vacuum-encapsulated magnetic cantilever beam sensor 400 can provide a high gradient magnetic field to the cells 200 captured in the single-cell capture microfluidic chip device 100. When the radio frequency magnetic field applied by the radio frequency coil 500 satisfies the electron spin magnetic resonance condition of the cell 200, the magnetic force generated between the cell 200 and the magnet 420 can modulate the radio frequency signal of the magnetic field modulating coil 600 to excite the resonance of the magnetic cantilever beam structure, so that the magnetic cantilever beam sensor 400 senses the generated electron spin resonance force and generates resonant motion. The fiber optic interferometer 700 is placed on the back of the vacuum-encapsulated magnetic cantilever beam sensor 400 (on one side of the glass substrate 430) to minimize the distance between the fiber optic interferometer 700 and the cantilever beam 410, thereby ensuring the sensitivity of the detection of the vibration of the magnetic cantilever beam structure; the lock-in amplifier 800 is connected to the fiber optic interferometer 700.

[0056] In the actual measurement process, the single-cell capture microfluidic chip device 100 needs to be placed on the displacement scanning platform 300 for initial positioning to ensure that the distance between the cell 200 and the small magnet 420 is small enough to ensure that the excited electron spin resonance force is large enough, thereby enabling the three-dimensional scanning measurement of free radicals inside the cell 200. This invention provides an effective capture method for single cells, capable of rapidly separating a single cell 200 from a cell population, constructing the ecological environment required for cell 200 survival, ensuring cell viability throughout the testing process, accurately matching the spatial positions of each component of the MRFM, and using an extremely thin silicon thin film window to effectively excite the ESR signal inside the cell 200, providing a reliable guarantee for the accurate measurement of the spatial distribution of free radical concentration.

[0057] The single-cell capture microfluidic chip device 100 provided by this invention has a small structure, which can meet the needs of SEM observation or MRFM detection, and solves the problem of long-term observation of live cells in suspension. This will promote the study of intracellular and external cell structures and elucidate the mechanisms of cell necrosis and apoptosis. By carefully examining the molecular processes occurring in dead cells and focusing on cell death outcomes that affect the development of inflammation and immune responses, we can better describe new cell death pathways and further understand the pathological mechanisms of various human health problems.

[0058] The background section of this invention may include background information about the problems or circumstances surrounding the invention, rather than a description of prior art by others. Therefore, the content included in the background section is not an admission of prior art by the applicant.

[0059] The above description provides a further detailed explanation of the present invention in conjunction with specific / preferred embodiments, and it should not be construed that the specific implementation of the present invention is limited to these descriptions. For those skilled in the art, various substitutions or modifications can be made to these described embodiments without departing from the concept of the present invention, and all such substitutions or modifications should be considered within the scope of protection of the present invention. In the description of this specification, the reference to terms such as "an embodiment," "some embodiments," "preferred embodiment," "example," "specific example," or "some examples," etc., indicates that the specific features, structures, materials, or characteristics described in connection with that embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials, or characteristics described can be combined in any suitable manner in one or more embodiments or examples. Furthermore, those skilled in the art can combine and integrate different embodiments or examples and features of different embodiments or examples described in this specification without contradiction. Although the embodiments of the present invention and their advantages have been described in detail, it should be understood that various changes, substitutions, and modifications can be made herein without departing from the scope defined by the appended claims.

Claims

1. A single-cell capture microfluidic chip device, characterized in that, This device, used for capturing and observing live single cells in a vacuum observation environment, includes a glass substrate and a silicon-based cap. The glass substrate has grooves forming a main channel and side channels, with inlets and outlets. The depth of the grooves, the width of the main channel, and the width of the side channels are all greater than the diameter of the cell. The main channel and side channels are respectively positioned between the inlet and outlet. The main channel is a straight channel, while the side channels are non-linear. A capture unit protrudes from the main channel for capturing cells. The side channels have a ring structure to facilitate the passage of other cells in the suspension during single-cell capture. The silicon-based cap is solid. A sensing window is fixedly connected to the glass substrate at the opening end of the groove, and a sensing window is formed on the silicon substrate cover above the main flow channel and the side flow channel. The silicon substrate cover is made of an SOI wafer including a substrate layer, an oxide layer and a device layer. The sensing window is a silicon thin film window formed by removing the substrate layer and the oxide layer at the position of the silicon substrate cover above the main flow channel and the side flow channel. The SOI wafer with the silicon thin film window is anoly bonded to the glass substrate to achieve encapsulation. It is used to realize the transmission of electron beam or magnetic field in a vacuum observation environment to realize the observation and detection of captured single cells and maintain the sealing of the internal liquid environment.

2. The single-cell capture microfluidic chip device according to claim 1, characterized in that, The thickness of the silicon-based cover at the sensing window is 0.5~2μm.

3. The single-cell capture microfluidic chip device according to claim 1, characterized in that, The length of the sensing window is 200~250μm and the width is 100~150μm.

4. The single-cell capture microfluidic chip device according to claim 1, characterized in that, The depth of the groove, the width of the main channel, and the width of the side channel are all 20~25μm.

5. The single-cell capture microfluidic chip device according to claim 1, characterized in that, The side channel is an annular channel.

6. The single-cell capture microfluidic chip device according to claim 1, characterized in that, The capture unit includes two symmetrically arranged arc-shaped cylinders to form a trap structure at the middle position of the main channel for capturing cells, and the gap between the two arc-shaped cylinders and the sidewall of the main channel is 2~5μm.

7. The single-cell capture microfluidic chip device according to claim 1, characterized in that, The capturing unit includes at least one cylinder or arc cylinder.

8. A method for manufacturing a single-cell capture microfluidic chip device according to any one of claims 1 to 7, characterized in that, Includes the following steps: S1: Pattern the photoresist on the substrate side of the SOI wafer, and use deep silicon etching process to etch a portion of the substrate layer at the location where the sensing window is to be opened on the SOI wafer. S2: Sputtered Cr and Au are used as seed layers for Ni electroplating on the glass substrate to form a mask layer. The seed layer is etched by Xe ion ball milling, and then the groove is etched by reactive ion etching to form the main channel, the side channel and the trapping unit. S3: Using dry molding compound, the underside of the glass substrate is sandblasted to form through holes; S4: The SOI wafer from step S1 is bonded to the glass substrate by anodic bonding. S5: Deep silicon etching is performed on the substrate layer of the SOI wafer until the oxide layer is exposed at the sensing window location, and wet etching is performed on the oxide layer at the sensing window location of the SOI wafer to form the silicon-based cap having the sensing window.

9. A cell-capturing microfluidic chip assembly, characterized in that, The invention includes a plurality of single-cell capture microfluidic chip devices according to any one of claims 1 to 7, wherein the outlet of the preceding single-cell capture microfluidic chip device is connected to the inlet of the following single-cell capture microfluidic chip device, such that the various single-cell capture microfluidic chip devices are connected in series.

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