Multi-channel high-precision microfluidic protein detection chip

Abstract

The utility model provides a kind of multi-channel high-precision microfluidic protein detection chip, belong to biological chip and diagnostic reagent technical field, the device includes upper layer mechanism, middle layer mechanism and base mechanism, when using, the light emitting condition of each detection area is observed, whether the corresponding target protein in blood sample exists and its approximate content can be judged quickly and accurately, by the design of multiple channels, so that one detection can be simultaneously directed to a variety of different proteins, greatly improve the detection efficiency, and, since each detection subchannel is independent, reduce the interference between different detections, further improve the detection accuracy, at the same time, the design of the chip also has certain flexibility, the number of detection subchannel can be adjusted according to actual demand, the fluorescence probe in storage cavity and nanometer antibody in detection area can also be customized according to the target protein to be detected, can be widely applied in the diagnosis and research of a variety of diseases.

Description

Technical Field

[0001] This invention relates to the field of biochip and diagnostic reagent technology, and in particular to a multi-channel high-precision microfluidic protein detection chip. Background Technology

[0002] Exosomes carry proteins and bioactive molecules that play important roles in tumor cell proliferation, invasion, immune escape, and epithelial-mesenchymal transition, thereby promoting the malignant progression of tumor cells. Tumor cell-derived exosomes can transfer oncoproteins to target cells in the form of peptides, leading to changes in the phenotype of target cells and tumorigenesis. Exosomes secreted by pancreatic cancer cells contain a variety of proteins that promote tumor growth and metastasis. These proteins enhance the proliferation and survival of cancer cells by regulating the tumor microenvironment and promoting interactions between cancer cells, interfering with the physiological activities of normal cells, thereby promoting the progression of pancreatic cancer.

[0003] Traditional percolation-based chips typically consist of multiple membrane layers, including a nitrocellulose membrane, a reverse osmosis layer, an absorbent layer, and a leak-proof layer. They are widely used in clinical testing because they allow for control of reaction time and process. However, the membrane layers of these traditional percolation-based chips are generally designed as rectangular or square, with the detection area at one end and the sample application area at the other. This results in a significant waste of sample solution during the flow to the sample application area.

[0004] A Chinese patent with authorization announcement number CN223501017U discloses a microporous permeation protein detection chip, belonging to the field of biochip and diagnostic reagent technology. It includes an upper shell and a lower shell, which are interlocked. A microporous membrane is disposed between the upper and lower shells. A recessed cavity is formed on the upper surface of the upper shell, and a sample injection window is provided in the cavity. An injection port is provided on the sample injection window, and the lower end of the injection port is connected to a sample injection channel. The bottom of the sample injection channel is connected to a reaction zone on the microporous membrane, and the lower end of the reaction zone is connected to an outlet channel. The lower end of the outlet channel is connected to an outlet port on the upper surface of the lower shell. This protein chip can reduce the amount of sample used, provides accurate measurement results, and does not cause environmental pollution.

[0005] The shortcomings of the existing technical solutions are as follows: Although the microporous permeation protein detection chip has certain advantages in reducing the amount of samples used, its structure is relatively simple and its support for multi-channel detection is not perfect. It is difficult to efficiently detect multiple different protein samples at the same time. In actual clinical testing scenarios, it is often necessary to detect multiple protein indicators at the same time to improve the accuracy and comprehensiveness of diagnosis. For example, exosomes secreted by pancreatic cancer cells contain a variety of proteins that promote tumor growth and metastasis, and this single-structure chip cannot meet such needs. Utility Model Content

[0006] To address the aforementioned problems, this invention provides a multi-channel, high-precision microfluidic protein detection chip, thus resolving the problems existing in the prior art.

[0007] This invention provides a multi-channel, high-precision microfluidic protein detection chip, comprising: The upper structure includes an upper shell, an injection pipe inside the upper shell, a storage cavity in the middle of the injection pipe, a fluorescent probe inside the storage cavity, and a micro valve below the storage cavity. The middle layer mechanism is located below the upper layer mechanism. The middle layer mechanism includes a middle shell, an enrichment channel connected to the sample injection pipe is opened inside the middle shell, an exosome capture coating is coated on the inner wall of the enrichment channel, and several detection sub-channels are opened at the lower part of the middle shell. The detection sub-channels are all connected to the enrichment channel, and a detection area is set at the end of the detection sub-channel. The detection area contains nanobodies for capturing proteins. The base mechanism includes a base support, which is located below the central housing. The base support has a discharge groove that communicates with several detection sub-channels.

[0008] As a further embodiment of this utility model: a liquid inlet cavity is provided on the upper end face of the upper shell, and a sample inlet is provided on the bottom surface of the liquid inlet cavity.

[0009] As a further embodiment of this utility model, the inlet is connected to the upper end of the inlet pipe.

[0010] As a further embodiment of this utility model, an inlet hole is provided on the upper end face of the middle shell.

[0011] As a further embodiment of this utility model: the injection port is connected to the lower end of the injection pipe, and the injection port is connected to the upper end of the enrichment channel.

[0012] As a further embodiment of this utility model: the lower end of the enrichment channel is connected to a connecting channel, and several detection sub-channels are all connected to the enrichment channel through the connecting channel.

[0013] As a further embodiment of this utility model: a connecting bracket is fixedly connected to the bottom surface of the middle shell, and the base bracket is fixedly connected to the connecting bracket.

[0014] As a further embodiment of this utility model, the miniature valve 16 is a one-way valve.

[0015] The beneficial effects of this utility model are: This invention allows for rapid and accurate determination of the presence and approximate content of target proteins in blood samples by observing the luminescence of each detection zone. Its multi-channel design enables simultaneous detection of multiple proteins, significantly improving efficiency. Furthermore, the independent nature of each detection sub-channel reduces interference between different detection methods, further enhancing accuracy. After detection, any remaining blood sample is discharged through a drain, facilitating subsequent cleaning and processing. The compact structure and tight, rational connections between components ensure the blood sample flows along a predetermined path, guaranteeing smooth detection. The chip also offers flexibility; the number of detection sub-channels can be adjusted to suit different detection scenarios and projects. Moreover, the fluorescent probes in the storage chamber and the nanobodies in the detection zones can be customized to target specific proteins, enabling the chip to be widely used in the diagnosis and research of various diseases. Attached Figure Description

[0016] Figure 1 This is a three-dimensional structural diagram of a multi-channel high-precision microfluidic protein detection chip according to the present invention. Figure 2 This is an exploded three-dimensional structural diagram of a multi-channel high-precision microfluidic protein detection chip according to the present invention; Figure 3 This is a side cross-sectional view of a multi-channel high-precision microfluidic protein detection chip according to the present invention.

[0017] List of reference numerals in the attached diagram: 1. Upper structure; 11. Upper shell; 12. Liquid inlet chamber; 13. Sample inlet; 14. Sample inlet pipe; 15. Storage chamber; 16. Miniature valve; 2. Middle structure; 21. Middle shell; 22. Sample inlet port; 23. Enrichment channel; 24. Exosome capture coating; 25. Connecting channel; 26. Detection sub-channel; 27. Connecting bracket; 3. Base structure; 31. Base bracket; 32. Discharge groove. Detailed Implementation

[0018] To make the objectives, technical solutions, and advantages of this utility model clearer, the embodiments of this utility model will be described in further detail below with reference to the accompanying drawings.

[0019] In the description of this utility model, it should be understood that the terms "center", "upper", "lower", "front", "rear", "left", "right", "vertical", "horizontal", "top", "bottom", "inner", "outer", etc., 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 this utility model 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 this utility model.

[0020] In the description of this utility model, it should be noted that, unless otherwise explicitly specified and limited, the terms "installation," "connection," and "joining" should be interpreted broadly. For example, they can refer to a fixed connection, a detachable connection, or an integral connection; they can refer to a mechanical connection or an electrical connection; they can refer to a direct connection or an indirect connection through an intermediate medium; and they can refer to the internal connection of two components. Those skilled in the art can understand the specific meaning of the above terms in this utility model based on the specific circumstances.

[0021] Reference Figures 1 to 3 This utility model provides a multi-channel high-precision microfluidic protein detection chip, comprising: an upper mechanism 1, a middle mechanism 2, and a base mechanism 3. The upper mechanism 1 includes an upper shell 11, within which a sample inlet channel 14 is provided. A storage cavity 15 is located in the middle of the sample inlet channel 14, and a fluorescent probe is disposed within the storage cavity 15. A micro-valve 16 is located below the storage cavity 15. The middle mechanism 2 is located below the upper mechanism 1 and includes a middle shell 21, within which a channel communicating with the sample inlet channel 14 is provided. The enrichment channel 23 has an exosome capture coating 24 coated on its inner wall. The lower part of the middle shell 21 has several detection sub-channels 26, which are connected to the enrichment channel 23. The end of the detection sub-channel 26 is provided with a detection area, in which nanoantibodies for capturing proteins are provided. The base mechanism 3 includes a base support 31, which is located below the middle shell 21. The base support 31 has a discharge groove 32 that is connected to the several detection sub-channels 26.

[0022] like Figure 2 and Figure 3 As shown, the upper shell 11 has a liquid inlet chamber 12 on its upper end face, and a sample inlet 13 on its bottom surface. The sample inlet 13 is connected to the upper end of the sample inlet pipe 14. The middle shell 21 has a sample inlet hole 22 on its upper end face, which is connected to the lower end of the sample inlet pipe 14. The sample inlet hole 22 is connected to the upper end of the enrichment channel 23, and the lower end of the enrichment channel 23 is connected to a connecting channel 25. Several detection sub-channels 26 are connected to the enrichment channel 23 through the connecting channel 25. Figure 3As shown, there are 5 detection sub-channels 26. In specific embodiments, the number of detection sub-channels 26 can be set according to specific needs, such as 8, 10, 20, etc.

[0023] like Figure 3 As shown, the diameter of the enrichment channel 23 is significantly smaller than that of the sample inlet pipe 14, and the length of the enrichment channel 23 is significantly longer than that of the sample inlet pipe 14, allowing the blood sample to flow in the enrichment channel 23 for a longer time. A connecting bracket 27 is fixedly connected to the bottom surface of the middle shell 21, and the base bracket 31 is fixedly connected to the connecting bracket 27. The blood sample can eventually be discharged through the discharge groove 32 in the base bracket 31. In use, 100 μL of peripheral blood is dripped into the sample inlet 13 of the upper shell 11. The blood sample will flow down the sample inlet pipe 14 and into the storage chamber 15. The fluorescent probes set in the storage chamber 15 mix with the blood sample. At the same time, due to the blood sample pressure, the micro valve 16 opens to allow the blood sample to pass through. The fluorescent probes are set for the target proteins to be captured, such as probes to capture VEGF protein or MMP9 protein. They are set in the storage chamber 15 in advance according to the ratio. After mixing, the blood sample carries all the fluorescent probes that can bind to the target proteins.

[0024] Micro-valves in microfabricated hard chips are mainly divided into two categories based on whether they require external energy to drive them: active valves and passive valves. Active valves rely on external signals (pneumatic, thermal, etc.) to control the opening / closing of the valve plate. The lightweight micro-valves 16 are one-way valves, which are essentially passive valves. They respond automatically based on the fluid's own pressure, channel structure, or material properties (such as hydrophobicity). Their structures are diverse, such as capillary valves, fishbone valves, and specific micro / nano structure surfaces.

[0025] Next, the blood sample mixed with the fluorescent probe flows into enrichment channel 23. Enrichment channel 23 is a thin tube, 3 cm long and 50 μm in inner diameter (like a thin straw). Because the inner wall of enrichment channel 23 is coated with an exosome-capturing coating 24, such as GPC1 antibody and CD44v6 antibody (both unique to pancreatic cancer exosomes), pancreatic cancer-related exosomes in the blood sample will bind to the exosome-capturing coating 24 as they pass through enrichment channel 23, remaining within the channel. Other substances in the blood will continue to flow forward and eventually be expelled. An exosome-capturing coating 24 is applied to capture exosomes in the blood sample. The captured exosomes release their internal proteins under continuous blood flow and enter the downstream detection subchannel 26. Since several detection subchannels 26 are connected to the lower end of the enrichment channel 23, each detection subchannel 26 has a detection zone at its end. This detection zone contains nanobodies for capturing proteins. Each detection zone captures only one protein; for example, the surface of "Detection Zone 1" is pre-positioned with a nanobodies for capturing VEGF protein; "Detection Zone 2" contains nanobodies for capturing MMP9 protein, and so on. The chip has 0 detection zones corresponding to 20 fluorescent probes for different proteins. VEGF protein is only captured by the hook in "Detection Zone 1" and remains in Zone 1. At the same time, the "VEGF fluorescent probe" (small green tag) mixed in the blood sample will immediately stick to the captured VEGF protein, which will then fluoresce green. MMP9 protein will fluoresce red (corresponding to its probe). Each detection zone will emit light of the corresponding color if it captures the target protein, and will not emit light if it does not capture it. By observing the luminescence of each detection zone, it is possible to quickly and accurately determine whether the corresponding target protein is present in the blood sample and its approximate content. The multi-channel design allows for simultaneous detection of multiple different proteins, greatly improving detection efficiency. Moreover, since each detection sub-channel is independent, interference between different detections is reduced, further improving detection accuracy. After detection, the remaining blood sample will be discharged through the discharge slot 32 for easy subsequent cleaning and processing. The entire detection chip has a compact structure, and the connections between various components are tight and reasonable, ensuring that the blood sample flows along the predetermined path and ensuring the smooth progress of the detection process.

[0026] Meanwhile, the chip's design also offers flexibility; the number of detection sub-channels can be adjusted according to actual needs to adapt to different detection scenarios and projects. Furthermore, the fluorescent probes within the storage chamber and the nanobodies within the detection area can be customized based on the specific target protein to be detected, enabling the chip to be widely used in the diagnosis and research of various diseases.

[0027] Furthermore, during the manufacturing process, the microfluidic chip is fabricated using polydimethylsiloxane and aldehyde-modified glass substrates, prepared by molding, and advanced microfluidic technology and microfabrication processes are employed to ensure the dimensional accuracy and surface quality of each channel and cavity, thereby further improving the chip's performance and reliability. The microfluidic chip in this application is a microfabricated hard chip, which uses micron-level channels and reaction cavities to be fabricated on hard substrates such as silicon and glass, achieving the integration of sample delivery and detection. The fabrication process typically includes the following steps: Photolithography: Photoresist (such as SU-8) is coated on a silicon wafer / glass, and then exposed and developed to form a mold.

[0028] Casting: The polymer prepolymer (such as PDMS) is poured into the mold and then heated to cure.

[0029] Plasma bonding: The cured polymer is subjected to plasma treatment with the glass sheet to permanently bond and seal it to form a closed microchannel.

[0030] Biomodification: Immobilizing probe molecules such as antibodies or aptamers within microchannels.

[0031] In practical use, operators only need to drip the blood sample into the inlet, and the chip will automatically complete the subsequent detection process. The operation is simple and convenient, reducing the requirements for the operator's professional skills.

[0032] It should be noted that not all steps and modules in the above processes and system structure diagrams are mandatory; some steps or modules can be omitted as needed. The execution order of each step is not fixed and can be adjusted as required. The system structure described in the above embodiments can be a physical structure or a logical structure. That is, some modules may be implemented by the same physical entity, or some modules may be implemented by multiple physical entities, or they may be jointly implemented by certain components in multiple independent devices.

[0033] In the above embodiments, the hardware modules can be implemented mechanically or electrically. The present invention has been described and illustrated in detail above with reference to the accompanying drawings and preferred embodiments. However, the present invention is not limited to these disclosed embodiments. Based on the above embodiments, those skilled in the art will understand that more embodiments of the present invention can be obtained by combining the code review methods in the different embodiments described above, and these embodiments are also within the protection scope of the present invention.

Claims

1. A multi-channel high-precision microfluidic protein detection chip, characterized in that, include: The upper structure (1) includes an upper shell (11), an injection pipe (14) is provided inside the upper shell (11), a storage cavity (15) is provided in the middle of the injection pipe (14), a fluorescent probe is provided inside the storage cavity (15), and a micro valve (16) is provided below the storage cavity (15). The middle layer (2) is located below the upper layer (1). The middle layer (2) includes a middle shell (21). An enrichment channel (23) connected to the sample injection pipe (14) is opened in the middle shell (21). An exosome capture coating (24) is coated on the inner wall of the enrichment channel (23). Several detection sub-channels (26) are opened at the lower part of the middle shell (21). Several detection sub-channels (26) are connected to the enrichment channel (23). A detection area is provided at the end of the detection sub-channel (26). Nanobodies for capturing proteins are provided in the detection area. The base mechanism (3) includes a base support (31), which is located below the central housing (21). The base support (31) has a discharge groove (32) that communicates with several detection sub-channels (26).

2. The multi-channel high-precision microfluidic protein detection chip according to claim 1, characterized in that, The upper end face of the upper shell (11) is provided with a liquid inlet cavity (12), and the bottom surface of the liquid inlet cavity (12) is provided with a sample inlet (13).

3. The multi-channel high-precision microfluidic protein detection chip according to claim 2, characterized in that, The inlet (13) is connected to the upper end of the inlet pipe (14).

4. The multi-channel high-precision microfluidic protein detection chip according to claim 1, characterized in that, An inlet hole (22) is provided on the upper end face of the middle shell (21).

5. A multi-channel high-precision microfluidic protein detection chip according to claim 4, characterized in that, The injection port (22) is connected to the lower end of the injection pipe (14), and the injection port (22) is connected to the upper end of the enrichment channel (23).

6. The multi-channel high-precision microfluidic protein detection chip according to claim 1, characterized in that, The enrichment channel (23) is connected to a connecting channel (25) at its lower end, and several detection sub-channels (26) are connected to the enrichment channel (23) through the connecting channel (25).

7. The multi-channel high-precision microfluidic protein detection chip according to claim 1, characterized in that, A connecting bracket (27) is fixedly connected to the bottom surface of the middle shell (21), and the base bracket (31) is fixedly connected to the connecting bracket (27).

8. The multi-channel high-precision microfluidic protein detection chip according to claim 1, characterized in that, The miniature valve (16) is a one-way valve.

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