Micro-fluidic chip for enriching exosome and preparation method of micro-fluidic chip
By designing microfluidic chips and utilizing specific binding of nucleic acid aptamers, the loss, time-consuming and specific challenges in exosome extraction and isolation are solved, and efficient enrichment and purification of exosomes are achieved, providing a new method for cancer diagnosis.
Patent Information
- Application Number
- CN202311802653.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2023-12-26
- Publication Date
- 2025-06-27
AI Technical Summary
The prior art has losses, time-consuming and specific challenges in the extraction and isolation of exosomes, making it difficult to effectively enrich exosomes for cancer diagnosis.
A microfluidic chip is designed to utilize the structure of upper and lower chips and filter membranes to combine the specific binding ability of nucleic acid aptamers to achieve exosome enrichment and purification.
The device can efficiently enrich exosomes from serum or cell culture supernatant, reduce losses, improve purification efficiency, and achieve precise capture of exosomes through specific binding.
Smart Images
Figure CN120209958A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the field of exosome cell extraction, in particular to a microfluidic chip for enriching exosomes and a preparation method thereof. Background Art
[0002] Pancreatic cancer, as one of the most malignant tumors, is characterized by insidious onset, rapid progression, low surgical resection rate, and poor efficacy, resulting in a 5-year overall survival rate of less than 5% for patients. Unfortunately, most patients with pancreatic cancer have lymphatic or hematogenous metastasis at the time of diagnosis, missing the best opportunity for radical surgical treatment and leading to extremely poor prognosis. Currently, the clinical diagnosis of pancreatic cancer mainly relies on techniques such as pathology, CT imaging, and serum tumor-associated antigen 19-9 (CA19-9). However, these methods still have limitations in the detection of early-stage cancer, easily causing patients to miss the golden period of treatment. In this context, exosomes have become a research hotspot for the diagnosis of various tumors due to their widespread presence in body fluids, simple extraction, and non-invasive nature. However, to study exosomes as potential biomarkers, a high-quality extraction method is required first. Since exosomes are relatively small in size and mixed with various other biomolecules, it is challenging to separate and purify them. Currently, widely used separation techniques include centrifugation, ultrafiltration, precipitation, and immunoaffinity methods. Currently, centrifugation is one of the most commonly used methods. For example, patent CN115466709B discloses a method for extracting and using exosomes from Sparganium stoloniferum, but centrifugation often leads to exosome loss and is relatively time-consuming in the operation process. Ultrafiltration has the advantages of speed and simplicity. For example, patent CN219449690U discloses a stable and efficient separation device for stem cell exosomes, and the exosomes in the device can be filtered by ultrafiltration. However, ultrafiltration poses certain challenges to specificity. Immunoaffinity methods, such as patent CN114113581B, disclose a method for detecting the content of KL-6 protein on exosomes and its application. Although the immunoassay has strong specificity, it is restricted by the selection of antibodies, thus affecting its wide application.
[0003] With the intervention of biomolecules such as nucleic acid aptamers, new possibilities have been explored in the scientific research field. Nucleic acid aptamers, a kind of molecule constructed from nucleotide-rich sequences, have a highly specific binding ability, similar to the selective binding of antibodies to antigens. This property makes nucleic acid aptamers widely used in biological detection and medical research. The Nature magazine reported a study in 2015 on glypican-1 (GPC1) on the surface of exosomes from pancreatic cancer cells as a non-invasive diagnostic indicator, providing new ideas for the application of exosomes in cancer diagnosis.
[0004] Therefore, there is an urgent need for a device that can specifically bind to GPCI on exosomes using nucleic acid aptamers, divert and purify exosomes, and finally obtain an exosome-enriched solution. Summary of the Invention
[0005] To overcome the above-mentioned defects of the prior art, a first aspect of the present invention provides a microfluidic chip for enriching exosomes. The microfluidic chip provided by the present invention includes an upper chip, a lower chip, and a glass slide arranged in sequence from top to bottom. The upper chip is provided with a first chamber with a downward opening, the lower chip is provided with a second chamber that penetrates up and down, and the first chamber is communicated with the second chamber; a filter membrane is arranged between the first chamber and the second chamber, and the area of the filter membrane is larger than that of the first chamber and the second chamber. The upper chip is provided with a first waste liquid channel and a first liquid injection channel, and the first liquid injection channel is communicated with the first chamber; the lower chip is provided with a second waste liquid channel, and the second chamber is communicated with the second waste liquid channel; the first waste liquid channel and the second waste liquid channel are communicated. The glass slide is provided with nucleic acid aptamers for enriching exosomes.
[0006] In a specific embodiment, the upper chip is further provided with a first dissociation solution channel; the lower chip is further provided with a second dissociation solution channel, and the second chamber is communicated with the second dissociation solution channel; the first dissociation solution channel is communicated with the second dissociation solution channel.
[0007] In a specific embodiment, the upper chip is further provided with a first microchannel, one end of the first microchannel is communicated with the first liquid injection channel, and the other end is communicated with the first chamber. The lower chip is further provided with a second microchannel and a third microchannel; one end of the second microchannel is communicated with the second dissociation solution channel, and the other end is communicated with the second chamber; one end of the third microchannel is communicated with the second waste liquid channel, and the other end is communicated with the second chamber.
[0008] In a specific embodiment, it further includes a fixture, and the fixture includes a detachable upper fixture and a lower fixture. The upper fixture includes an upper frame and an upper fixing cavity arranged in the upper frame; the lower fixture includes a bottom plate, a lower frame arranged along the edge of the bottom plate, and a lower fixing cavity arranged in the lower frame. When the upper fixture is connected to the lower fixture, the upper chip abuts against the upper frame; the lower chip is arranged on the bottom plate and abuts against the lower frame.
[0009] In a feasible embodiment, the lower frame is provided with a notch.
[0010] In some specific embodiments, the thicknesses of the upper chip and the lower chip are both 5 - 15 mm; the pore diameter of the filter holes of the filter element is 30 - 70 nm; the inner diameters of the first chamber and the second chamber are both 10 - 30 mm; the inner diameters of the first dissociation solution channel, the first waste liquid channel, the first liquid injection channel, the second dissociation solution channel, and the second waste liquid channel are all 2 - 8 mm; the upper chip, the lower chip, and the glass slide are connected by plasma bonding.
[0011] In a specific embodiment, the material of the filter membrane is a polycarbonate membrane.
[0012] In a specific embodiment, the materials of the upper chip and the lower chip are both PDMS.
[0013] The second aspect of the present invention provides a method for preparing the microfluidic chip described in the first aspect of the present invention. The preparation method at least includes the following steps:
[0014] 1) Provide an upper chip, a lower chip, an aminated glass slide and a filter membrane.
[0015] 2) Couple the aminated glass slide with the nucleic acid aptamer to prepare a glass slide. The nucleic acid aptamer is a carboxyl-modified nucleic acid aptamer that specifically binds to exosomes.
[0016] 2-1) Activate the carboxyl-modified nucleic acid aptamer: Adjust the pH of the MES buffer to 6.0 - 7.0, add NHS, EDC and the carboxyl-modified nucleic acid aptamer, and activate for 30 min - 1 h to obtain an activated carboxyl-modified nucleic acid aptamer solution.
[0017] 2-2) Couple the carboxyl-modified nucleic acid aptamer and the aminated glass slide: Adjust the pH of the activated carboxyl-modified nucleic acid aptamer solution in step 2-1) to 7.1 - 7.5, then drop it onto the aminated glass slide, and incubate at 20 - 35 °C for 6 - 24 h to obtain a glass slide 3 carrying the nucleic acid aptamer 31.
[0018] 3) Bond the upper chip, the lower chip, the glass slide and the filter membrane to obtain a microfluidic chip.
[0019] The third aspect of the present invention provides an application of the microfluidic chip described in the first aspect in exosome enrichment.
[0020] Beneficial effects
[0021] This device is sequentially provided with an upper chip, a lower chip and a glass slide. A first chamber is opened in the upper chip and a second chamber is opened in the lower chip. At the same time, a first liquid injection channel, a first dissociation solution channel and a first waste liquid channel are also provided in the upper chip, and a second dissociation solution channel and a second waste liquid channel are also provided in the lower chip. A filter membrane is also provided between the upper chip and the lower chip. A nucleic acid aptamer is also provided on the glass slide. When serum or cell culture supernatant is introduced, the serum or cell culture supernatant flows in from the first liquid injection channel and passes through the first liquid injection channel, the first chamber, the filter membrane, the second chamber, the second waste liquid channel and the first waste liquid channel, and finally flows out through the first waste liquid channel, thereby realizing the enrichment of exosomes in the serum or cell culture supernatant on the glass slide. When the dissociation solution is introduced, the dissociation solution flows in from the first dissociation solution channel and passes through the first dissociation solution channel, the second chamber, the second waste liquid channel and the first waste liquid channel, and finally flows out through the first waste liquid channel, thereby realizing the collection of the exosome enrichment solution. In short, this device can enrich exosomes in serum or cell culture supernatant and finally obtain an exosome enrichment solution. BRIEF DESCRIPTION OF THE DRAWINGS
[0022] Figure 1 is a schematic diagram of the overall structure of the present invention.
[0023] Figure 2 is a sectional view of the upper chip of the present invention.
[0024] Figure 3 is a sectional view of the lower chip of the present invention.
[0025] Figure 4 is a schematic diagram of the structure of the fixture of the present invention.
[0026] REFERENCE NUMERALS
[0027] Upper chip 1
[0028] First chamber 11
[0029] First dissociation solution channel 12
[0030] First waste liquid channel 13
[0031] First liquid injection channel 14
[0032] First microchannel 15
[0033] Lower chip 2
[0034] Second chamber 21
[0035] Second dissociation solution channel 22
[0036] Second waste liquid channel 23
[0037] Second microchannel 24
[0038] The third microchannel 25
[0039] The glass slide 3
[0040] The aptamer 31
[0041] The filter membrane 4
[0042] The clamp 5
[0043] The upper clamp 51
[0044] The upper frame 511
[0045] The upper fixing cavity 512
[0046] The lower clamp 52
[0047] The lower frame 521
[0048] The fixing cavity 522
[0049] The bottom plate 523 Detailed implementation manners
[0050] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention. In the description of the present invention, it should be noted that the orientation or positional relationship indicated by the terms "left side", "right side", "upper side", "lower side", "above", "below", "top surface", "bottom surface", etc. is based on the orientation or positional relationship shown in the accompanying drawings, and is only for the convenience of describing the present invention and simplifying the description, rather than indicating or implying that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and therefore cannot be understood as a limitation of the present invention. In addition, the terms "first" and "second" are only used for descriptive purposes and cannot be understood as indicating or implying relative importance.
[0051] In the description of the present invention, it should be noted that unless otherwise clearly defined and limited, the terms "installed", "connected", and "connected" should be understood in a broad sense. For example, it can be a fixed connection, a detachable connection, or an integral connection; it can be a mechanical connection or an electrical connection; it can be directly connected or indirectly connected through an intermediate medium, and it can be the communication inside two elements. For those of ordinary skill in the art, the specific meanings of the above terms in the present invention can be understood according to specific circumstances.
[0052] In addition, in the description of the present invention, unless otherwise stated, the meaning of "a plurality of" is two or more.
[0053] Example 1
[0054] Refer to Figure 1 , this embodiment provides a microfluidic chip for enriching exosomes, which includes an upper chip 1, a lower chip 2 and a glass slide 3 arranged in sequence from top to bottom. Specifically, the materials of the upper chip 1 and the lower chip 2 are both PDMS. More specifically, the thicknesses of the upper chip 1 and the lower chip 2 are both 5 - 15 mm. Refer to Figure 2 , the upper chip 1 is provided with a first chamber 11 with a downward opening, the lower chip 2 is provided with a second chamber 21 that penetrates up and down, and the first chamber 11 communicates with the second chamber 21. Generally speaking, the cross-sectional shapes of the first chamber 11 and the second chamber 21 are both circular, coaxially aligned and have the same radius. Specifically, the inner diameters of the first chamber 11 and the second chamber 21 are both 10 - 30 mm. Continue to refer to Figure 1 , a filter membrane 4 is provided between the first chamber 11 and the second chamber 21, and the area of the filter membrane 4 is larger than that of the first chamber 11 and the second chamber 21. It should be noted that the function of the filter membrane 4 is to intercept macromolecules with a particle size larger than the pore diameter of the filter membrane 4 on the filter membrane 4 in the serum sample or cell culture supernatant. Specifically, the filter membrane 4 is a polycarbonate membrane. More specifically, the pore diameter of the filter element 4 is 30 - 70 nm. Continue to refer to Figure 1 , the upper chip 1 is provided with a first waste liquid channel 13 and a first liquid injection channel 14, and the first liquid injection channel 14 communicates with the first chamber 11. Specifically, the inner diameters of the first waste liquid channel 13 and the first liquid injection channel 14 are both 2 - 8 mm. Continue to refer to Figure 1, a second waste liquid channel 23 is provided in the lower chip 2, and the second chamber 21 communicates with the second waste liquid channel 23. The first waste liquid channel 13 communicates with the second waste liquid channel 23. Generally, the cross-sectional shapes of the first waste liquid channel 13 and the second waste liquid channel 23 are both circular, coaxially aligned and have the same radius. Specifically, the inner diameter of the second waste liquid channel 23 is 2-8 mm. A nucleic acid aptamer 31 is provided on the glass slide 3, and the nucleic acid aptamer 31 is used for enriching exosomes. Specifically, the nucleic acid aptamer 31 is a carboxyl-modified nucleic acid aptamer 31 that specifically binds to exosomes. It should be noted that the upper chip 1, the lower chip 2 and the glass slide 3 are connected by plasma bonding. Specifically, the 5'-end of the nucleic acid aptamer 31 is carboxyl-modified and is bonded to the glass slide (made of glass) through a condensation reaction. More specifically, the sequence of the nucleic acid aptamer is 5'-COOH-CATCCCCCTTTTG-3', which is used for specific capture of exosomes. It should be noted that the plasma bonding technology is completed by means of a plasma bonder. After the plasma bonder is turned on, some gases are sealed, and the electric field will excite the gas molecules to generate plasma. These plasmas can interact with the material surface, and reactive groups will be introduced on the material surface, making the material more adhesive. Finally, when it is placed together with another material and pressed, the bonding of the two materials can be achieved.
[0055] In a specific embodiment, refer to Figure 1 and Figure 2 , the upper chip 1 is further provided with a first dissociation solution channel 12. The lower chip 2 is further provided with a second dissociation solution channel 22, and the second chamber 21 communicates with the second dissociation solution channel 22. The first dissociation solution channel 12 communicates with the second dissociation solution channel 22. Generally, the cross-sectional shapes of the first dissociation solution channel 12 and the second dissociation solution channel 22 are both circular, coaxially aligned and have the same radius. Specifically, the inner diameters of the first dissociation solution channel 12 and the second dissociation solution channel 22 are both 2-8 mm.
[0056] In a specific embodiment, refer to Figure 2 , the upper chip 1 is further provided with a first microchannel 15. One end of the first microchannel 15 communicates with the first liquid injection channel 14, and the other end communicates with the first chamber 11. Refer to Figure 3 , the lower chip 2 is further provided with a second microchannel 24 and a third microchannel 25. One end of the second microchannel 24 communicates with the second dissociation solution channel 22, and the other end communicates with the second chamber 21. One end of the third microchannel 25 communicates with the second waste liquid channel 23, and the other end communicates with the second chamber 21.
[0057] In a specific embodiment, refer to Figure 4, further comprising a fixture 5, the fixture 5 includes a detachable upper fixture 51 and a lower fixture 52. The upper fixture 51 includes an upper frame 511 and an upper fixing cavity 512 provided in the upper frame 511. The lower fixture 52 includes a bottom plate 523, a lower frame 521 arranged along the edge of the bottom plate 523, and a lower fixing cavity 522 provided in the lower frame 521. Generally, the inner diameter of the upper frame 511 is slightly smaller than the inner diameter of the lower frame 521 to be able to abut against the upper surface of the upper layer chip 1. When the upper fixture 51 is connected to the lower fixture 52, the upper layer chip 1 abuts against the upper frame 511, the lower layer chip 2 is arranged on the bottom plate 523 and abuts against the lower frame 521. Here, a specific solution for the detachable connection between the upper fixture 51 and the lower fixture 52 is provided: a plurality of screw holes are provided on the upper frame 511, and a plurality of screw holes are also provided on the lower frame 521. The number of screw holes provided on the upper frame 511 is the same as that on the lower frame 521, and the positions of the screw holes in the upper frame 511 can correspond to the positions of the screw holes in the lower frame 521. When the upper fixture 51 and the lower fixture 52 need to be connected, screws are respectively passed through the screw holes in the upper frame 511 and the lower frame 521 for fixation.
[0058] In addition, in another specific embodiment, refer to Figure 4 , a notch is further provided on the lower frame 521. The notch can enable the experimenter to more easily place the microfluidic chip on the bottom plate 523. Continuing to refer to Figure 4 , four fixing corners can also be provided in the upper frame 511, and the above-mentioned fixing corners can better abut against the upper surface of the upper layer chip 1.
[0059] This embodiment also provides a method for using a microfluidic chip, and the specific usage method is as follows: 1) Block the first dissociation solution channel 12, and introduce serum or cell culture supernatant containing exosomes into the first injection channel 14; 2) The serum or cell culture supernatant flows into the first chamber 11 through the first injection channel 14 and continues to flow in the direction of the second chamber 21; 3) The serum or cell culture supernatant passes through the filter membrane 4, and the filter membrane 4 intercepts the macromolecules in the serum or cell culture supernatant on the filter membrane 4, so that most exosomes can pass through the filter membrane 4 and continue to flow in the direction of the glass slide 3; 4) When the serum or cell culture supernatant filtered by the filter membrane 4 flows onto the glass slide 3, since the glass slide 3 is loaded with carboxyl-modified nucleic acid aptamers 31 that specifically bind to exosomes, the exosomes specifically bind to the nucleic acid aptamers 31, so that the exosomes are enriched on the glass slide 3; 5) The waste liquid in the serum or cell culture supernatant flows out of the microfluidic chip through the second waste liquid channel 23 and the first waste liquid channel 13 and flows into an external waste liquid collection device; 6) After being processed by the above steps 1) to 5), the exosomes are enriched on the glass slide 3. Subsequently, open the first dissociation solution channel 12 and introduce the dissociation solution into the first dissociation solution channel 12; 7) The dissociation solution flows into the second chamber 21 through the first dissociation solution channel 12 and continues to flow in the direction of the glass slide 3; 8) When the dissociation solution flows onto the glass slide 3, the specific binding between the exosomes and the nucleic acid aptamers 31 is disconnected by the dissociation solution. Therefore, the exosomes flow out of the microfluidic chip through the second waste liquid channel 23 and the first waste liquid channel 13 with the dissociation solution and flow into an external exosome collection device, and finally an exosome enrichment solution concentrated within a certain particle size range is obtained.
[0060] It should be noted that in the above step 1), blocking the first dissociation solution channel 12 is to prevent the waste liquid of the serum or cell culture supernatant passing through the filter membrane 4 from flowing out through the first dissociation solution channel 12. In addition, the dissociation solution generally uses urea and methanol. The interaction forces between nucleic acid aptamers and exosomes are mainly hydrogen bonds and van der Waals forces. There are various methods to separate the target substance from the nucleic acid aptamer, including temperature, pH, ionic strength, etc., and there are also special dissociating agents such as urea and methanol. Urea and methanol can reduce the water activity and destroy hydrogen bonds and electrostatic interactions, thereby promoting the dissociation of nucleic acid aptamers from the target substance.
[0061] Embodiment Two
[0062] This embodiment provides a preparation method of the microfluidic chip in Embodiment One, and the specific preparation method is as follows:
[0063] 1) Provide an upper chip 1 made of PDMS material and a lower chip 2 made of PDMS material, and punch holes accordingly. Provide a commercially available amino-functionalized glass slide and a commercially available filter membrane 4 (with a pore size of 50 nm);
[0064] 2) Couple the aminated glass slide with the nucleic acid aptamer 31 to prepare slide 3. The nucleic acid aptamer 31 is a carboxyl-modified nucleic acid aptamer that specifically binds to exosomes. Specifically, it includes the following steps
[0065] 2-1) Activate the carboxyl-modified nucleic acid aptamer: Take 2 mL of 0.1 M MES buffer, adjust the pH = 6.5 with 0.1 M NaOH, add 0.76 mg of NHS, 1.77 μL of EDC, and 1 μL of DNA, and activate for 30 min to 1 h to obtain an activated DNA solution. It should be noted that the nucleic acid aptamer (i.e., DNA) is the nucleic acid aptamer strand corresponding to the typical GPC1 protein on exosomes (the sequence is 5’-COOH-CATCCCCCTTTTG-3’), and this nucleic acid aptamer strand can be directly ordered.
[0066] 2-2) Couple the carboxyl-modified nucleic acid aptamer and the aminated glass slide: Adjust the pH of the solution to 7.3 with 0.1 M NaOH; drop 10 μL of the activated DNA solution on the aminated glass slide and incubate at room temperature for 12 h to obtain slide 3 carrying the nucleic acid aptamer 31. It should be noted that the aminated glass slide can be directly ordered.
[0067] 3) Bond the upper chip 1, the lower chip 2, the slide 3, and the filter membrane 4 using conventional plasma technology to obtain a microfluidic chip. The upper chip 1, the lower chip 2, the slide 3, and the filter membrane 4 are subjected to plasma treatment for 1 min to bond them into a microfluidic chip.
[0068] It should be noted that the DNA solution in step 2-1) is a DNA fragment that can specifically bind to the GPC1 protein, and the concentration is 25 - 75 μM. In addition, the DNA sequence can be customized.
[0069] The above are only the preferred embodiments of the present invention. It should be pointed out that for those of ordinary skill in the art, without departing from the technical principle of the present invention, several improvements and replacements can be made, and these improvements and replacements should also be regarded as the protection scope of the present invention.
Claims
1. A microfluidic chip for enriching exosomes, characterized in that: It includes an upper chip (1), a lower chip (2) and a glass slide (3) arranged successively from top to bottom; The upper chip (1) is provided with a first chamber (11) with a downward opening, the lower chip (2) is provided with a second chamber (21) penetrating up and down, and the first chamber (11) communicates with the second chamber (21); A filter membrane (4) is arranged between the first chamber (11) and the second chamber (21), and the area of the filter membrane (4) is larger than that of the first chamber (11) and the second chamber (21); A first waste liquid channel (13) and a first liquid injection channel (14) are arranged in the upper chip (1), and the first liquid injection channel (14) communicates with the first chamber (11); A second waste liquid channel (23) is arranged in the lower chip (2), and the second chamber (21) communicates with the second waste liquid channel (23); The first waste liquid channel (13) communicates with the second waste liquid channel (23); The glass slide (3) is provided with a nucleic acid aptamer (31), and the nucleic acid aptamer (31) is used for enriching exosomes.
2. The microfluidic chip according to claim 1, characterized in that: The upper chip (1) is further provided with a first dissociation solution channel (12); The lower chip (2) is further provided with a second dissociation solution channel (22), and the second chamber (21) communicates with the second dissociation solution channel (22); The first dissociation solution channel (12) communicates with the second dissociation solution channel (22).
3. The microfluidic chip according to claim 2, characterized in that: The upper chip (1) is further provided with a first microchannel (15), one end of the first microchannel (15) communicates with the first liquid injection channel (14), and the other end communicates with the first chamber (11); The lower chip (2) is further provided with a second microchannel (24) and a third microchannel (25); One end of the second microchannel (24) communicates with the second dissociation solution channel (22), and the other end communicates with the second chamber (21); One end of the third microchannel (25) communicates with the second waste liquid channel (23), and the other end communicates with the second chamber (21).
4. The microfluidic chip according to claim 1 or 2 or 3, characterized in that: It further includes a fixture (5), and the fixture (5) includes a detachable upper fixture (51) and a lower fixture (52); The upper fixture (51) includes an upper frame (511) and an upper fixing cavity (512) arranged in the upper frame (511); The lower fixture (52) includes a bottom plate (523), a lower frame (521) arranged along the edge of the bottom plate (523) and a lower fixing cavity (522) arranged in the lower frame (521); When the upper fixture (51) is connected to the lower fixture (52), the upper chip (1) abuts against the upper frame (511); The lower chip (2) is arranged on the bottom plate (523) and abuts against the lower frame (521); And / or, the lower frame (521) is provided with a notch.
5. The microfluidic chip according to claim 4, characterized in that, It further includes any one of the following technical features: a) The thicknesses of the upper chip (1) and the lower chip (2) are both 5 - 15 mm; b) The pore diameter of the filter holes of the filter element (4) is 30 - 70 nm; c) The inner diameters of the first chamber (11) and the second chamber (21) are both 10 - 30 mm; d) The inner diameters of the first dissociation solution channel (12), the first waste liquid channel (13), the first liquid injection channel (14), the second dissociation solution channel (22), and the second waste liquid channel (23) are all 2 - 8 mm; e) The upper chip (1), the lower chip (2), and the glass slide (3) are bonded together by plasma bonding.
6. The microfluidic chip according to claim 1, wherein: The material of the filter membrane (4) is a polycarbonate membrane; and / or, the materials of the upper chip (1) and the lower chip (2) are both PDMS.
7. The preparation method of the microfluidic chip according to any one of claims 1 - 6 includes at least the following steps: 1) Provide an upper chip (1), a lower chip (2), an aminated glass slide, and a filter membrane (4); 2) Couple the aminated glass slide with the nucleic acid aptamer (31) to prepare the glass slide (3), and the nucleic acid aptamer (31) is a carboxyl - modified nucleic acid aptamer that specifically binds to exosomes; 3) Bond the upper chip (1), the lower chip (2), the glass slide (3), and the filter membrane (4) to obtain the microfluidic chip.
8. According to the preparation method of the microfluidic chip described in claim 7, in step 2), the following steps are further included: 2 - 1) Activate the carboxyl - modified nucleic acid aptamer: Adjust the pH of the MES buffer to 6.0 - 7.0, add NHS, EDC, and the carboxyl - modified nucleic acid aptamer, and activate for 30 min - 1 h to obtain an activated nucleic acid aptamer solution; 2 - 2) Couple the carboxyl - modified nucleic acid aptamer and the aminated glass slide: Adjust the pH of the activated nucleic acid aptamer solution in step 2 - 1) to 7.1 - 7.5, then drop it onto the aminated glass slide, and incubate at 20 - 35 °C for 6 - 24 h to obtain the glass slide (3) carrying the nucleic acid aptamer (31).
9. Application of a microfluidic chip according to any one of claims 1 - 6 in exosome enrichment.