A microfluidic chip and a method for using the same

Abstract

The application discloses a micro-fluidic chip and a use method of the micro-fluidic chip, which are characterized in that the upper layer channel and the lower layer channel are independently arranged, the outlet ends of the upper layer channel and the lower layer channel are located on the two sides of the capture mixing cavity or the detection cavity, the gas-phase mixing mode is used, the energy generated by the generation and rupture of the bubbles is used to provide power for the mixing incubation of the original sample and the elution of the exosomes, the gas enters the cavity from the two sides, and the mixing effect is improved, the gas-phase mixing process on the chip can avoid the pipetting operation, the requirement for the sample amount is reduced, the loss of the magnetic beads and the exosomes in the liquid transfer process is reduced, the recovery rate of the exosomes is improved, the gas flow rates of the inner upper layer gas channel and the outer lower layer gas channel can be adjusted respectively to meet different mixing requirements, the mixing efficiency is extremely high, the long-time mixing incubation can be realized, the device can realize the sample adding to the exosome purification recovery on the chip and the sample adding to the microRNA detection on the chip.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of exosome separation and purification and detection, and relates to a microfluidic chip and a use method of the microfluidic chip. BACKGROUND

[0002] Exosomes are a kind of nanoscale biomarkers secreted by cells and widely exist in human body fluids. Exosomes have a similar phospholipid bilayer structure as cells, and a variety of specific functional proteins and nucleic acids, such as CD series proteins and various microRNAs, exist on the membrane and inside the exosomes. The content of a variety of proteins and nucleic acids changes with the occurrence and development of related diseases, so quantitative detection of the proteins and nucleic acids can realize monitoring and diagnosis of diseases.

[0003] However, due to the special physical and chemical properties of exosomes, it is difficult to obtain high-purity exosomes directly from human body fluids, and it is also difficult to quantitatively detect the nucleic acids inside the exosomes. The gold standard currently applied in the field of exosome separation and purification is the ultracentrifugation method, which has the advantage of high separation purity, but has low recovery rate, and the ultracentrifugation action can cause damage to exosomes. The immunomagnetic method based on the specific recognition principle of antigens and antibodies has high specificity, and can realize higher recovery efficiency while ensuring high separation purity. The method of modifying antibodies and other capture tools to magnetic microspheres is called immunomagnetic method.

[0004] The common detection methods for microRNAs include EXPAR, RCA, CRISPR / Cas13a, etc. The CRISPR / Cas13a is an amplification-free detection method, which is simple in principle and operation and is convenient for on-chip integration. The detection principle is to use the specific recognition of microRNAs to crRNA on Cas13a to activate the transcleavage activity of Cas13a protein, cut the fluorescence quenching group FQ reporter in the solution, and release the fluorescence signal, so as to detect the fluorescence intensity and quantify the microRNAs.

[0005] The immunomagnetic purification method and the CRISPR / Cas13a detection method operated manually in the laboratory need to use multiple specific instruments and devices to complete the separation and purification and detection of exosomes, which is complicated and responsible. The microfluidic chip as a reaction platform can integrate the main separation and purification operation steps on the chip by reasonable design of the chip structure, reduce the dependence on equipment, and facilitate automation and integration.

[0006] At present, microfluidic chips for exosome purification and detection have been developed, and the main steps that can be completed on-chip include mixing of immunomagnetic beads and raw samples, impurity removal, liquid transfer, magnetic bead transfer enrichment, detection area temperature control, negative pressure driving, etc. The current widely used mixing method on-chip is achieved through a long mixing channel, and the mixing effect is difficult to guarantee, and long-term mixing incubation function cannot be achieved. In addition, the existing exosome chip only has the single function of purification or detection, and the whole process of purification and detection is not integrated. SUMMARY

[0007] The purpose of the present application is to solve the problem in the prior art that the mixing method on-chip is achieved through a long mixing channel, and the mixing effect is difficult to guarantee, and long-term mixing incubation function cannot be achieved, and part of the steps cannot be integrated on one chip, and multiple devices need to be coordinated. A microfluidic chip and a use method of the microfluidic chip are provided.

[0008] To achieve the above purpose, the following technical solutions are adopted:

[0009] A microfluidic chip, comprising a chip body, wherein a capture mixing cavity, a filter cavity, an elution lysis cavity and a detection cavity are sequentially arranged on the chip body and are in communication with each other.

[0010] The detection cavity is connected with a detection cavity gas channel.

[0011] The capture mixing cavity and the elution lysis cavity are both connected with an external gas channel, and the external gas channel comprises an upper channel and a lower channel which are independent of each other, and the outlet ends of the upper channel and the lower channel are located on both sides of the capture mixing cavity or the elution lysis cavity.

[0012] Further improvements of the present application are as follows:

[0013] The outlet ends of the upper channel and the lower channel are both located at the bottom of the capture mixing cavity or the detection cavity.

[0014] The filter cavity is sequentially distributed with three.

[0015] The chip body is in a cylindrical structure, and the inlet ends of the upper channel and the lower channel are both located at the middle part of the chip body.

[0016] The mixing cavity, the filter cavity, the elution lysis cavity and the detection cavity are distributed along the circumference of the chip body.

[0017] A clamping groove is arranged on the chip body, and the clamping groove is used for connecting external devices.

[0018] The detection cavity gas channel is located on one side of the detection cavity.

[0019] The upper layer channel and the lower layer channel are both distributed with several upper layer channels and lower layer channels, and the several upper layer channels and lower layer channels are uniformly distributed along the two sides of the capture mixing cavity and the elution lysis cavity.

[0020] The detection cavity gas channel and the external gas channel are both sealed by steel needles.

[0021] A use method of the microfluidic chip comprises the following steps:

[0022] The original sample and the immunomagnetic beads are added into the capture mixing cavity;

[0023] Corresponding reaction reagents are added into the filter cavity, the elution lysis cavity and the detection cavity respectively;

[0024] The gas is sequentially delivered into the capture mixing cavity through the upper layer channel and the lower layer channel, the gas generates bubbles after entering the mixing cavity, the bubbles drive the sample and the magnetic beads to mix, and the exosomes are adsorbed on the magnetic beads;

[0025] The magnet is placed at the bottom of the chip, the magnetic beads move along with the magnet, sequentially pass through the filter cavity and the elution lysis cavity, and are finally transferred to the detection cavity, the sample reacts with the CRISPR detection reagent stored in the detection cavity in advance, and the fluorescence can be measured after the reaction is completed.

[0026] Compared with the prior art, the microfluidic chip has the following beneficial effects:

[0027] The application discloses a microfluidic chip, which is provided with independent upper layer channels and lower layer channels, the outlet ends of the upper layer channels and the lower layer channels are located on the two sides of a capture mixing cavity or a detection cavity, through the introduction of external gas, a gas phase mixing method, the generation and rupture of bubbles provide power for the mixing incubation of the original sample and the elution of exosomes, without the aid of external driving and shaking devices, and the gas enters the chamber from both sides, improving the mixing effect, the gas phase mixing process on the chip can avoid liquid transfer operation, reduce the requirement for the sample amount, and is beneficial to reducing the loss of magnetic beads and exosomes in the liquid transfer process and improving the recovery rate of exosomes; meanwhile, the gas flow rates of the inner upper layer gas channel and the outer lower layer gas channel can be adjusted to meet different mixing requirements, achieve extremely high mixing efficiency, and realize long-time mixing incubation, the device integrates the exosome purification and detection processes, can realize sample addition on the chip to exosome purification and recovery, and can realize sample addition on the chip to microRNA detection.

[0028] Further, in the application, the chip body is a cylindrical structure, and the chambers and channels distributed along the circumference facilitate the circumferential transfer of the magnetic beads under the driving of the magnet carried by the external motor.

[0029] Further, in the present application, the upper channels and the lower channels are evenly distributed along the two sides of the capture mixing cavity 1 and the elution lysis cavity, the outlet gas is uniform, and the mixing effect inside is improved. BRIEF DESCRIPTION OF DRAWINGS

[0030] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings needed in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present application, and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can also be obtained without creative labor on the basis of these drawings.

[0031] Figure 1 It is a schematic diagram of the upper layer of the chip body of the present application;

[0032] Figure 2 It is a schematic diagram of the upper layer of the chip body of the present application;

[0033] Figure 3 It is a schematic diagram of the upper layer of the chip body of the present application;

[0034] Figure 4 It is a schematic diagram of the lower layer of the chip body of the present application;

[0035] Figure 5 It is a schematic diagram of the lower layer of the chip body of the present application.

[0036] 1 - capture mixing cavity; 2 - first filter cavity; 3 - second filter cavity; 4 - third filter cavity; 5 - elution lysis cavity; 6 - detection cavity; 7 - upper channel; 8 - lower channel; 9 - clamping slot; 10 - separation and purification zone sealing element; 11 - detection zone sealing element; 12 - steel needle. DETAILED DESCRIPTION

[0037] In order to make the purpose, technical solutions and advantages of the embodiments of the present application more clear, the technical solutions in the embodiments of the present application will be described clearly and completely below in conjunction with the drawings in the embodiments of the present application. Obviously, the described embodiments are some embodiments of the present application, but not all embodiments. The components of the embodiments of the present application described and shown in the drawings can be arranged and designed in various different configurations.

[0038] Therefore, the following detailed description of the embodiments of the present application provided in the drawings is not intended to limit the scope of the claimed present application, but only represents selected embodiments of the present application. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor are within the scope of protection of the present application.

[0039] It should be noted that similar reference numerals and letters refer to like items in the several figures, and that, once an item is defined in one figure, it need not be further defined and explained in subsequent figures.

[0040] In the description of the embodiments of the present application, it should be noted that the orientation or position relationship indicated by the terms "upper", "lower", "horizontal", "inner" and the like is based on the orientation or position relationship shown in the drawings or the orientation or position relationship in which the product of the present application is usually placed, and is only for the convenience of describing the present application and simplifying the description, and therefore cannot be understood as indicating or implying that the device or element referred to must have a particular orientation, be constructed and operated in a particular orientation, and therefore cannot be understood as limiting the present application. In addition, the terms "first", "second" and the like are only used to distinguish the description and cannot be understood as indicating or implying relative importance.

[0041] In addition, if the term "horizontal" appears, it does not mean that the component must be absolutely horizontal, but can be slightly inclined. For example, "horizontal" only means that its direction is relatively more horizontal than "vertical", and does not mean that the structure must be completely horizontal, but can be slightly inclined.

[0042] In the description of the embodiments of the present application, it should also be noted that, unless otherwise explicitly specified and limited, if the terms "arrangement", "installation", "connection", "connection" appear, they should be understood in a broad sense, for example, they can be fixedly connected, or can be detachably connected, or integrally connected, can be mechanically connected, or can be electrically connected, can be directly connected, or indirectly connected through an intermediate medium, or can be connected inside two elements. For those skilled in the art, the specific meaning of the above terms in the present application can be understood according to the specific circumstances.

[0043] The present application will be described in further detail below with reference to the accompanying drawings:

[0044] Referring to Figure 1 The embodiment of the present application discloses a kind of microfluidic chip, microfluidic chip includes seven liquid chambers, and microchannel is connected between chamber. Seven chambers can be divided into 2 functional areas, purification area and detection area, wherein purification area can be subdivided into 3 areas, magnetic bead exosome mixing incubation area, water-oil two-phase filtration area and exosome elution / cleavage area, specific structure is as follows:

[0045] A kind of microfluidic chip, including chip body, the capture mixing cavity 1, filter cavity, elution cleavage cavity 5 and detection cavity 6 are opened in the chip body once communication;The detection cavity 6 connects detection cavity gas passage;The capture mixing cavity 1 and elution cleavage cavity 5 are all connected with external gas passage, the external gas passage includes mutually independent upper passage 7 and lower passage 8, the outlet end of upper passage 7 and lower passage 8 is located on the two sides of capture mixing cavity 1 or detection cavity 6.

[0046] Further, in this embodiment, the specific structure of the upper layer channel 7 and the lower layer channel 8 is as follows:

[0047] In this embodiment, the outlet end of the upper layer channel 7 is located on the inner side wall of the capture mixing cavity 1 or the elution lysis cavity 5, and the outlet end of the lower layer channel 8 is located on the outer side wall of the capture mixing cavity 1 or the elution lysis cavity 5. At the same time, the height of the outlet end of the upper layer channel 7 and the lower layer channel 8 is the same as the height of the bottom surface of the capture mixing cavity 1 or the elution lysis cavity 5, which ensures that the external gas enters from bottom to top, so that the gas bubbles generate power to mix from bottom to top along the bottom of the mixing cavity.

[0048] Further, the inlet end of the upper layer channel 7 and the lower layer channel 8 is located between the capture mixing cavity 1 and the elution lysis cavity 5, wherein the lower layer channel 8 is located below the upper layer channel 7. After passing through the lower part of the capture mixing cavity 1 or the elution lysis cavity 5, the lower layer channel 8 enters the corresponding cavity from the outer side wall of the capture mixing cavity 1 or the elution lysis cavity 5. The vertical distance between the upper layer channel 7 and the lower layer channel 8 is 0.5 mm.

[0049] Further, the longitudinal section of the upper layer channel 7 and the lower layer channel 8 is square, and the section size is 0.1 mm x 0.1 mm (length x width). The gas channels are distributed in a fan shape, and the interval angle between adjacent channels is 5°. All channel intersections are located at the center of the chip.

[0050] Further, the upper layer channel 7 and the lower layer channel 8 are independently supplied with gas, and the bottom surface of the upper layer channel is 0.5 mm away from the top surface of the lower layer channel. The inlet of the upper layer gas channel is located at the center of the top surface of the chip, and the inlet of the lower layer gas channel is located on the symmetry plane of the fan-shaped channel, which is 10 mm away from the center. The diameter of the channel inlet is 1 mm.

[0051] The mixing method and mixing structure are specially designed to improve the mixing efficiency and achieve long-time mixing. First, the gas-phase mixing method is selected, which uses the energy generated by the generation and rupture of gas bubbles to provide power for the mixing incubation of the original sample and the elution of exosomes, without the need for external driving and shaking devices. Meanwhile, the capture mixing chamber and the elution recovery / cleavage chamber on the chip are connected to the gas input channels on the inner and outer sides of the bottom surface. The inner and outer double-layer gas channels are independently supplied with gas and do not affect each other. The outlets of the double-layer gas channels are evenly distributed along the side wall of the chamber bottom surface. The inlet of the upper channel is located at the center of the chip, and the inlet of the lower channel is located on the symmetric plane of the channel 10 mm away from the center. The inlet and outlet are connected by a rectangular channel. The distance between the inlet and each outlet is equal to ensure that the flow resistance during gas input is equal, and the gas outlet of each outlet is uniform. The on-chip gas-phase mixing process can avoid pipetting operations, reduce the sample volume requirement, and also help to reduce the loss of magnetic beads and exosomes during liquid transfer, thereby improving the recovery rate of exosomes. At the same time, the gas flow rates of the inner upper and outer lower double-layer gas channels can be adjusted to meet different mixing requirements, achieving extremely high mixing efficiency and long-time mixing incubation.

[0052] Further, in this embodiment, the chip body is a cylindrical structure, and the chip as a whole is disc-shaped. The circumferentially distributed chambers and channels facilitate the circumferential transfer of magnetic beads driven by the magnet carried by the external motor. The rotation speed and number of rotations of the motor determine the final transfer efficiency of the magnetic beads. As the motor rotates, the magnetic beads are continuously moved from the initial chamber to the end chamber, and the transfer efficiency of the magnetic beads is ensured. At the same time, the entire process avoids liquid transfer operations, which is conducive to improving the recovery rate of exosomes.

[0053] Further, in this embodiment, the filter chamber includes a first filter chamber 2, a second filter chamber 3, and a third filter chamber 4 connected in sequence.

[0054] The first filter chamber 2 and the third filter chamber 4 are used to place oil phase solutions, and the second filter chamber 3 is used to place aqueous buffer. Non-miscible phase interface filtration technology is selected to remove impurities. The chambers spaced apart by oil phase solutions or aqueous solutions are arranged in sequence in the channel for magnetic bead transfer. When the magnetic beads are transferred through the non-miscible phase interface under the drive of the magnet, the non-specifically adsorbed impurities on the surface of the magnetic beads are filtered and removed under the action of surface tension, thereby eliminating the need for additional on-chip washing and purification operation steps. The non-miscible phase impurity removal method avoids liquid flow and reduces the amount of reagents required. At the same time, the various reagents have no intersection in space, which can reduce the source of pollution and help to improve the purity of the recovered exosomes.

[0055] Further, in this embodiment, two detection cavities 6 are provided, which are cylindrical cavities with a diameter of 0.3-0.5 mm and a depth of 5-8 mm, and the top of each cavity is provided with a chamfer of 0.5 mm. The end detection cavity is connected to the external gas through a detection cavity gas channel with a diameter of 0.7 mm and a height of 7 mm. In this embodiment, the arrangement of the detection cavities integrates the exosome purification and detection processes, which can realize on-chip sample addition for exosome purification recovery and on-chip sample addition for microRNA detection. The end 5th cavity in the purification area plays different roles in different application scenarios. In the exosome purification and recovery, the 5th cavity stores the eluent, and the exosomes are eluted and released through gas phase mixing. In the microRNA detection, the 5th cavity stores the exosome-specific lysis solution, and the exosomes captured on the magnetic beads are directly lysed. Then, the liquid containing microRNA is transferred to the detection cavity by external negative pressure driving, reacts with the detection reagent stored in the detection cavity in advance, and the appropriate reaction temperature is adjusted by the external temperature control system. After the detection reaction is completed, the target can be quantitatively detected by detecting the fluorescence intensity.

[0056] Further, in this embodiment, the capture mixing cavity 1, the first filter cavity 2, the second filter cavity 3, the third filter cavity 4, and the elution and lysis cavity 5 are connected through microchannels. The microchannels have a trapezoidal structure with dimensions of 0.8 mm x 3 mm x 3 mm (upper base x lower base x height), and the channel height is 0.5 mm.

[0057] Further, in this embodiment, the two detection cavities and the detection cavity and the lysis cavity are connected through microchannels. The channels are cylindrical pipes with a diameter of 0.5-1 mm. The microchannels are distributed along the central circular orbit of the cavities, and the channel height is 3-5 mm.

[0058] Further, in this embodiment, the connection channel height from the elution and lysis cavity 5 to the detection cavity 6 is 3-5 mm from the bottom surface of the cavity, which can prevent the magnetic beads from being transferred to the detection cavity.

[0059] Further, in this embodiment, the material of the chip body is organic glass (PMMA), which has a four-layer structure. The upper layer includes the upper gas channel, the cavities, and the microchannels, with a thickness of 8 mm. The middle upper layer is the lower gas channel, with a thickness of 0.5 mm. The middle lower layer is the lower gas input channel, with a thickness of 0.5 mm. The bottom layer is the chip base, with a thickness of 0.5 mm.

[0060] Further, in this embodiment, the upper channel 7 and the lower channel 8 are sealed by a steel needle 12 with an outer diameter of 1 mm and an inner diameter of 0.7 mm. The center height of the steel needle is 7-8 mm from the bottom surface of the cavity.

[0061] Further, the embodiment also includes separation and purification zone seal 10 and detection zone seal 11, the separation and purification zone seal 10 is used to seal the capture mixing cavity 1, the filter cavity, the elution and lysis cavity 5 and the detection cavity 6, and the detection zone seal 11 is used to seal the detection cavity 6.

[0062] Further, in the embodiment, the capture mixing cavity 1, the filter cavity, the elution and lysis cavity 5 are designed with equal-width sealing steps at the top, which are used to cooperate with the sealing elements, wherein the separation and purification zone seal 10 cooperates with the corresponding cavity gap, the detection zone seal 11 cooperates with the corresponding cavity interference, and the gas outlet hole on the separation and purification zone seal 10 has a diameter of 1mm, and each gas outlet hole is evenly distributed at a circumferential interval of 15°.

[0063] Further, in the embodiment, the volumes of the capture mixing cavity 1, the first filter cavity 2, the second filter cavity 3, the third filter cavity 4 and the elution and lysis cavity 5 are respectively 1500-2000ul, 150-180ul, 150-180ul, 150-180ul and 2000-2500ul.

[0064] Further, in the embodiment, the chip can be used to recover high-purity exosomes only by using the five chambers of the separation and purification zone, and can be used to directly lyse in the No.5 chamber after the exosome purification is completed, and then transferred to the detection zone to complete the microRAN detection.

[0065] The chip disclosed in the application can be used in combination with any immunomagnetic separation method, and has a wide range of applications. The circumferentially distributed chambers and channels facilitate the circumferential transfer of the magnetic beads driven by the magnet carried by the external motor. The number of rotations of the motor determines the transfer efficiency of the magnetic beads. With the rotation of the motor, the magnetic beads are continuously moved from the initial chamber to the terminal chamber, and the transfer efficiency of the magnetic beads is ensured. The whole process avoids liquid transfer operation, which is conducive to improving the recovery rate of exosomes. The air input by the air pump passes through the double-layer gas channel to realize liquid mixing in the capture and elution stages. The gas channels are evenly distributed, the distance between each gas outlet and the inlet is equal, the flow resistance is equal, the air outlet is uniform, and the mixing effect is good. The detection chamber is provided, liquid transfer from the separation and purification zone to the detection zone is realized by negative pressure driving, the whole chip integrates the separation, purification and detection processes, reduces the intermediate operation links, realizes the "sample in and result out", and greatly shortens the exosome purification and detection time.

[0066] Since the detection steps basically include the purification steps, only the specific detection operation steps are introduced below.

[0067] The specific microRNA detection operation steps are as follows:

[0068] First, in the 5 liquid chambers, in turn, add the appropriate amount of PBS buffer, 10% silicone oil solution, PBS buffer, 10% silicone oil solution, lysis solution. Add CRISPR / Cas13a detection reagent system to two detection chambers.

[0069] Step 1, immune magnetic bead capture of exosomes:

[0070] After pretreatment of the cell supernatant and other original samples, the cell culture supernatant sample and immune magnetic beads are added to the 0.01M PBS buffer in the capture mixing chamber 1, and the external air pump is used to input gas for mixing and incubation for 20 minutes;

[0071] The volume ratio of the pretreated cell culture supernatant sample to the mass of the immune magnetic beads is 100 μl:0.1 mg. The speed of the gas injected by the syringe pump during mixing and incubation is preferably 1000 μl / min, which can produce uniform bubbles, and at the same time, it will not cause liquid overflow, and can achieve good mixing and incubation effect of exosomes and magnetic beads;

[0072] Step 2, magnetic bead transfer, i.e. impurity removal:

[0073] The magnetic beads that captured exosomes in step 1 are transferred through the first filter chamber 2, the second filter chamber 3 and the third filter chamber 4 in turn by using an external magnetic field, and finally enriched in the elution lysis chamber 5. In this process, the non-specifically adsorbed impurities (mainly small proteins) on the magnetic beads will be removed under the filtration of 4 times surface tension;

[0074] Step 3, exosome lysis:

[0075] The magnetic beads are transferred to the lysis solution in the elution lysis chamber 5, and after simple mixing by gas phase, they are left to stand for 10 minutes for lysis, which destroys the cell-like membrane structure of the exosomes and releases the microRAN inside the exosomes into the solution;

[0076] Step 4, lysis solution transfer:

[0077] The lysis solution in the elution lysis chamber 5 is transferred to the No. 6 detection chamber by external negative pressure driving, and the gas flow rate driven by negative pressure is 1000 μl / min;

[0078] Step 5, microRNA detection reaction:

[0079] Under the adjustment of the external temperature control system, the optimal reaction temperature of 37℃ is set, and the lysis solution containing the detection target and the detection system are fully reacted. After 30 minutes, fluorescence intensity detection can be performed, and the fluorescence intensity has a linear relationship with the concentration of microRNA, so that the microRNA can be quantitatively detected.

[0080] The specific composition and volume ratio of the CRISPR / Cas13a detection reagent system are as follows: Cas13a (500 nM): crRNA (500 nM): FQ reporter (200 nM): buffer (1x): enzyme-free water = 2:1:5:5:27.

[0081] Further, when the gas is input, the injection pump inputs the gas at a rate of 1000-2000 μl / min, the incubation mixing rate is preferably 1000 μl / min, and the elution rate is preferably 1200 μl / min.

[0082] The chip disclosed in the embodiments of the present application is used for exosome separation and purification, and the required time is less than 60 minutes, the capture efficiency of the exosome can reach about 80%, the release efficiency can reach about 90%, the final exosome recovery rate can reach about 75%, and the morphology and biological function of the exosome are not changed. The detection limit of microRNA detection can reach 0.63 fM.

[0083] The present application uses a gas pump to input gas, and uniform bubbles are generated on the inner and outer sides of the two chambers after passing through the double-layer gas channel, which can provide mixing power for the mixing incubation of on-chip magnetic beads and raw samples and the elution and release of exosomes. Compared with the traditional on-chip mixing method, the mixing method can meet different mixing requirements by independently adjusting the gas injection speed of the inner and outer gas channels as needed, and the mixing efficiency is higher and the effect is better.

[0084] The present application uses the surface tension between the two non-miscible phase stable interfaces of water and oil to realize the filtration and removal of non-specific adsorption impurities during magnetic bead transfer, which saves the additional washing operation step; at the same time, the microchannel structure ensures the stable existence of the interfacial tension, greatly reducing the difficulty of chip design and manufacturing.

[0085] Based on the mature immunomagnetic bead method and the corresponding elution method and detection method, the whole process from exosome capture to elution / release / nucleic acid detection can be realized automatically on the chip. Compared with manual extraction operation, the randomness and uncertainty of the operation process are reduced, and the repeatability and stability of the experiment are greatly improved.

[0086] Compared with the elution method of rotating and blowing under the chip, the gas-phase mixing elution release method on the chip can reduce the loss of magnetic beads and exosomes during liquid transfer, which is conducive to improving the recovery rate of exosomes.

[0087] In the on-chip purification method, the liquid in each chamber is in a static state, compared with the traditional on-chip positive / negative pressure liquid driving, the chip can reduce the demand amount of samples and reagents, and various reagents without intersection in space reduce the source and risk of pollution, which is conducive to improving the purity of exosomes.

[0088] Finally, the chip realizes liquid transfer from the purification area to the detection area by negative pressure driving, integrates the purification of exosomes and the downstream detection, and completes the on-chip detection of microRNA by providing a suitable detection temperature through an external temperature control module.

[0089] The preferred embodiments of the present application have been described above with the preferred embodiments, but the present application is not limited to the above examples, and various modifications and changes can be made by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A microfluidic chip, characterized by, Including chip body, the chip body is provided with capture mixing cavity (1), filter cavity, elution lysis cavity (5) and detection cavity (6) that are sequentially communicated; The detection cavity (6) is connected with a detection cavity gas channel; The capture mixing cavity (1) and the elution lysis cavity (5) are both connected with an external gas channel, and the external gas channel comprises an upper channel (7) and a lower channel (8) that are independent of each other, the outlet end of the upper channel (7) is located at the inner side wall of the capture mixing cavity (1) or the elution lysis cavity (5), and the outlet end of the lower channel (8) is located at the outer side wall of the capture mixing cavity (1) or the elution lysis cavity (5); The outlet end of the upper channel (7) and the lower channel (8) is located at the bottom of the capture mixing cavity (1) or the detection cavity (6); The chip body is in a cylindrical structure, and the inlet end of the upper channel (7) and the lower channel (8) is located at the middle part of the chip body; The mixing cavity (1), the filter cavity, the elution lysis cavity (5) and the detection cavity (6) are distributed along the circumference of the chip body; The upper channel (7) and the lower channel (8) are both distributed with a plurality of channels, and the plurality of upper channels (7) and lower channels (8) are evenly distributed along the two sides of the capture mixing cavity (1) and the elution lysis cavity (5), and the plurality of upper channels (7) and lower channels (8) are both distributed in a fan shape.

2. The microfluidic chip according to claim 1, wherein, The filter cavity is sequentially provided with three filter cavities.

3. The microfluidic chip of claim 1, wherein, A clamping groove (9) is formed in the chip body, and the clamping groove (9) is used for connecting an external device.

4. The microfluidic chip of claim 1, wherein, The detection cavity gas channel is located at one side of the detection cavity (6).

5. The microfluidic chip of claim 1, wherein, The detection cavity gas channel and the external gas channel are both sealed by a steel needle (12).

6. A method of using the microfluidic chip of claim 1, wherein, The method comprises the following steps: The original sample and the immunomagnetic beads are added into the capture mixing cavity (1); Corresponding reaction reagents are added into the filter cavity, the elution lysis cavity (5) and the detection cavity (6) respectively; Gas is sequentially delivered into the capture mixing cavity (1) through the upper channel (7) and the lower channel (8), and after the gas enters the mixing cavity (1), bubbles are generated, which drive the sample and the magnetic beads to mix, so that the exosomes are adsorbed on the magnetic beads; A magnet is placed at the bottom of the chip, and the magnetic beads move with the magnet, sequentially pass through the filter cavity and the elution lysis cavity (5) and are finally transferred to the detection cavity (6), the sample reacts with the CRISPR detection reagent stored in the detection cavity (6) in advance, and after the reaction is completed, the fluorescence can be measured.

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