Microfluidic chip and operation method and application thereof

Abstract

The invention relates to biological detection, in particular to a microfluidic chip and an operation method and application thereof. The microfluidic chip includes: a lower housing including one or more microwell arrays formed on an inner surface of the lower housing; the upper shell comprises one or more reaction tanks which are formed on the inner surface of the upper shell and correspond to the corresponding micropore arrays, one or more waste liquid tanks which are formed on the inner surface of the upper shell and are communicated with the corresponding reaction tanks, and one or more sample injection holes which penetrate through the upper shell and are communicated with the corresponding reaction tanks; the air holes penetrate through the upper shell and are communicated with the corresponding waste liquid pools; wherein the inner surface of the lower shell and the inner surface of the upper shell are opposite to each other and are in sealing fit with each other, so that one or more independent reaction units are formed. The device has the advantages of high detection flux, wide dynamic range, anti-pollution function, reliable, sensitive and rapid detection, simple and compact structure and low manufacturing cost.

Description

Technical Field

[0001] The present invention relates to biological detection, and in particular to a microfluidic chip and an operation method and application thereof. Background Art

[0002] With the development of micro-nano manufacturing technology, high-density fL-volume microwell reaction arrays can be manufactured to construct tens of thousands of independent microreaction units. Microfluidic chips integrated with microwell arrays have been widely used in high-throughput detection of cells, ultrasensitive detection of proteins and nucleic acids, and other fields.

[0003] In order to achieve efficient, rapid, and high-throughput detection, microfluidic chips are required to have multiple groups of reaction units and an effective way to manipulate the fluid in the reaction units in parallel. Although related products on the market contain multiple groups of reaction units, the reaction units usually do not contain structures such as liquid storage tanks and waste liquid tanks. Instead, they rely on a single pump to pump the waste liquid in the reaction unit into a waste liquid barrel one by one. This requires a waste liquid barrel and connected fluid pipelines, which increases complexity. At the same time, the waste liquid is stored in the waste liquid barrel, which increases the risk of environmental pollution. Summary of the invention

[0004] In order to solve the above problems in the prior art, the present invention provides a microfluidic chip and an operation method and use thereof, which have the advantages of high detection flux and wide dynamic range, and at the same time have anti-pollution function, reliable, sensitive and rapid detection, simple and compact structure, and low manufacturing cost.

[0005] The present invention provides a microfluidic chip, the microfluidic chip comprising:

[0006] a lower housing, the lower housing comprising one or more microwell arrays formed on an inner surface of the lower housing;

[0007] An upper shell, the upper shell comprising one or more reaction pools formed on the inner surface of the upper shell and corresponding to the corresponding microwell arrays, one or more waste liquid pools formed on the inner surface of the upper shell and connected to the corresponding reaction pools, one or more injection holes penetrating the upper shell and connected to the corresponding reaction pools, and one or more air holes penetrating the upper shell and connected to the corresponding waste liquid pools;

[0008] The inner surface of the lower shell and the inner surface of the upper shell are opposite to each other and sealed to form one or more independent reaction units.

[0009] In one embodiment of the present invention, each waste liquid pool is divided into two areas in the flow direction of the reaction sample, the bottoms of the two areas are not connected to each other, and the tops are connected to each other.

[0010] In one embodiment of the present invention, the two areas are separated by supporting ribs.

[0011] In one embodiment of the present invention, a water absorbing membrane is provided in each waste liquid pool.

[0012] In one embodiment of the present invention, the reaction tank and the corresponding waste liquid tank are connected to each other via a guide groove, and a check valve is provided at one end of the guide groove close to the waste liquid tank.

[0013] In one embodiment of the present invention, the microwell array occupies 1%-100% of the area directly below the corresponding reaction pool.

[0014] In one embodiment of the present invention, the microwell array occupies 30%-100% of the area directly below the corresponding reaction pool.

[0015] In one embodiment of the present invention, the microwell array occupies 60%-100% of the area directly below the corresponding reaction pool.

[0016] In one embodiment of the present invention, the one or more micro-hole arrays and the lower shell are integrally injection molded or spliced ​​and assembled into one body.

[0017] In one embodiment of the present invention, the inner surface of the lower shell and the inner surface of the upper shell are sealed and matched via adhesive film or laser welding.

[0018] In one embodiment of the present invention, the microfluidic chip further comprises one or more sealing gaskets disposed on the corresponding injection holes.

[0019] In one embodiment of the present invention, the microfluidic chip further comprises one or more guide grooves arranged on the corresponding sealing gasket.

[0020] In one embodiment of the present invention, the microfluidic chip further comprises one or more gas permeable membranes disposed on the corresponding gas permeable holes.

[0021] In one embodiment of the present invention, the lower shell and the upper shell are circular or fan-shaped, and the multiple independent reaction units are spaced apart from each other and each extends along a radial direction.

[0022] In one embodiment of the present invention, through holes are provided at the centers of the lower shell and the upper shell, and a pressing platform is provided on the outer surface of the upper shell and close to the through holes.

[0023] In one embodiment of the present invention, a return-to-zero mark is provided at the periphery of the outer surface of the upper shell.

[0024] The present invention further provides an operating method of the microfluidic chip described above, the operating method comprising:

[0025] Add one or more reaction samples into corresponding injection holes in a single or batch manner;

[0026] The reaction sample added each time enters the corresponding reaction pool and the corresponding microwell array by self-absorption, centrifugation or pressure injection;

[0027] Centrifuging the microfluidic chip so that the reaction samples enter the corresponding waste liquid pool from the corresponding reaction pool, while the reaction samples in the corresponding microwell array remain therein;

[0028] Add the split oil into the corresponding injection hole;

[0029] The split oil enters the corresponding reaction pool by self-absorption, centrifugation or pressure injection to split the reaction sample in the corresponding microwell array;

[0030] Waiting for the reaction sample in the corresponding microwell array to perform a biochemical reaction;

[0031] The corresponding microwell arrays are imaged, detected and digitally analyzed.

[0032] The present invention further provides a use of the microfluidic chip described above in digital ELISA detection.

[0033] The present invention further provides a use of the microfluidic chip described above in digital PCR detection.

[0034] As described above, the present invention has the following beneficial effects:

[0035] In the embodiment of the present invention, the microfluidic chip integrates the injection chamber, the reaction chamber and the waste liquid chamber, has a simple and compact structure, and has a low manufacturing cost. The reaction liquid is centrifuged into the liquid storage tank by centrifugation, and no additional negative pressure pump and waste liquid bucket and connecting joints and fluid pipelines are required. The microfluidic chip can be a disposable consumable. After the reaction is completed, the chip containing the waste liquid is stored in a specific area for unified treatment to prevent contamination.

[0036] Secondly, the waste liquid pool is divided into two areas, or a water-absorbing membrane is set in the waste liquid pool to prevent the waste liquid from flowing back into the reaction pool. In addition, the microwell array occupies most of the area directly below the corresponding reaction pool, and all samples in the reaction pool can participate in the detection. At the same time, the number of microwells increases, which increases the sample loading rate and improves the sensitivity and dynamic range of the detection. The microwell array and the lower shell of the microfluidic chip are integrally injection molded or spliced ​​and assembled into one. The integral injection molding not only ensures the flatness but also reduces the assembly process, while the splicing assembly has lower manufacturing requirements. In addition, the microfluidic chip can provide multiple independent reaction units, which improves the detection throughput.

[0037] In an embodiment of the present invention, by selecting a microfluidic chip including a suitable microwell array and by adding a suitable reaction sample, the microfluidic chip can be operated to achieve different detections, such as digital ELISA detection and digital PCR detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 is a schematic diagram of the overall structure of a microfluidic chip according to an embodiment of the present invention;

[0039] Figure 2 is an exploded schematic diagram of a microfluidic chip according to one embodiment of the present invention;

[0040] Figure 3 is a top view of the inner surface of the lower housing of a microfluidic chip according to one embodiment of the present invention;

[0041] Figure 4A and Figure 4B They are scanning electron microscope images of a microwell array of a microfluidic chip according to an embodiment of the present invention;

[0042] Figure 5A is a top view of the inner surface of the upper housing of a microfluidic chip according to one embodiment of the present invention, Figure 5B It is a partial enlarged view of the outer surface of the upper shell. Figure 5C is a partial enlarged view of the inner surface of the upper shell;

[0043] Figure 6 is an assembly diagram of a reaction pool and a microwell array in a microfluidic chip according to an embodiment of the present invention;

[0044] Figure 7 is a side view of a microfluidic chip according to one embodiment of the present invention;

[0045] Figure 8 4 is a flow chart of a method for operating a microfluidic chip according to an embodiment of the present invention. DETAILED DESCRIPTION

[0046] Embodiments of the present invention are described below with reference to the accompanying drawings.

[0047] Exemplary embodiments will be described in detail herein, examples of which are shown in the accompanying drawings. When the following description refers to the drawings, the same numbers in different drawings represent the same or similar elements unless otherwise indicated. The implementations described in the following exemplary embodiments do not represent all implementations consistent with the present application. Instead, they are merely examples of devices and methods consistent with some aspects of the present application as detailed in the appended claims.

[0048] The terms used in this application are only for the purpose of describing specific embodiments, and are not intended to limit this application. The singular forms of "a", "said" and "the" used in this application and the appended claims are also intended to include plural forms, unless the context clearly indicates other meanings. It should also be understood that the term "and / or" used in this article refers to and includes any or all possible combinations of one or more associated listed items. "Include" or "comprising" and similar words mean that the elements or objects appearing in front of "include" or "comprising" cover the elements or objects listed after "include" or "comprising" and their equivalents, and do not exclude other elements or objects.

[0049] Figure 1 is a schematic diagram of the overall structure of a microfluidic chip according to an embodiment of the present invention, Figure 2 Schematic diagram of an exploded view of a microfluidic chip according to an embodiment of the present invention.

[0050] like Figure 1 and Figure 2 As shown, the microfluidic chip 100 includes a lower housing 110. The lower housing 110 can be made of one or more plastic materials such as polycarbonate (PC), polystyrene (PS), polymethyl methacrylate (PMMA), cyclo olefin copolymer (COC) and cyclo olefin polymer (COP), etc. using conventional injection molding technology, and the minimum processing accuracy is 100nm-50μm. The lower housing 110 can also be made of one or more semiconductor materials such as silicon and glass using semiconductor processing technology, and the minimum processing accuracy is 100nm-50μm. As an example, the lower housing 110 is made of PC material using conventional injection molding technology.

[0051] In the depth direction, the lower housing 110 may have a first surface and a second surface opposite to each other, wherein the first surface located inside the microfluidic chip 100 is an inner surface, and the second surface located outside the microfluidic chip 100 is an outer surface.

[0052] Figure 3 FIG. 4 is a top view of the inner surface of the lower housing of the microfluidic chip according to one embodiment of the present invention.

[0053] like Figure 3 As shown, and reference Figure 1 and Figure 2, the lower housing 110 includes one or more micro-hole arrays 112 formed on its inner surface. As an example, the one or more micro-hole arrays 112 and the lower housing 110 are integrally injection molded, which not only ensures flatness but also reduces the assembly process. As another example, the one or more micro-hole arrays 112 and the lower housing 110 are spliced ​​and assembled into one body, which has lower manufacturing requirements.

[0054] Each micropore array 112 includes 5,000 to 10 million micropores 113 , each micropore 113 has a diameter of 1 μm to 120 μm, each micropore 113 has a depth of 1 μm to 120 μm, and the center distance between adjacent micropores 113 is 3 μm to 180 μm.

[0055] Each micropore 113 may be circular in shape, and depending on the detection requirements, the micropore 113 may have different shapes, such as a hexagon. In the case where the micropore 113 is non-circular in shape, the diameter of the micropore 113 may refer to the diameter of the circumscribed circle of the micropore 113. The micropores 113 may be arranged in rows and columns, or in a hexagonal stacking manner.

[0056] Figure 4A and Figure 4B They are scanning electron microscope images of a microwell array of a microfluidic chip according to an embodiment of the present invention.

[0057] like Figure 4A As shown, each microwell 113 is circular, has a diameter of 4 μm, a depth of 4 μm, and a center distance of 8 μm between adjacent microwells 113. The microwell array 112 composed of such microwells 113 is particularly suitable for digital ELISA detection.

[0058] like Figure 4B As shown, each microwell 113 is hexagonal, the circumscribed circle diameter of each microwell 113 is 70 μm, the depth of each microwell 113 is 70 μm, and the center distance between adjacent microwells 113 is 105 μm. The microwell array 112 composed of such microwells 113 is particularly suitable for digital PCR detection.

[0059] Back to Figures 1 to 3 The lower housing 110 may be circular in shape, and when the lower housing 110 includes a plurality of micropore arrays 112, the plurality of micropore arrays 112 are spaced apart from each other and extend in a radial direction, so that they can be used in conjunction with a centrifugal control device. Depending on the detection requirements, the lower housing 110 may have different shapes (such as a fan shape, etc.), and the plurality of micropore arrays 112 extend outwardly in a radial shape from the center (such as the center of a circle, etc.) of the lower housing 110.

[0060] A through hole 111 penetrating the lower housing 110 is provided at the center of the lower housing 110 . The through hole 111 can be tightly matched with a rotor (not shown) of a centrifugal control device.

[0061] Continue as Figure 1 and Figure 2 As shown, the microfluidic chip 100 also includes an upper housing 120. The upper housing 120 can be made of one or more plastic materials such as polycarbonate (PC), polystyrene (PS), polymethyl methacrylate (PMMA), cyclo olefin copolymer (COC), and cyclo olefin polymer (COP), etc. using conventional injection molding technology, and the minimum processing accuracy is above 20μm. As an example, the material of the upper housing 120 is PC material.

[0062] In the depth direction, the upper housing 120 may have a first surface and a second surface opposite to each other, wherein the first surface located inside the microfluidic chip 100 is an inner surface, and the second surface located outside the microfluidic chip 100 is an outer surface.

[0063] Figure 5A is a top view of the inner surface of the upper housing of a microfluidic chip according to one embodiment of the present invention, Figure 5B It is a partial enlarged view of the outer surface of the upper shell. Figure 5C It is a partial enlarged view of the inner surface of the upper shell.

[0064] like Figure 5A As shown, and reference Figures 1 to 3 The upper shell 120 includes one or more reaction pools 123 formed on its inner surface and corresponding to the corresponding micropore arrays 112, one or more waste liquid pools 124 formed on its inner surface and connected to the corresponding reaction pools 123, one or more injection holes 125 penetrating the upper shell 120 and connected to the corresponding reaction pools 123, and one or more air holes 126 penetrating the upper shell 120 and connected to the corresponding waste liquid pools 124.

[0065] The number of reaction pools 123 is equal to the number of microwell arrays 112 and their positions correspond. The number of injection holes 125 and waste liquid pools 124 are both equal to the number of reaction pools 123 . The number of vent holes 126 is equal to or greater than the number of waste liquid pools 124 .

[0066] Each waste liquid pool 124 extends from the inner surface of the upper housing 120 toward the outside of the microfluidic chip 100 , wherein the end close to the inner surface is the bottom, and the end away from the inner surface is the top.

[0067] like Figure 5B As shown, for each injection hole 125, it is connected to the corresponding reaction pool 123 via the guide groove 127A, and the corresponding reaction pool 123 is connected to the corresponding waste liquid pool 124 via the guide groove 127B. The reaction sample will enter from the injection port 125 and pass through the reaction pool 123 and the waste liquid pool 124 in sequence. The vent hole 126 is used to release the negative pressure generated during the injection. In order to increase the vent area, each waste liquid pool 124 can correspond to a plurality of vent holes 126.

[0068] A check valve 1211 is provided at one end of the guide groove 127B for connecting the reaction pool 123 and the waste liquid pool 124, near the waste liquid pool 124. As an example, the width of the guide groove 127B is 1.0 mm, and the depth is 0.6 mm, while the width of one end of the waste liquid pool 124 connected to the guide groove 127B is 18 mm, and the depth is 2 mm. The check valve 1211 is formed and acts by the sudden change in the size of the guide groove 127B and the waste liquid pool 124. The check valve 1211 can generate capillary resistance to prevent the fluid from flowing in, and can also prevent the waste liquid that has flowed to the waste liquid pool 124 from flowing back to the reaction pool 123.

[0069] like Figure 5C As shown, each waste liquid pool 124 is divided into two areas 124A and 124B in the flow direction of the reaction sample, and the bottoms of the two areas 124A and 124B are not connected to each other, and the tops are connected to each other. As an example, the two areas 124A and 124B are separated by a support rib 128, and the height of the support rib 128 is lower than the height of the waste liquid pool 124, so that the bottoms of the two areas 124A and 124B of the waste liquid pool 124 are not connected to each other, and the tops are connected to each other. The fluid in the reaction pool first enters the area 124A, and then enters the area 124B, and the waste liquid that has flowed into the area 124B is difficult to cross the support rib 128 and the area 124A and communicate with the guide groove 127B, thereby preventing the waste liquid from flowing back to the reaction pool 123. In the case where the upper shell 120 is circular or fan-shaped, the support rib 128 extends in the tangential direction, and the waste liquid pool 124 is divided into two areas 124A and 124B in the radial direction.

[0070] A water-absorbing film 170 is provided in each waste liquid pool 124. The water-absorbing film 170 can effectively absorb the waste liquid that has flowed into the waste liquid pool 124, and can prevent the waste liquid from forming a liquid film on the side wall of the waste liquid pool 124 and communicating with the guide groove 127B, thereby flowing back to the reaction pool 123 under the capillary action to cause pollution. The material of the water-absorbing film 170 can be cellulose acetate (NC), polyvinyl pyrrolidone (PVP) film, super absorbent resin polymerized from acrylic acid derivatives, etc., and has the functions of strong water absorption, good biocompatibility, and no chip shedding.

[0071] The support ribs 128 and the water-absorbing membrane 170 can be used in the waste liquid pool 124 alone or in combination. In the case of combined application, the water-absorbing membrane 170 can be arranged in the area 124B.

[0072] Back to Figure 1 , Figure 2 and Figure 5A The upper shell 120 may be circular in shape, and when the upper shell 120 includes a plurality of reaction pools 123, the plurality of reaction pools 123 are spaced apart from each other and extend in a radial direction, so that they can be used in conjunction with a centrifugal control device. Depending on the detection needs, the upper shell 120 may have different shapes (such as a fan shape, etc.), and the plurality of reaction pools 123 extend outward in a radial shape from the center (such as the center of a circle, etc.) of the upper shell 120. A plurality of injection holes 125 are arranged in a circle and close to the center of the upper shell 120, a plurality of waste liquid pools 124 are arranged in a circle and close to the outer periphery of the upper shell 120, a guide groove 127A connects the corresponding injection hole 125 and the reaction pool 123, a guide groove 127B connects the corresponding reaction pool 123 and the waste liquid pool 124, and one or more air holes 126 are arranged at the top of each waste liquid pool 124.

[0073] A through hole 121 penetrating the upper shell 120 is provided at the center of the upper shell 120, and the through hole 121 can be closely matched with the rotor (not shown) of the centrifugal control device. A semi-concave pressing platform 122 is provided on the outer surface of the upper shell 120 and near the through hole 121. The through hole 121 can be in the shape of an elongated strip, and the pressing platform 122 can be provided on both sides of the elongated through hole 121. The rotor of the centrifugal control device is provided with a loading component (not shown) adapted to the structure of the through hole 121 and the pressing platform 122, and the loading component is parallel to the structure of the through hole 121 to load or unload the chip, and is fixed and pressed when it is perpendicular to the structure of the through hole 121.

[0074] Continue as Figure 1 and Figure 2 As shown, and reference Figure 3 and Figure 5A The inner surface of the lower housing 110 and the inner surface of the upper housing 120 are opposite to each other and sealed to form one or more independent reaction units. Each reaction unit includes a microwell array 112, a reaction pool 123 above the microwell array 112, an injection hole 125 connected to the reaction pool 123 via a guide groove 127A, a waste liquid pool 124 connected to the reaction pool 123 via a guide groove 127B, and one or more air holes 126 at the top of the waste liquid pool 124. In the case where the microfluidic chip 100 is circular or fan-shaped, multiple independent reaction units are spaced apart from each other and each extends in a radial direction.

[0075] As an example, the inner surface of the lower shell 110 and the inner surface of the upper shell 120 are sealed and matched via the adhesive film 130. The inner surface of the lower shell 110 is opposite to the inner surface of the upper shell 120, the micropore array 112 of the lower shell 110 corresponds to the position of the reaction pool 123 of the upper shell 120, and the adhesive film 130 is provided with through holes at positions corresponding to the through holes 111, the guide grooves 127A and the reaction pool 123. As another example, the inner surface of the lower shell 110 and the inner surface of the upper shell 120 are sealed and matched via laser welding.

[0076] After the lower housing 110 and the upper housing 120 are sealed and matched, the microwell array 112 occupies a certain proportion of the area directly below the corresponding reaction pool 123. In one example, the microwell array 112 occupies 1%-100% of the area directly below the corresponding reaction pool 123. In another example, the microwell array 112 occupies 30%-100% of the area directly below the corresponding reaction pool 123. In yet another example, the microwell array 112 occupies 60%-100% of the area directly below the corresponding reaction pool 123.

[0077] Figure 6 1 is an assembly diagram of a reaction pool and a microwell array in a microfluidic chip according to an embodiment of the present invention.

[0078] like Figure 6 As described above, the length and width of the micropore array 112 are 17.5 mm and 5 mm, respectively, and the number of micropores 113 is 1,034,000. The length and width of the reaction pool 123 are 16.5 mm and 4 mm, respectively, and the volume is 40 μL. The length and width of the micropore array 112 are slightly larger than the length and width of the reaction pool 123, so after the lower shell 110 and the upper shell 120 are sealed together, the effective area of ​​the micropore array 112 is the area formed in the chamber of the reaction pool 123, that is, the effective area of ​​the micropore array 112 is 16.5 mm×4 mm (length×width), and the micropore array 112 occupies 100% of the area directly below the reaction pool 123.

[0079] 500,000 magnetic beads were dispersed in 45 μL solution, then added to the microfluidic chip 100, left to stand for 3 minutes, and magnetically attracted for 20 seconds. The excess magnetic bead solution in the reaction pool 123 was separated by centrifugation at 600 rpm, and the magnetic beads falling into the micropores 113 were retained therein. The number of magnetic beads falling into the micropores 113 was 233,909 by microscopic observation, and the loading rate (loading rate = number of magnetic beads falling into the micropores / number of magnetic beads added to the microfluidic chip 100) was 46.78%.

[0080] Since only the reaction samples located in the area directly above the microwell array 112 can fall into the microwell 113 for detection under the action of gravity sedimentation or magnetic attraction, when the absolute number of microwells remains unchanged, the larger the area directly below the corresponding reaction pool 123 occupied by the microwell array 112, the higher the sample loading rate and the higher the dynamic range in theory.

[0081] Depending on the detection requirements, the size of the microwell array 112 can be adjusted so that the microwell array 112 occupies less than 100% of the area directly below the corresponding reaction pool 123 .

[0082] Back to Figure 1 and Figure 2 , the upper shell 120 can be made of transparent material or translucent material. Additionally or alternatively, a detection window 129 can be opened on the outer surface of the upper shell 120 corresponding to the reaction pool 123. Through such an arrangement, the biochemical reaction can be observed from the outer surface of the upper shell 120, and the micropore array 112 can be imaged and detected. In addition, a zeroing mark 1210 with a groove structure is also provided at the periphery of the outer surface of the upper shell 120. The zeroing mark 1210 can be used as a position recognition area of ​​an external photoelectric recognition device. By identifying the position of the zeroing mark 1210, the R-axis position of the microfluidic chip 100 can be accurately positioned.

[0083] Figure 7 is a side view of a microfluidic chip according to one embodiment of the present invention.

[0084] like Figure 7 As shown, and reference Figure 1 and Figure 2 The microfluidic chip 100 further includes one or more sealing pads 140 disposed on the corresponding injection holes 125. The sealing pads 140 may be silicone pads, and the size of the sealing pads 140 is adapted to the injection holes 125. It is understood that the sealing pads 140 may be fixed by a sealant.

[0085] The microfluidic chip 100 further includes one or more guide grooves 150 disposed on the corresponding sealing gasket 140. The guide groove 150 is used to assist the external sample injection needle (not shown) to cooperate with the position of the injection hole 125, guide the sample injection needle, and ensure that the fluid can be added to the injection hole 125. It is understood that the guide groove 150 can be fixed to the outer surface of the upper housing 120 by barbs. The guide groove 150 can be a whole piece structure or a multi-piece independent structure, so as to be disposed on the sealing gasket 140.

[0086] like Figure 1 and Figure 2As shown, the microfluidic chip 100 also includes one or more breathable membranes 160 disposed on the corresponding breathable holes 126. The breathable membrane 160 isolates the breathable holes 126 from contact with the outside world to prevent foreign objects from entering the microfluidic chip 100 and contaminating the microfluidic chip 100. The breathable membrane 160 can be a nitrocellulose membrane, and the size of the breathable membrane 160 is adapted to the breathable holes 126. The breathable membrane 160 can be a whole piece structure or a multi-piece independent structure to be disposed on the breathable holes 126. It is understandable that the breathable membrane 160 can be further fixed by a sealant.

[0087] In the embodiment of the present invention, the microfluidic chip integrates the injection chamber, the reaction chamber and the waste liquid chamber, has a simple and compact structure, and has a low manufacturing cost. The reaction liquid is centrifuged into the liquid storage tank by centrifugation, and no additional negative pressure pump and waste liquid bucket and connecting joints and fluid pipelines are required. The microfluidic chip can be a disposable consumable. After the reaction is completed, the chip containing the waste liquid is stored in a specific area for unified treatment to prevent contamination.

[0088] Secondly, the waste liquid pool is divided into two areas, or a water-absorbing membrane is set in the waste liquid pool to prevent the waste liquid from flowing back into the reaction pool. In addition, the microwell array occupies most of the area directly below the corresponding reaction pool, and all samples in the reaction pool can participate in the detection. At the same time, the number of microwells increases, which increases the sample loading rate and improves the sensitivity and dynamic range of the detection. The microwell array and the lower shell of the microfluidic chip are integrally injection molded or spliced ​​and assembled into one. The integral injection molding not only ensures the flatness but also reduces the assembly process, while the splicing assembly has lower manufacturing requirements. In addition, the microfluidic chip can provide multiple independent reaction units, which improves the detection throughput.

[0089] Figure 8 4 is a flow chart of a method for operating a microfluidic chip according to an embodiment of the present invention.

[0090] like Figure 8 As shown, in step 81, one or more reaction samples are added to the corresponding injection holes in a single or batch manner. Since the microfluidic chip provides one or more independent reaction units, one or more reaction samples can be detected at the same time, thereby improving the detection throughput. Depending on the type and content of the detection, the reaction samples may be different. For example, for digital ELISA detection, the reaction sample may include a protein sample molecule to be detected and magnetic beads or microspheres, and for digital PCR detection, the reaction sample may include a nucleic acid sample molecule to be detected and a PCR reaction reagent. In addition, for digital ELISA detection, the reaction sample may also include a fluorescent substrate, which may be added to the injection hole together with the protein sample molecule to be detected and the magnetic beads or microspheres in a single time, or may be added to the injection hole in batches, for example, the protein sample molecule to be detected and the magnetic beads or microspheres are first added, and then the fluorescent substrate is added.

[0091] In step 82, each added reaction sample can be further sent into the corresponding reaction pool through the injection hole under the positive pressure of the sample injection needle and the connected external injection pump. After standing for a specific time (for example, 3 minutes), the reaction sample is settled into the micropores of the micropore array below the reaction pool. It is understandable that self-priming or centrifugal injection and other methods can also be used to allow each added reaction sample to enter the corresponding reaction pool and the corresponding micropore array.

[0092] In step 83, centrifugation is applied (e.g., 600 rpm for 10 seconds), at which speed, the excess reaction sample in the corresponding reaction pool enters the corresponding waste liquid pool through the guide groove, while the reaction sample in the micropores of the corresponding micropore array is retained. During the centrifugation process, the volume of the corresponding waste liquid cavity decreases, the gas pressure increases, and the gas pressure is released to the outside of the microfluidic chip through the corresponding air holes and the air permeable membrane.

[0093] In step 84, split oil is added to the corresponding injection hole. Depending on the type and content of the test, the split oil can be different. For example, for digital ELISA detection and digital PCR detection, the split oil can include fluorine oil or silicone oil with high viscosity coefficient.

[0094] In step 85, the split oil can also be further delivered to the corresponding reaction pool from the injection hole under the positive pressure of the sample injection needle and the connected external injection pump. The hydrophobic split oil can fully infiltrate the corresponding microwell array surface, further remove the reaction samples that have not fallen into the microwells, and at the same time isolate the reaction samples in the microwells from each other, and the fluorescent product is difficult to diffuse, and the thermal stability of a single microwell is excellent. It is understandable that self-priming or pressure injection can also be used to allow the split oil to enter the corresponding reaction pool to split the reaction samples in the corresponding microwell array.

[0095] In step 86, the reaction samples in the corresponding microwell array are waited for biochemical reaction. Depending on the type and content of the test, the waiting time and the required biochemical reaction temperature are different. For example, for digital ELISA detection, one may wait for 1 minute at room temperature, and for digital PCR detection, one may wait for 15 seconds at 95°C, 50 seconds at 60°C, and repeat 35 times.

[0096] In step 87, the corresponding microwell array is imaged and detected and digitally analyzed. An imaging system is used to capture the image of the microwell array, and the imaging system may include components such as a mercury lamp light source, a filter, an objective lens, and a CCD camera. The microfluidic chip can be fixed on a mobile platform, and the mobile platform moves the microfluidic chip so that the lens is aligned with different positions of the microwell array to capture images of multiple areas. During the image capture process, a precise autofocus platform can adjust the distance between the lens and the microwell to ensure the capture of the clearest image. A 20x objective lens can be used, and each microwell array can capture images of multiple fields of view (for example, for digital ELISA detection, 35 fields of view are captured, and for digital PCR detection, 256 fields of view are captured). For the image of each field of view, a fluorescent image (for example, 577nm excitation, 620nm emission, and exposure time 600ms) and a bright field image (for example, a mercury lamp light source, and exposure time 50ms) are obtained respectively. According to the fluorescent image and the bright field image, digital analysis is performed.

[0097] The microfluidic chip can be a disposable consumable. After the reaction is completed, the chip containing the waste liquid is stored in a specific area for unified treatment to prevent contamination. It is understandable that a cleaning liquid can be added to the injection hole, and the microfluidic chip is centrifuged so that the reaction sample after the reaction enters the waste liquid pool, thereby achieving the cleaning of the microfluidic chip and the harmless treatment of the waste liquid.

[0098] In an embodiment of the present invention, by selecting a microfluidic chip including a suitable microwell array and by adding a suitable reaction sample, the microfluidic chip can be operated to achieve different detections, such as digital ELISA detection and digital PCR detection.

[0099] According to one embodiment of the present invention, the microfluidic chip is used in a digital ELISA assay, and according to one embodiment of the present invention, the microfluidic chip is used in a digital PCR assay.

[0100] Although the present invention has been shown and described with reference to certain preferred embodiments, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention.

Claims

1. A microfluidic chip, characterized in that: The microfluidic chip comprises: a lower housing, the lower housing comprising one or more microwell arrays formed on an inner surface of the lower housing; An upper shell, the upper shell comprising one or more reaction pools formed on the inner surface of the upper shell and corresponding to the corresponding microwell arrays, one or more waste liquid pools formed on the inner surface of the upper shell and connected to the corresponding reaction pools, one or more injection holes penetrating the upper shell and connected to the corresponding reaction pools, and one or more air holes penetrating the upper shell and connected to the corresponding waste liquid pools; The inner surface of the lower shell and the inner surface of the upper shell are opposite to each other and sealed to form one or more independent reaction units.

2. The microfluidic chip according to claim 1, characterized in that: Each waste liquid pool is divided into two areas in the flow direction of the reaction sample, the bottoms of the two areas are not connected to each other, and the tops of the two areas are connected to each other.

3. The microfluidic chip according to claim 2, characterized in that: The two areas are separated by supporting ribs.

4. The microfluidic chip according to any one of claims 1 to 3, characterized in that: A water absorbing membrane is arranged in each waste liquid pool.

5. The microfluidic chip according to claim 1, characterized in that: The reaction tank and the corresponding waste liquid tank are connected to each other via a guide groove, and a check valve is arranged at one end of the guide groove close to the waste liquid tank.

6. The microfluidic chip according to claim 1, characterized in that: The microwell array occupies 1% to 100% of the area directly below the corresponding reaction pool.

7. The microfluidic chip according to claim 6, characterized in that: The microwell array occupies 30% to 100% of the area directly below the corresponding reaction pool.

8. The microfluidic chip according to claim 7, characterized in that: The microwell array occupies 60%-100% of the area directly below the corresponding reaction pool.

9. The microfluidic chip according to claim 1, characterized in that: The one or more micro-hole arrays and the lower shell are integrally formed by injection molding or splicing and assembly.

10. The microfluidic chip according to claim 1, characterized in that: The inner surface of the lower shell and the inner surface of the upper shell are sealed and matched via adhesive film or laser welding.

11. The microfluidic chip according to claim 1, characterized in that: The microfluidic chip further comprises one or more sealing pads disposed on the corresponding injection holes.

12. The microfluidic chip according to claim 11, characterized in that: The microfluidic chip further comprises one or more guide grooves arranged on the corresponding sealing gasket.

13. The microfluidic chip according to claim 1, characterized in that: The microfluidic chip further comprises one or more gas permeable membranes arranged on the corresponding gas permeable holes.

14. The microfluidic chip according to claim 1, characterized in that: The lower shell and the upper shell are circular or fan-shaped, and the plurality of independent reaction units are spaced apart from each other and each extends along a radial direction.

15. The microfluidic chip according to claim 14, characterized in that: A through hole is provided at the center of the lower shell and the upper shell, and a pressing platform is provided on the outer surface of the upper shell and close to the through hole.

16. The microfluidic chip according to claim 14, characterized in that: A zero return mark is arranged at the periphery of the outer surface of the upper shell.

17. A method for operating a microfluidic chip according to any one of claims 1 to 16, characterized in that: The operation method comprises: Add one or more reaction samples into corresponding injection holes in a single or batch manner; The reaction sample added each time enters the corresponding reaction pool and the corresponding microwell array by self-absorption, centrifugation or pressure injection; Centrifuging the microfluidic chip so that the reaction samples enter the corresponding waste liquid pool from the corresponding reaction pool, while the reaction samples in the corresponding microwell array remain therein; Add the split oil into the corresponding injection hole; The split oil enters the corresponding reaction pool by self-absorption, centrifugation or pressure injection to split the reaction sample in the corresponding microwell array; Waiting for the reaction sample in the corresponding microwell array to perform a biochemical reaction; The corresponding microwell arrays are imaged, detected and digitally analyzed.

18. Use of the microfluidic chip according to any one of claims 1 to 16 in digital ELISA detection.

19. Use of the microfluidic chip according to any one of claims 1 to 16 in digital PCR detection.

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