Integrated micro-droplet chip

Abstract

The present invention provides an integrated micro-droplet chip, including a chip body, which is provided with a reaction chamber, a sample adding chamber, a micro-droplet generation structure, an oil-liquid interface, a gas-liquid interface and a fluorescence detection area; when droplets are generated, a pressure difference is formed between the sample adding chamber and the gas-liquid interface and between the oil-liquid interface and the gas-liquid interface, and the pressure difference drives the sample in the sample adding chamber and the generated oil of the oil-liquid interface to enter the micro-droplet generation structure to generate micro-droplets and enter the reaction chamber; when droplets are detected, external pressure drives the detection to push the oil into the reaction chamber, so that the micro-droplets flow out of the reaction chamber to the micro-droplet generation structure, and the external pressure drives the detection separation oil to enter the micro-droplet generation structure, and the detection separation oil will flow out of the reaction chamber to the micro-droplet separation in the micro-droplet generation structure to form a queue and enter the fluorescence detection area. The present invention integrates droplet generation, amplification and detection in one chip through time-sharing multiplexing of the micro-droplet generation structure, thereby realizing a fully integrated and fully enclosed digital PCR process.

Description

Technical Field

[0001] The invention belongs to the technical field of digital PCR analyzers, and in particular relates to an integrated micro-droplet chip. Background Art

[0002] Droplet-based microfluidics is a technology platform for manipulating tiny volumes of liquids developed on microfluidic chips in recent years. Its principle is: two immiscible liquids, one of which is an oil phase and the other is an aqueous phase, enter the microchannel at the same time. Under the action of the microchannel, the aqueous phase is distributed in the oil phase in the form of tiny volume units, forming a series of discrete microdroplets. Each droplet acts as a microreactor to complete a set of chemical or biological reactions.

[0003] Digital PCR technology is known as the third generation of PCR technology. It has the advantages of absolute quantification and single-molecule detection sensitivity, and has important application prospects in the field of molecular diagnosis. A mainstream technical route of digital PCR technology uses droplet microfluidic chips to divide the reaction system into tens of thousands or even millions of droplets of uniform size, and completes generation, amplification and fluorescence detection. The exact copy number of the target molecule in the sample is calculated using a mathematical model based on the fluorescence detection results.

[0004] In droplet digital PCR technology, the structure in the droplet generation chip is often used to complete the droplet generation, and then the droplets are transferred to the reaction tube for amplification. Finally, the droplets are formed into a droplet queue with a certain interval using the structure in the droplet detection chip. Each droplet passes through the fluorescence detection area in turn to stimulate and detect the fluorescence signal in the droplet. This method of using droplet microfluidics to achieve digital PCR has the advantages of uniform droplet size, less restricted droplet number, and high fluorescence detection signal-to-noise ratio. However, it also has the disadvantages of complex chip structure, generation and detection completed in different chips, low integration, and difficulty in automation. Summary of the invention

[0005] Therefore, the technical problem to be solved by the present invention is to provide an integrated micro-droplet chip to overcome the shortcomings of the prior art that droplet generation and detection are completed in different droplet chips, resulting in low integration and automation.

[0006] In order to solve the above problems, the present invention provides an integrated micro-droplet chip, including a chip body, the chip body having a reaction chamber and a sample adding chamber, the chip body is configured with a micro-droplet generation structure, an oil-liquid interface, a gas-liquid interface and a fluorescence detection area, the gas-liquid interface is connected to the reaction chamber, the sample adding chamber is connected to the micro-droplet generation structure, and the oil-liquid interface is connected to the micro-droplet generation structure;

[0007] When droplets are generated, a first pressure difference is formed between the sample adding chamber and the gas-liquid interface, and a second pressure difference is formed between the oil-liquid interface and the gas-liquid interface. The first pressure difference and the second pressure difference respectively drive the sample in the sample adding chamber and the generated oil of the oil-liquid interface to enter the micro-droplet generation structure, and the generated micro-droplets enter and are stored in the reaction chamber;

[0008] During droplet detection, external pressure drives the detection oil to enter the reaction chamber from the gas-liquid interface, so that the micro-droplets in the reaction chamber flow out of the reaction chamber to the micro-droplet generation structure, and external pressure drives the detection separation oil to enter the micro-droplet generation structure from the oil-liquid interface. The detection separation oil separates the micro-droplets flowing out of the reaction chamber to the micro-droplet generation structure into a queue and enters the fluorescence detection area.

[0009] Preferably, the micro-droplet generation structure includes an oil pipeline and a connecting pipeline, the oil pipeline and the connecting pipeline form a cross, the connecting pipeline includes a first pipeline located on the first side of the cross intersection and connected to the reaction chamber and a second pipeline located on the second side of the cross intersection and connected to the sample addition chamber, and the oil interface is connected to the oil pipeline.

[0010] Preferably, with the first side surface of the chip body being in a horizontal position as a reference, the reaction chamber and the sample loading chamber are located on the first side surface, and the connection interface between the reaction chamber and the first side surface of the chip body extends upward and forms a trumpet mouth that is smaller at the bottom and larger at the top.

[0011] Preferably, a gas-liquid pipeline extending from bottom to top is also constructed in the reaction chamber, the lower end of the gas-liquid pipeline is communicated with the gas-liquid interface, and the upper end of the gas-liquid pipeline is higher than the upper end of the connection interface.

[0012] Preferably, a micro-droplet observation area is provided between the first pipe and the connection interface.

[0013] Preferably, the sample adding chamber comprises an open chamber and a sealing cover sealedly connected to the opening of the open chamber.

[0014] Preferably, the sample adding chamber is provided with a filter membrane or an exhaust hole.

[0015] Preferably, the fluorescence detection area is located on the second channel.

[0016] Preferably, the sample adding chamber is located on the first side surface, the reaction chamber is located on the second side surface, and the second side surface and the first side surface are two opposite sides of the chip body.

[0017] Preferably, before the micro-droplets enter the reaction chamber, light oil is pre-placed in the reaction chamber.

[0018] The present invention provides an integrated micro-droplet chip, wherein a sample addition chamber, a reaction chamber, a micro-droplet generation structure and a fluorescence detection area are integrated on the chip body. By time-sharing multiplexing of the micro-droplet generation structure, droplet generation, amplification and detection are integrated into one chip, thereby realizing a fully integrated and fully closed digital PCR process. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 Schematic diagram of the three-dimensional structure of the integrated micro-droplet chip according to an embodiment of the present invention;

[0020] Figure 2 It is a structural schematic diagram of a micro-droplet generation structure in an integrated micro-droplet chip according to an embodiment of the present invention;

[0021] Figure 3 Schematic diagram of the internal structure of the reaction chamber in the integrated micro-droplet chip according to an embodiment of the present invention;

[0022] Figure 4 Schematic diagram of the microdroplet generation process;

[0023] Figure 5 Schematic diagram of microdroplets being stored in a reaction chamber after being generated;

[0024] Figure 6 This is a schematic diagram of the state inside the reaction chamber after the integrated micro-droplet chip is flipped 180°;

[0025] Figure 7 for Figure 6 The state after the oil is introduced into the reaction chamber is shown;

[0026] Figure 8 The diagram is a schematic diagram of the state of the micro-droplets after being forced out of the reaction chamber (the arrows in the figure show the flow direction of the micro-droplets and the oil);

[0027] Fig. 9 FIG. 4 is a schematic diagram of the three-dimensional structure of an integrated micro-droplet chip according to another embodiment of the present invention.

[0028] The reference numerals are:

[0029] 1. Chip body; 11. Reaction chamber; 111. Connection interface; 112. Gas-liquid pipeline; 12. Sample adding chamber; 121. Opening chamber; 122. Sealing cover; 21. Oil-liquid pipeline; 22. First pipeline; 23. Second pipeline; 31. Oil-liquid interface; 32. Gas-liquid interface; 33. Fluorescence detection area; 34. Micro-droplet observation area; 4. Micro-droplet; 5. Detection driving oil. DETAILED DESCRIPTION

[0030] See also Figures 1 to 9As shown, according to an embodiment of the present invention, an integrated micro-droplet chip is provided, comprising a chip body 1, wherein the chip body 1 has a reaction chamber 11 and a sample adding chamber 12, wherein a micro-droplet generating structure, an oil-liquid interface 31, a gas-liquid interface 32 and a fluorescence detection area 33 are constructed in the chip body 1, wherein the gas-liquid interface 32 is communicated with the reaction chamber 11, the sample adding chamber 12 is communicated with the micro-droplet generating structure, and the oil-liquid interface 31 is communicated with the micro-droplet generating structure;

[0031] When droplets are generated, a first pressure difference is formed between the sample adding chamber 12 and the gas-liquid interface 32, and a second pressure difference is formed between the oil-liquid interface 31 and the gas-liquid interface 32. The first pressure difference and the second pressure difference respectively drive the sample in the sample adding chamber 12 and the generated oil of the oil-liquid interface 31 to enter the micro-droplet generation structure, and the generated micro-droplets 4 enter and are stored in the reaction chamber 11.

[0032] During droplet detection, external pressure drives the detection oil 5 to enter the reaction chamber 11 from the gas-liquid interface 32, so that the micro-droplets 4 in the reaction chamber 11 flow out of the reaction chamber 11 to the micro-droplet generation structure, and external pressure drives the detection separation oil to enter the micro-droplet generation structure from the oil-liquid interface 31. The detection separation oil separates the micro-droplets 4 flowing out of the reaction chamber 11 to the micro-droplet generation structure into a queue and enters the fluorescence detection area 33.

[0033] In this technical solution, the chip body 1 is integrated with a sample loading chamber 12, a reaction chamber 11, a micro-droplet generation structure and a fluorescence detection area 33, so that the generation and detection of the micro-droplets are integrated on the same chip, and the degree of integration and automation can be improved; more importantly, through time-sharing multiplexing of the micro-droplet generation structure (with the flipping of the integrated micro-droplet chip as the time-sharing boundary), droplet generation, amplification and detection are integrated into one chip, realizing a fully integrated and fully closed digital PCR process, which not only inherits the advantages of uniform droplet size, unrestricted droplet number, and high fluorescence detection signal-to-noise ratio, but also overcomes the difficulties of the original chip structure being complex, generation and detection being completed in different chips, low integration, and difficulty in automation. It is an important technical breakthrough in the field of digital PCR.

[0034] As a specific implementation of the micro-droplet generation structure, the micro-droplet generation structure includes an oil pipeline 21 and a connecting pipeline, the oil pipeline 21 and the connecting pipeline form a cross, the connecting pipeline includes a first pipeline 22 located on the first side of the cross intersection and connected to the reaction chamber 11 and a second pipeline 23 located on the second side of the cross intersection and connected to the sample addition chamber 12, and the oil interface 31 is connected to the oil pipeline 21.

[0035] In some embodiments, see Figure 1 As shown, with the first side of the chip body 1 in a horizontal position as a reference, the reaction chamber 11 and the sample loading chamber 12 are on the first side, and the connection interface 111 between the reaction chamber 11 and the first side of the chip body 1 extends upward and forms a trumpet mouth with a small bottom and a large top. The connection interface 111 forming a trumpet mouth can facilitate the micro-droplets 4 to enter the reaction chamber 11 from the first pipe 22, and also facilitate the micro-droplets 4 to enter the first pipe 22 from the reaction chamber 11, thereby preventing the micro-droplets 4 from being detained. It should be noted that at this time, the reaction chamber 11 and the sample loading chamber 12 are both on the first side (specifically the top surface) of the chip body 1, and the micro-droplets 4 entering the reaction chamber 11 are all gathered at the connection interface 111. When performing PCR amplification, the chip body 1 needs to be inverted as a whole, that is, flipped 180°, so that the micro-droplets 4 can be in the reaction area of ​​the reaction chamber 11.

[0036] In some embodiments, a gas-liquid pipeline 112 extending from bottom to top is further constructed in the reaction chamber 11, and the lower end of the gas-liquid pipeline 112 is connected to the gas-liquid interface 32, and the upper end of the gas-liquid pipeline 112 is higher than the upper end of the connection interface 111. This can prevent the micro-droplets 4 generated by the micro-droplet generation structure from further flowing out of the gas-liquid pipeline 112 after entering the reaction chamber 11 when the reaction chamber 11 is under negative pressure.

[0037] In some embodiments, a micro-droplet observation area 34 is provided between the first pipe 22 and the connection interface 111, and the flow area of ​​the micro-droplet observation area 34 is much larger than the flow area of ​​the first pipe 22, that is, the micro-droplet observation area 34 is an area enlarged on the first pipe 22 (the width becomes larger), so that the flow rate of the micro-droplets 4 entering the area is reduced, which can facilitate external camera imaging, record the droplet morphology, and determine whether the state of the droplet generation process is normal.

[0038] As a specific implementation, the sample loading chamber 12 includes an open chamber 121 and a sealing cover 122 sealed to the opening of the open chamber 121, so that the operator can add the sample into the sample loading chamber 12. Furthermore, the sample loading chamber 12 is provided with a filter membrane or an exhaust hole, and when the sample loading chamber 12 becomes a waste liquid pool (that is, when the droplet chip is turned upside down), a certain amount of air is exhausted to prevent pressure accumulation in the sample loading chamber 12.

[0039] In one embodiment, the fluorescence detection area 33 is located on the second pipe 23. On the second pipe 23, the micro-droplets 4 flowing out of the reaction chamber 11 can be separated into droplet queues with appropriate spacing under the action of the detection oil in the oil pipe 21 when passing through the cross point, thereby completing the fluorescence detection under the action of the external system.

[0040] like Fig. 9 As shown, another implementation of the integrated micro-droplet chip is given. Figure 1 The difference of the integrated micro-droplet chip shown is that the reaction chamber 11 and the sample addition chamber 12 are respectively located on two opposite sides of the chip body 1. Specifically, the sample addition chamber 12 is located on the first side, and the reaction chamber 11 is located on the second side. The second side and the first side are opposite sides of the chip body 1. At this time, the working principle and process of the integrated micro-droplet chip are basically the same as the integrated micro-droplet chip mentioned above. The difference is that during the droplet generation process, since the reaction chamber 11 is located on the bottom side of the chip body 1 (the sample addition chamber 12 is on the top side), the micro-droplet 4 will directly fall into the reaction area at the bottom of the reaction chamber 11 when entering the reaction chamber 11, and be collected in the reaction area. Therefore, after the droplet generation is completed, the integrated micro-droplet chip does not need to be flipped 180° and can directly enter the subsequent amplification link.

[0041] In some embodiments, before the micro-droplets 4 enter the reaction chamber 11, light oil (i.e., oil with a lower density) is pre-placed in the reaction chamber 11 to ensure that the light oil can always be on the top of the micro-droplets 4 in the reaction chamber 11, thereby solving the problem of micro-droplet evaporation during amplification and realizing heat-cover-free PCR.

[0042] According to an embodiment of the present invention, a digital PCR method for an integrated micro-droplet chip is also provided. The integrated micro-droplet chip is as described above, comprising a chip body 1, wherein the chip body 1 has a reaction chamber 11 and a sample addition chamber 12, wherein a micro-droplet generation structure, an oil-liquid interface 31, a gas-liquid interface 32 and a fluorescence detection area 33 are constructed in the chip body 1, wherein the gas-liquid interface 32 is connected to the reaction chamber 11, the sample addition chamber 12 is connected to the micro-droplet generation structure, and the oil-liquid interface 31 is connected to the micro-droplet generation structure.

[0043] The digital PCR method comprises the following steps:

[0044] The step of segmenting and generating micro-droplets is to control the formation of a first pressure difference between the sample adding chamber 12 and the gas-liquid interface 32, and a second pressure difference between the oil-liquid interface 31 and the gas-liquid interface 32, so that the first pressure difference and the second pressure difference respectively drive the sample in the sample adding chamber 12 and the generated oil of the oil-liquid interface 31 to enter the micro-droplet generating structure, and the generated micro-droplets 4 enter and are stored in the reaction chamber 11. Specifically, oil is provided to the oil-liquid interface 31, and negative pressure is provided to the gas-liquid interface 32. Under the action of the negative pressure, the sample in the sample adding chamber 12 The sample and the oil at the oil interface 31 are driven to converge along the second pipe 23 and the oil pipe 21 to the cross intersection of the micro-droplet generation structure. Under the action of the shear force and surface tension of the oil fluid, the sample forms micro-droplets 4 (water-in-oil droplets) of uniform size, and finally, under the action of the negative pressure, the micro-droplets 4 enter the reaction chamber 11 through the first pipe 22 for storage. It should be noted that when the micro-droplets enter the micro-droplet observation area 34, the flow rate of the micro-droplets 4 forms a dense droplet community, which is convenient for camera imaging and recording;

[0045] In the amplification reaction step, the reaction chamber 11 is placed in a heating module (not shown) and amplified by heating according to a preset cycle. The heating module can be an existing heating module.

[0046] The micro-droplet detection step is to control the external pressure to drive the detection oil 5 to enter the reaction chamber 11 from the gas-liquid interface 32, so that the micro-droplets 4 in the reaction chamber 11 flow out of the reaction chamber 11 to the micro-droplet generation structure, and the external pressure drives the detection separation oil to enter the micro-droplet generation structure from the oil-liquid interface 31. The detection separation oil separates the micro-droplets 4 flowing out of the reaction chamber 11 to the micro-droplet generation structure into a queue, and enters the fluorescence detection area 33 to complete the fluorescence detection. Specifically, the gas-liquid interface 32 Oil (i.e., detection driving oil 5, also called floating oil) is provided, and the micro-droplets 4 after the amplification reaction in the reaction chamber 11 are floated by the buoyancy of the oil. Under the buoyancy of the oil, the micro-droplets 4 can flow out of the reaction chamber 11 through the connection interface 111 and enter the first pipe 22, and flow through the micro-droplet observation area 34 to enter the cross intersection, and enter the second pipe 23, and are detected in the fluorescence detection area 33, and then finally enter the sample addition chamber 12. At this time, the sample addition chamber 12 is a waste liquid pool.

[0047] In this technical solution, droplet generation, amplification and detection are integrated into one chip through time-sharing multiplexing of the micro-droplet generation structure (with the flipping of the integrated micro-droplet chip as the time-sharing boundary), thereby realizing a fully integrated and fully closed digital PCR process. This not only inherits the advantages of uniform droplet size, unrestricted droplet number, and high fluorescence detection signal-to-noise ratio, but also overcomes the difficulties of the original chip structure being complex, generation and detection being completed in different chips, low integration, and difficulty in automation. This is an important technological breakthrough in the field of digital PCR.

[0048] In some embodiments, with the first side of the chip body 1 being in a horizontal position as a reference, the reaction chamber 11 and the sample loading chamber 12 are on the first side, and before the amplification reaction step and after the micro-droplet segmentation generation step, it also includes: a chip flipping step, controlling the chip body 1 to flip up and down 180°, at this time, the micro-droplets 4 in the reaction chamber 11 are flipped from the side close to the connection interface 111 to the side away from the connection interface 111, and the position of the reaction chamber 11 corresponding to the micro-droplet 4 is the reaction zone of the reaction chamber 11, and the reaction zone is in contact with the heating module for temperature adjustment to realize the temperature regulation reaction of the sample.

[0049] The following combination Figures 1 to 8 The operation process of the integrated micro-droplet chip of the present invention is further described as follows:

[0050] First, 30 microliters of the system (i.e., the aforementioned sample) is added to the sample chamber 12. The 30 microliters of PCR system includes 10 microliters of Bio-Rad's ddPCR Supermix for Probes, 5 microliters of GJB2 gene upstream and downstream primer reagents, and 5 microliters of template containing 1 ng of genomic DNA. Figure 1 As shown, a chip includes 8 parallel independent droplet chip structures, each of which includes a sample addition chamber 12, an oil-liquid interface 31, a gas-liquid interface 32, a micro-droplet generation structure, and a reaction chamber 11.

[0051] Then, the sealing cover 122 is tightly closed or bonded to seal the sample adding chamber 12. The sample adding chamber 12 is preferably provided with a filter membrane or a small-caliber exhaust hole.

[0052] Then, the Bio-Rad Generation Oil required for droplet generation is provided to the oil-liquid interface 31, and the generated oil contains a surfactant that can stabilize the droplets; a negative pressure of -200 mBar is provided to the gas-liquid interface 32, so that a pressure difference is formed between the sample addition chamber 12 and the oil-liquid interface 31. Among them, the sample addition chamber 12 is connected to the second pipeline 23 in the micro-droplet generation structure, the oil-liquid interface 31 is connected to the oil-liquid pipeline 21 in the micro-droplet generation structure, and the first pipeline 22 and the gas-liquid interface 32 are both connected to the reaction chamber 11, wherein the first pipeline 22 is connected to the connection interface 111, and the gas-liquid interface 32 is connected to the gas-liquid pipeline 112. The various parts of the micro-droplet generation structure are as follows Figure 2 As shown, the oil pipeline 21 has two branches, which are located on both sides of the second pipeline 23 and the first pipeline 22, and are both connected to the oil interface 31. The first pipeline 22 may include a micro-droplet observation area 34. The pipeline in the observation area becomes wider, and the flow rate of the droplets decreases after entering, which can facilitate external camera imaging, record the droplet morphology, and determine whether the state of the droplet generation process is normal.

[0053] Driven by the pressure difference, the reaction system enters the second pipe 23, and the generated oil enters the oil pipe 21 and intersects at the cross structure (i.e., the aforementioned cross intersection). Under the action of fluid shear force and surface tension, uniform-sized oil-in-water micro-droplets 4 are formed. The pipe depth at the cross is about 70 microns, the width is 80 microns, and the droplet size is about 100 microns. The micro-droplets 4 enter the first pipe 22, and after entering the micro-droplet observation area 34, the flow rate is reduced to form a dense droplet community, which is convenient for camera imaging and recording. The schematic diagram of the droplet generation process is shown in the figure. Figure 4 shown.

[0054] The generated droplets flow through the first pipe 22 and reach the connection interface 111 of the reaction chamber 11. The bottom of the connection interface 111 has a slope structure (i.e., the aforementioned bell mouth), and the bottom of the slope is connected to the first pipe 22. When the droplet generation process is completed, the pressure difference applied to the chip interface is removed, and the droplets should still be located below the gas-liquid pipe 112.

[0055] Afterwards, the chip is turned upside down to transfer the droplets from the connection interface 111 to the reaction area (ie, away from the connection interface 111). Figure 5As shown. The structure of the reaction zone should be designed with high heat transfer efficiency, such as a flat design with high depth and thin thickness, so that the external system (i.e., the heating module) can heat and cool the reaction zone from the left and right sides, which not only ensures a short distance for temperature conduction, but also ensures a large contact area, thereby achieving efficient heat transfer. In this embodiment, the temperature cycle process is to first perform a pre-denaturation at 95°C for 10 minutes, followed by 40 temperature cycles, with 95°C for 5 seconds, 60°C for 15 seconds, and finally 4°C insulation in each cycle. In order to reduce evaporation, 30 microliters of low-density anti-volatile reagent can be placed in the reaction chamber 11 in advance.

[0056] After the temperature cycle is completed, the amplification reaction in the droplet containing the template is also completed accordingly, and the droplet fluorescence detection stage needs to be entered. The detection oil (that is, the detection driving oil 5 mentioned above) is injected into the gas-liquid interface 32 to continuously fill the reaction chamber 11. In this process, the liquid level of the droplet continues to rise, and then is guided by the slope of the connection interface 111 and enters the first pipeline 22. This process is as follows Figure 7 shown.

[0057] When the droplets pass through the observation area in the first pipe 22, the camera can also be used to perform bright field imaging of the droplets, so as to evaluate the state of the droplets after the amplification reaction. At the same time, the detection oil is injected into the oil interface 31. The detection oil passes through the oil pipe 21 and merges with the droplet queue at the cross pipe, and separates the closely arranged droplets into droplet queues with appropriate spacing. The droplet queues pass through the fluorescence detection area 33 located in the second pipe 23 in turn, such as Figure 8 As shown. The position corresponding to the fluorescence detection area 33 is the fluorescence detection focus of the external system. The external system focuses the excitation light, such as laser or LED narrow-band light with wavelengths of 488nm and 532nm, to the detection focus. As the droplets pass through the detection focus in sequence, the fluorescence excited in the droplets will also be received by the collection light path of the external system, thereby obtaining the fluorescence information of each droplet. Using the fluorescence information of the droplets, the signal threshold is defined to distinguish the positive and negative nature of the droplets, and the Poisson distribution model is used to calculate the copy number of the target molecule in the sample.

[0058] Finally, the droplets that have completed the fluorescence detection enter the sample loading chamber 12. Since the sample loading chamber 12 has been sealed by the sealing cover 122, it will not come into contact with the environment outside the chip, eliminating the possibility of aerosol contamination and realizing a fully closed digital PCR process.

[0059] In this embodiment, a method of time-division multiplexing of the micro-droplet generation structure is pioneered. When generating droplets, the micro-droplet generation structure is used to achieve droplet generation. When detecting droplet fluorescence, the micro-droplet generation structure completes the separation of the droplet queue, ensuring the detection of droplet fluorescence signals. Using this time-division multiplexing method, a fully integrated and fully closed digital PCR process of droplet generation, amplification, and detection is realized on a single chip for the first time in the flow-like digital PCR technology route, which is an important technological breakthrough in the field of digital PCR.

[0060] In another embodiment, when the droplets pass through the observation area in the first pipe 22, a camera is used to perform fluorescence imaging of the droplets, and the imaged photos are analyzed to obtain fluorescence intensity information of each droplet in the photo, thereby completing the droplet fluorescence signal detection.

[0061] It is easy for those skilled in the art to understand that, under the premise of no conflict, the above-mentioned advantageous methods can be freely combined and superimposed.

[0062] The above are only preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention shall be included in the protection scope of the present invention. The above are only preferred embodiments of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and variations can be made without departing from the technical principles of the present invention, and these improvements and variations should also be regarded as the protection scope of the present invention.

Claims

1. An integrated micro-droplet chip, characterized in that: The chip body (1) comprises a reaction chamber (11) and a sample adding chamber (12), the chip body (1) having a micro-droplet generation structure, an oil-liquid interface (31), a gas-liquid interface (32) and a fluorescence detection area (33), the gas-liquid interface (32) being in communication with the reaction chamber (11), the sample adding chamber (12) being in communication with the micro-droplet generation structure, and the oil-liquid interface (31) being in communication with the micro-droplet generation structure; When droplets are generated, a first pressure difference is formed between the sample adding chamber (12) and the gas-liquid interface (32), and a second pressure difference is formed between the oil-liquid interface (31) and the gas-liquid interface (32), the first pressure difference and the second pressure difference respectively drive the sample in the sample adding chamber (12) and the generated oil in the oil-liquid interface (31) to enter the micro-droplet generation structure, and the generated micro-droplets (4) enter and are stored in the reaction chamber (11); During droplet detection, external pressure drives the detection pushing oil (5) to enter the reaction chamber (11) from the gas-liquid interface (32), so that the micro-droplets (4) in the reaction chamber (11) flow out of the reaction chamber (11) to the micro-droplet generation structure, and external pressure drives the detection separation oil to enter the micro-droplet generation structure from the oil-liquid interface (31), and the detection separation oil separates the micro-droplets (4) flowing out of the reaction chamber (11) to the micro-droplet generation structure into a queue, and enters the fluorescence detection area (33); The micro-droplet generation structure comprises an oil pipeline (21) and a connecting pipeline, wherein the oil pipeline (21) and the connecting pipeline form a cross, and the connecting pipeline comprises a first pipeline (22) located at a first side of the cross point and connected to the reaction chamber (11) and a second pipeline (23) located at a second side of the cross point and connected to the sample addition chamber (12), and the oil interface (31) is connected to the oil pipeline (21); with the first side surface of the chip body (1) being in a horizontal orientation as a reference, the reaction chamber (11) and the sample addition chamber (12) are located on the first side surface, with the first side surface of the chip body (1) being in a horizontal orientation as a reference, the reaction chamber (11) and the sample addition chamber (12) are located on the first side surface, and the connection interface (111) between the reaction chamber (11) and the first side surface of the chip body (1) extends upward; A gas-liquid pipeline (112) extending from bottom to top is also constructed in the reaction chamber (11), the lower end of the gas-liquid pipeline (112) is in communication with the gas-liquid interface (32), and the upper end of the gas-liquid pipeline (112) is higher than the upper end of the connection interface (111).

2. The integrated micro-droplet chip according to claim 1, characterized in that: A micro-droplet observation area (34) is provided between the first pipeline (22) and the connection interface (111).

3. The integrated micro-droplet chip according to claim 1, characterized in that: The sample adding chamber (12) comprises an open chamber (121) and a sealing cover (122) sealedly connected to the opening of the open chamber (121).

4. The integrated micro-droplet chip according to claim 3, characterized in that: The connection interface (111) extends upward to form a bell mouth that is smaller at the bottom and larger at the top.

5. The integrated micro-droplet chip according to claim 1, characterized in that: The sample adding chamber (12) is provided with a filter membrane or an exhaust hole.

6. The integrated micro-droplet chip according to claim 1, characterized in that: The fluorescence detection area (33) is located on the second pipe (23).

7. The integrated micro-droplet chip according to claim 1, characterized in that: The sample addition chamber (12) is located on the first side surface, and the reaction chamber (11) is located on the second side surface; the second side surface and the first side surface are two opposite sides of the chip body (1).

8. The integrated micro-droplet chip according to claim 1, characterized in that: Before the micro-droplets (4) enter the reaction chamber (11), light oil is pre-placed in the reaction chamber (11).

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