Integrated microdroplet chip

Abstract

The present invention provides an integrated micro-droplet chip. The integrated micro-droplet chip includes a chip body. The chip body has a reaction chamber, a sample addition chamber, a micro-droplet generating structure, an oil-liquid port, a gas-liquid port, and a fluorescence detection area. During droplet generation, a pressure difference is formed between the sample addition chamber and the gas-liquid port, and a pressure difference is formed between the oil-liquid port and the gas-liquid port. The pressure difference drives the sample in the sample addition chamber and the generated oil of the oil-liquid port to enter the micro-droplet generating structure, respectively, so that the micro-droplets are generated and enter the reaction chamber. During droplet detection, the external pressure drives the detection pushing oil to enter the reaction chamber, causing the micro-droplets to flow out from the reaction chamber to the micro-droplet generating structure, and the external pressure drives the detection separation oil to flow out to the micro-droplet generating structure, and the detection separation oil separates the micro-droplets flowing out from the reaction chamber into the micro-droplet generating structure to form a row and enter the fluorescence detection area. The present invention integrates droplet generation, amplification and detection all onto one chip through time division multiplexing of microdroplet generation structures, realizing a fully integrated and completely enclosed digital PCR process.

Description

[Technical Field]

[0001] The present invention relates to the technical field of digital PCR analysis devices, and more particularly to integrated micro-droplet chips. [Background technology]

[0002] Droplet-based microfluidics is a technology platform recently developed on microfluidics chips for controlling minute volumes of liquid. Its principle is as follows: When two incompatible liquids, for example, one oil phase and the other aqueous phase, enter a microchannel simultaneously, the aqueous phase is distributed into the oil phase in the form of minute volume units under the action of the microchannel, 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, known as the third-generation PCR technology, has the advantages of absolute quantification and single-molecule detection sensitivity, and is expected to be of important application in the field of molecular diagnostics. Digital PCR technology is the mainstream technology route, which uses droplet microfluidic chips to divide the reaction system into uniform droplets of tens of thousands to millions of sizes, and completes the generation, amplification, and fluorescence detection. According to the fluorescence detection results, a mathematical model is used to calculate the exact copy number of the target molecule in the sample.

[0004] In droplet digital PCR technology, droplets are generated using a chip-based structure, then transferred to a reaction tube for amplification. Finally, a droplet detection chip is used to form a droplet train at regular intervals. Each droplet passes through a fluorescent detection area in sequence, exciting and detecting the fluorescent signal within the droplet. This method of using droplet microfluidics to achieve digital PCR has advantages such as uniform droplet size, minimal droplet number limitations, and a high signal-to-noise ratio for fluorescent detection. However, it also has disadvantages such as a complex chip structure, generation and detection being completed on separate chips, low integration, and difficulty in automation. Summary of the Invention

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

[0006] To solve the above problems, the present invention provides an integrated micro-droplet chip, which includes a chip body having a reaction chamber and a sample addition chamber, a micro-droplet generation structure, an oil-liquid port, a gas-liquid port, and a fluorescence detection region configured within the chip body, the gas-liquid port communicating with the reaction chamber, the sample addition chamber communicating with the micro-droplet generation structure, and the oil-liquid port communicating with the micro-droplet generation structure.

[0007] When droplets are generated, a first pressure difference is formed between the sample addition chamber and the gas-liquid port, and a second pressure difference is formed between the oil-liquid port and the gas-liquid port. The first pressure difference and the second pressure difference drive the sample in the sample addition chamber and the generated oil in the oil-liquid port, respectively, to enter the microdroplet generation structure, and the generated microdroplets enter the reaction chamber and are stored.

[0008] During droplet detection, the external pressure drives the detection oil to enter the reaction chamber through the gas-liquid port, causing the microdroplets in the reaction chamber to flow out from the reaction chamber to the microdroplet generating structure, and the external pressure drives the detection separation oil to enter the microdroplet generating structure through the oil-liquid port, and the detection separation oil separates the microdroplets that have flowed out from the reaction chamber into the microdroplet generating structure to form a line, which then enters the fluorescence detection area.

[0009] Preferably, the microdroplet generating structure includes an oil-liquid conduit and a communicating conduit, the oil-liquid conduit and the communicating conduit intersect in a cross shape, the communicating conduit includes a first conduit located on a first side of the cross intersection and communicating with the reaction chamber, and a second conduit located on a second side of the cross intersection and communicating with the sample addition chamber, and the oil-liquid port communicates with the oil-liquid conduit.

[0010] Preferably, the first side of the chip body is positioned in a horizontal direction, the reaction chamber and the sample addition chamber are positioned on the first side, and the connection port between the reaction chamber and the first side of the chip body extends upward and forms a trumpet-shaped mouth that is small at the bottom and large at the top.

[0011] Preferably, a gas-liquid conduit extending from bottom to top is configured within the reaction chamber, the lower opening of the gas-liquid conduit communicates with the gas-liquid port, and the upper opening of the gas-liquid conduit is higher than the upper opening of the connection port.

[0012] Preferably, there is a microdroplet observation region between the first conduit and the connection port.

[0013] Preferably, the sample addition chamber includes an open chamber and a sealing cover hermetically connected to the opening of the open chamber.

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

[0015] Preferably, the fluorescence detection region is located in the second conduit.

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

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

[0018] The integrated microdroplet chip provided by the present invention has a sample addition chamber, a reaction chamber, a microdroplet generation structure, and a fluorescence detection area integrated into the chip body. Through time-division multiplexing of the microdroplet generation structure, droplet generation, amplification, and detection are all integrated into a single chip, realizing a fully integrated and fully sealed digital PCR process. [Brief explanation of the drawings]

[0019] [Figure 1] 1 is a schematic diagram of the three-dimensional structure of an integrated micro-droplet chip according to an embodiment of the present invention; [Figure 2] 1 is a structural schematic diagram of a micro-droplet generating structure in an integrated micro-droplet chip according to an embodiment of the present invention; [Figure 3] 1 is a schematic diagram showing the internal structure of a reaction chamber in an integrated micro-droplet chip according to an embodiment of the present invention; [Figure 4] FIG. 1 is a schematic diagram of a microdroplet generation process. [Figure 5] FIG. 1 is a schematic diagram of microdroplets being generated and then stored in a reaction chamber. [Figure 6] FIG. 1 is a schematic diagram of the state inside the reaction chamber after the integrated microdroplet chip is turned over by 180°. [Figure 7] FIG. 7 is a schematic diagram of the state after oil liquid has been introduced into the reaction chamber in the state shown in FIG. 6. [Figure 8] This is a schematic diagram of the state after the microdroplets are pushed out of the reaction chamber (the arrows in the figure indicate the flow direction of the microdroplets and oil liquid). [Figure 9] 1 is a schematic diagram of the three-dimensional structure of an integrated micro-droplet chip according to another embodiment of the present invention; DETAILED DESCRIPTION OF THE INVENTION

[0020] 1 to 9, an embodiment of the present invention provides an integrated micro-droplet chip. This integrated micro-droplet chip includes a chip body 1, which has a reaction chamber 11 and a sample addition chamber 12. The chip body 1 is configured with a micro-droplet generation structure, an oil / liquid port 31, a gas / liquid port 32, and a fluorescence detection region 33. The gas / liquid port 32 communicates with the reaction chamber 11, the sample addition chamber 12 communicates with the micro-droplet generation structure, and the oil / liquid port 31 communicates with the micro-droplet generation structure.

[0021] When droplets are generated, a first pressure difference is formed between the sample addition chamber 12 and the gas-liquid port 32, and a second pressure difference is formed between the oil-liquid port 31 and the gas-liquid port 32. The first pressure difference and the second pressure difference drive the sample in the sample addition chamber 12 and the generated oil in the oil-liquid port 31, respectively, to enter the microdroplet generation structure, and the generated microdroplets 4 enter the reaction chamber 11 and are stored.

[0022] When detecting droplets, external pressure drives the detection oil 5 to enter the reaction chamber 11 through the gas-liquid port 32, causing the microdroplets 4 in the reaction chamber 11 to flow out from the reaction chamber 11 to the microdroplet generation structure, and external pressure drives the detection separation oil to enter the microdroplet generation structure through the oil-liquid port 31. The detection separation oil separates the microdroplets 4 that have flowed out from the reaction chamber 11 into the microdroplet generation structure to form a line, and causes them to enter the fluorescence detection area 33.

[0023] This technical solution integrates the sample addition chamber 12, reaction chamber 11, droplet generation structure, and fluorescence detection region 33 into the chip body 1, allowing droplet generation and detection to be completed on the same chip, improving the degree of integration and automation. More importantly, time-division multiplexing of the droplet generation structure (using the time division boundaries before and after the flip of the integrated droplet chip) allows droplet generation, amplification, and detection to be integrated on a single chip, achieving a fully integrated and fully sealed digital PCR process. While retaining advantages such as uniform droplet size, fewer droplet number limitations, and a high fluorescence detection signal-to-noise ratio, it also overcomes the original challenges of complex chip structure, generation and detection completed on separate chips, resulting in low integration and difficulty in automation. This represents a significant technological advancement in the field of digital PCR.

[0024] In one specific embodiment of the microdroplet generation structure, the microdroplet generation structure includes an oil-liquid pipe line 21 and a connecting pipe line, the oil-liquid pipe line 21 and the connecting pipe line intersect in a cross shape, the connecting pipe line includes a first pipe line 22 located on a first side of the cross intersection and connecting with the reaction chamber 11, and a second pipe line 23 located on a second side of the cross intersection and connecting with the sample addition chamber 12, and the oil-liquid port 31 is connected with the oil-liquid pipe line 21.

[0025] 1, in the integrated micro-droplet chip, the first side surface of the chip body 1 is positioned horizontally, the reaction chamber 11 and the sample addition chamber 12 are positioned on the first side surface, and the connection port 111 between the reaction chamber 11 and the first side surface of the chip body 1 extends upward and forms a horn-shaped opening that is small at the bottom and large at the top. The horn-shaped connection port 111 can facilitate the entry of the micro-droplets 4 from the first conduit 22 into the reaction chamber 11 and can also facilitate the entry of the micro-droplets 4 from the reaction chamber 11 into the first conduit 22, preventing the micro-droplets 4 from stagnating. At this time, the reaction chamber 11 and the sample addition chamber 12 are both located on the first side (specifically, the top surface) of the chip body 1, and all the microdroplets 4 that enter the reaction chamber 11 are collected at the connection port 111. During PCR amplification, the entire chip body 1 needs to be turned upside down, i.e., flipped 180°, to position the microdroplets 4 in the reaction area of ​​the reaction chamber 11.

[0026] In some embodiments, a gas-liquid conduit 112 extending from bottom to top is further configured in the reaction chamber 11, the lower opening of the gas-liquid conduit 112 communicates with the gas-liquid port 32, and the upper opening of the gas-liquid conduit 112 is higher than the upper opening of the connection port 111. Thus, when the reaction chamber 11 is under negative pressure, the microdroplets 4 generated by the microdroplet generating structure can be prevented from flowing out of the gas-liquid conduit 112 after entering the reaction chamber 11.

[0027] In some embodiments, a microdroplet observation region 34 is located between the first conduit 22 and the connection port 111. The flow area of ​​the microdroplet observation region 34 is much larger than the flow area of ​​the first conduit 22. That is, the microdroplet observation region 34 is an enlarged (wide) region on the first conduit 22, which reduces the flow rate of the microdroplets 4 entering this region, making it easier to image the microdroplets 4 with an external camera, record the droplet morphology, and determine whether the droplet generation process is normal.

[0028] In a specific embodiment, the sample addition chamber 12 includes an open chamber 121 and a sealed cover 122 sealed to the opening of the open chamber 121, making it easy for an operator to add sample to the sample addition chamber 12. Furthermore, the sample addition chamber 12 is provided with a filter membrane or an exhaust hole. When the sample addition chamber 12 becomes a waste liquid reservoir (i.e., when the droplet chip is turned upside down), a certain amount of air is exhausted to prevent pressure buildup in the sample addition chamber 12.

[0029] In one embodiment, the fluorescence detection area 33 is located in the second pipeline 23, and in the second pipeline 23, the microdroplets 4 flowing out of the reaction chamber 11 can be separated into a series of droplets with appropriate spacing when passing through the cross-junction under the action of the detection oil in the oil-liquid pipeline 21, thereby completing fluorescence detection under the action of an external system.

[0030] As shown in FIG. 9, another embodiment of the integrated micro-droplet chip is proposed, which differs from the integrated micro-droplet chip shown in FIG. 1 in the following respects: The reaction chamber 11 and the sample addition chamber 12 are 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, with the second side and the first side being opposite sides of the chip body 1. In this case, the operating principle and process of the integrated micro-droplet chip are basically the same as those of the integrated micro-droplet chip described above, with the following differences: During the droplet generation process, the reaction chamber 11 is located on the bottom side of the chip body 1 (the sample addition chamber 12 is located on the top side). Therefore, when the micro-droplets 4 enter the reaction chamber 11, they fall directly into the reaction area at the bottom of the reaction chamber 11 and are collected in the reaction area. Therefore, the integrated micro-droplet chip does not need to be turned over 180° after droplet generation is completed, and can directly enter the subsequent amplification process.

[0031] In some embodiments, before the microdroplets 4 enter the reaction chamber 11, a light oil (i.e., an oil liquid with a relatively low density) is pre-placed in the reaction chamber 11 to ensure that the light oil is always located at the top of the microdroplets 4 in the reaction chamber 11, thereby solving the problem of evaporation of the microdroplets during amplification and realizing heat-free lid PCR.

[0032] An embodiment of the present invention further provides a digital PCR method for an integrated micro-droplet chip. As described above, the integrated micro-droplet chip includes a chip body 1. The chip body 1 has a reaction chamber 11 and a sample addition chamber 12. The chip body 1 is configured with a micro-droplet generation structure, an oil / liquid port 31, an air / liquid port 32, and a fluorescence detection region 33. The air / liquid port 32 communicates with the reaction chamber 11, the sample addition chamber 12 communicates with the micro-droplet generation structure, and the oil / liquid port 31 communicates with the micro-droplet generation structure.

[0033] The digital PCR method includes the following steps of dividing and generating a microdroplet, amplifying the microdroplet, and detecting the microdroplet. In the microdroplet division and generation step, a first pressure difference is formed between the sample addition chamber 12 and the gas-liquid port 32, and a second pressure difference is formed between the oil-liquid port 31 and the gas-liquid port 32. The first pressure difference and the second pressure difference drive the sample in the sample addition chamber 12 and the oil generated at the oil-liquid port 31 to enter the microdroplet generation structure, respectively, and the generated microdroplets 4 enter and are stored in the reaction chamber 11. Specifically, oil is supplied to the oil-liquid port 31, and negative pressure is applied to the gas-liquid port 32. Under the action of the negative pressure, the sample in the sample addition chamber 12 and the oil at the oil-liquid port 31 are driven along the second conduit 23 and the oil-liquid conduit 21, respectively, to collect at the cross-intersection port of the microdroplet generation structure. The sample forms uniformly sized microdroplets 4 (water-in-oil droplets) due to the action of the fluid shear force and surface tension of the oil liquid, and finally, due to the action of the negative pressure, the microdroplets 4 enter and are stored in the reaction chamber 11 via the first conduit 22. When the microdroplets enter the microdroplet observation region 34, they form a dense droplet community at the flow rate of the microdroplets 4, which makes it easy to image and record them with a camera. In the amplification reaction step, the reaction chamber 11 is placed in a heating module (not shown) to heat and amplify the reaction mixture according to a preset cycle, and an existing heating module can be used as the heating module. In the microdroplet detection step, the external pressure drives the detection oil 5 to enter the reaction chamber 11 through the gas-liquid port 32, thereby causing the microdroplets 4 in the reaction chamber 11 to flow out from the reaction chamber 11 to the microdroplet generation structure, and the external pressure drives the detection separation oil to enter the microdroplet generation structure through the oil-liquid port 31. The detection separation oil separates the microdroplets 4 that have flowed out from the reaction chamber 11 into the microdroplet generation structure to form a line, which then enters the fluorescence detection area 33, completing the fluorescence detection. Specifically, oil (i.e., detection push oil 5, also called floating oil) is supplied to the gas-liquid port 32, and the buoyancy of the oil causes the microdroplets 4 after the amplification reaction in the reaction chamber 11 to float. Furthermore, due to the action of the buoyancy of the oil, the microdroplets 4 flow out of the reaction chamber 11 through the connection port 111 and enter the first pipe line 22, pass through the microdroplet observation region 34, enter the cross-intersection port, enter the second pipe line 23, are detected in the fluorescence detection region 33, and finally enter the sample addition chamber 12. At this time, the sample addition chamber 12 becomes a waste liquid reservoir.

[0034] This technical solution utilizes time-division multiplexing of the droplet generation structure (the time division boundaries are before and after the flip of the integrated droplet chip), integrating droplet generation, amplification, and detection into a single chip, achieving a fully integrated and fully enclosed digital PCR process. It not only inherits the advantages of uniform droplet size, fewer droplet limitations, and a high fluorescence detection signal-to-noise ratio, but also overcomes the original challenges of complex chip structure, generation and detection completed on separate chips, low integration, and difficulty in automation. This represents a significant technological advancement in the field of digital PCR.

[0035] In some embodiments, the first side of the chip body 1 is positioned horizontally, and the reaction chamber 11 and the sample addition chamber 12 are positioned on this first side. A chip inversion step is further included before the amplification reaction step and after the droplet division and generation step. In the chip inversion step, the chip body 1 is controlled to be inverted 180° up and down. At this time, the droplets 4 in the reaction chamber 11 are inverted from the side closer to the connection port 111 to the side away from the connection port 111. At this time, the position of the reaction chamber 11 corresponding to the droplets 4 is the reaction region of the reaction chamber 11. The reaction region is in contact with the heating module to adjust the temperature, thereby achieving a temperature-regulated reaction.

[0036] The operation process of employing the integrated micro-droplet chip of the present invention will be further described below with reference to FIGS. First, 30 microliters of the system (i.e., the aforementioned sample) was added to the sample addition chamber 12. The 30 microliter PCR system contained 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. The entire chip is shown in Figure 1. One chip contains eight parallel and independent droplet chip structures, each of which includes a sample addition chamber 12, an oil-liquid port 31, an air-liquid port 32, a microdroplet generation structure, and a reaction chamber 11.

[0037] Next, the sealing cover 122 is sealed or glued to seal the sample addition chamber 12. The sample addition chamber 12 preferably has a filter membrane or a small-diameter exhaust hole.

[0038] Next, Bio-Rad Generation Oil, the oil required for droplet generation, is supplied to the oil-liquid port 31. The oil-liquid contains a surfactant that can stabilize the droplets. A negative pressure of -200 mBar is applied to the gas-liquid port 32. As a result, a pressure difference is created between the gas-liquid port 32 and the sample addition chamber 12 and the oil-liquid port 31. The sample addition chamber 12 is connected to the second conduit 23 within the microdroplet generation structure, the oil-liquid port 31 is connected to the oil-liquid conduit 21 within the microdroplet generation structure, and the first conduit 22 and the gas-liquid port 32 are both connected to the reaction chamber 11. Here, the first conduit 22 is connected to the connection port 111, and the gas-liquid port 32 is connected to the gas-liquid conduit 112. The components of the microdroplet generation structure are shown in Figure 2. The oil-liquid conduit 21 has two branches. The two branches are located on either side of the second conduit 23 and the first conduit 22, respectively, and both are connected to the oil-liquid port 31. The first conduit 22 may include a microdroplet observation area 34. The observation area conduit is wider, the flow rate of the droplets after they enter is slower, and it is easier to image them with an external camera, record the droplet morphology, and determine whether the droplet generation process is normal.

[0039] Driven by the pressure difference, the reaction system enters the second conduit 23, and the resulting oil enters the oil-liquid conduit 21. They meet at a cross structure (i.e., the aforementioned cross-junction port). Fluid shear and surface tension combine to form uniformly sized water-in-oil microdroplets 4. The cross-junction conduit is approximately 70 micrometers deep and 80 micrometers wide, with droplets approximately 100 micrometers in size. The microdroplets 4 enter the first conduit 22 and then enter the microdroplet observation region 34, where their flow rate slows, forming a dense droplet community that can be easily imaged and recorded by a camera. A diagram of the droplet generation process is shown in Figure 4.

[0040] The generated droplets flow through the first conduit 22 and then reach the connection port 111 of the reaction chamber 11. The bottom of the connection port 111 has a slope structure (i.e., the aforementioned trumpet mouth), and the bottom of the slope communicates with the first conduit 22. When the droplet generation process is completed, the pressure difference applied to the chip port is removed. At this time, the droplets should still be located below the gas-liquid conduit 112.

[0041] Then, as shown in Figure 5, the chip is flipped upside down to transfer the droplets from the connection port 111 to the reaction region (i.e., away from the connection port 111). The structure of the reaction region should be designed for high heat transfer efficiency, such as a flat design with a deep depth and a thin thickness, so that an external system (i.e., a heating module) can heat and cool the reaction region from both the left and right sides. This ensures not only a short heat conduction distance but also a large contact area, thereby achieving efficient heat transfer. In this example, the temperature cycling process involves a 10-minute pre-denaturation at 95°C, followed by 40 temperature cycles, each cycle consisting of 5 seconds at 95°C, 15 seconds at 60°C, and a final incubation at 4°C. To reduce evaporation, 30 microliters of a low-density volatile inhibitor can be pre-placed in the reaction chamber 11.

[0042] After the temperature cycle is completed, the amplification reaction in the template-containing droplets is completed accordingly, and the droplet fluorescence detection process must proceed. Detection oil (i.e., the detection oil 5 described above) is injected into the gas-liquid port 32 to continuously fill the reaction chamber 11. During this process, the droplet surface continues to rise and, guided by the slope of the connection port 111, enters the first conduit 22. This process is shown in Figure 7.

[0043] As the droplets pass through the observation area in the first channel 22, a camera can be used to perform bright-field imaging of the droplets to evaluate their status after the amplification reaction. At the same time, detection oil is injected into the oil-liquid port 31. The detection oil passes through the oil-liquid channel 21 and merges with the droplet train at the cross channel, separating the closely spaced droplets into separate droplet trains with appropriate spacing. The droplet train then passes through the fluorescence detection area 33 in the second channel 23, as shown in Figure 8. The position corresponding to the fluorescence detection area 33 is the fluorescence detection focus of the external system. The external system focuses excitation light, such as a laser or LED narrowband light with wavelengths of 488 nm and 532 nm, onto the detection focus. As the droplets pass through the detection focus, the fluorescence excited within the droplets is also received by the collection optical path of the external system, thereby obtaining fluorescence information for each droplet. The fluorescence information of the droplets is used to determine a signal threshold to distinguish between negative and positive droplets, and a Poisson distribution model is used to calculate the copy number of the target molecule in the sample.

[0044] Finally, the droplets for which fluorescence detection has been completed enter the sample addition chamber 12. The sample addition chamber 12 is sealed by a sealing cover 122, so that it does not come into contact with the environment outside the chip, eliminating the possibility of aerosol contamination and realizing a completely sealed digital PCR process.

[0045] This embodiment employs a groundbreaking method of implementing time-division multiplexing in the microdroplet generation structure. During droplet generation, the microdroplet generation structure is used to achieve droplet generation. During droplet fluorescence detection, the microdroplet generation structure completes droplet train separation, ensuring the detection of droplet fluorescence signals. This time-division multiplexing method is the first in the flow-through digital PCR technology route to achieve a fully integrated and fully enclosed digital PCR process that completes droplet generation, amplification, and detection on a single chip, marking an important technological advancement in the field of digital PCR.

[0046] In another embodiment, when the droplets pass through the observation area in the first pipe 22, a camera is used to perform fluorescent imaging of the droplets, and the imaged photograph is analyzed to obtain the fluorescent intensity information of each droplet in the photograph, thereby completing the detection of the droplet fluorescent signal.

[0047] Those skilled in the art will readily understand that the above advantageous methods can be freely combined or superimposed as long as no contradiction occurs.

[0048] The above is only a preferred embodiment of the present invention, and the present invention is not limited thereto. As long as it falls within the spirit and principle of the present invention, all modifications, equivalent replacements, improvements, etc., made should be included within the protection scope of the present invention. It is obvious to those skilled in the art that the above is only a preferred embodiment of the present invention, and some improvements and modifications can be made without departing from the technical principles of the present invention, and these improvements and modifications are also included within the protection scope of the invention. [Explanation of symbols]

[0049] 1, chip body; 11, reaction chamber; 111, connection port; 112, gas-liquid channel; 12, sample addition chamber; 121, opening chamber; 122, sealing cover; 21, oil-liquid channel; 22, first channel; 23, second channel; 31, oil-liquid port; 32, gas-liquid port; 33, fluorescence detection area; 34, microdroplet observation area; 4, microdroplet; 5, detection push oil.

Claims

1. An integrated microdroplet chip comprising a chip body (1), the chip body (1) having a reaction chamber (11) and a sample addition chamber (12), the chip body (1) being configured with a microdroplet generation structure, an oil-liquid port (31), a gas-liquid port (32), and a fluorescence detection region (33), the gas-liquid port (32) communicating with the reaction chamber (11), the sample addition chamber (12) communicating with the microdroplet generation structure, and the oil-liquid port (31) communicating with the microdroplet generation structure; When droplets are generated, a first pressure difference is formed between the sample addition chamber (12) and the gas-liquid port (32), and a second pressure difference is formed between the oil-liquid port (31) and the gas-liquid port (32). The first pressure difference and the second pressure difference drive the sample in the sample addition chamber (12) and the generated oil in the oil-liquid port (31), respectively, to enter the microdroplet generating structure, and the generated microdroplets (4) enter the reaction chamber (11) and are stored. During droplet detection, the external pressure drives the detection oil (5) to enter the reaction chamber (11) through the gas-liquid port (32), causing the microdroplets (4) in the reaction chamber (11) to flow out from the reaction chamber (11) to the microdroplet generating structure; the external pressure drives the detection separation oil to enter the microdroplet generating structure through the oil-liquid port (31); the detection separation oil separates the microdroplets (4) that flow out from the reaction chamber (11) into the microdroplet generating structure to form a line, and causes them to enter the fluorescence detection area (33); The microdroplet generating structure includes an oil-liquid conduit (21) and a communicating conduit, the oil-liquid conduit (21) and the communicating conduit intersect, the communicating conduit includes a first conduit (22) located on a first side of the intersection and communicating with the reaction chamber (11), and a second conduit (23) located on a second side of the intersection and communicating with the sample addition chamber (12), the oil-liquid port (31) communicates with the oil-liquid conduit (21), An integrated micro-droplet chip, characterized in that a gas-liquid conduit (112) is configured in the reaction chamber (11), and the gas-liquid conduit (112) communicates with the gas-liquid port (32).

2. An integrated micro-droplet chip as described in claim 1, characterized in that the oil / liquid pipeline (21) and the connecting pipeline intersect in a cross shape, the first pipeline (22) is located on a first side of the cross intersection, and the second pipeline (23) is located on a second side of the cross intersection.

3. The integrated micro-droplet chip of claim 2, characterized in that, with reference to the first side of the chip body (1) being positioned in a horizontal orientation, the reaction chamber (11) and the sample addition chamber (12) are positioned on the first side, and the connection port (111) between the reaction chamber (11) and the first side of the chip body (1) extends upward and forms a trumpet-shaped mouth that is small at the bottom and large at the top.

4. An integrated micro-droplet chip as described in claim 3, characterized in that the gas-liquid conduit (112) extends from bottom to top within the reaction chamber (11), the lower opening of the gas-liquid conduit (112) is connected to the gas-liquid port (32), and the upper opening of the gas-liquid conduit (112) is higher than the upper opening of the connection port (111).

5. 5. The integrated micro-droplet chip according to claim 4, wherein a micro-droplet observation region (34) is located between the first conduit (22) and the connection port (111).

6. The integrated micro-droplet chip according to claim 1, characterized in that the sample addition chamber (12) includes an open chamber (121) and a sealing cover (122) hermetically connected to the opening of the open chamber (121).

7. 2. The integrated micro-droplet chip according to claim 1, wherein the sample addition chamber (12) is provided with a filter membrane or an exhaust hole.

8. 3. The integrated micro-droplet chip according to claim 2, wherein the fluorescence detection region (33) is located in the second channel (23).

9. The integrated micro-droplet chip according to claim 2, characterized in that the sample addition chamber (12) is located on a first side, the reaction chamber (11) is located on a second side, and the second side and the first side are opposite sides of the chip body (1).

10. 2. The integrated micro-droplet chip according to claim 1, wherein light oil is pre-placed in the reaction chamber (11) before the micro-droplet (4) enters the reaction chamber (11).

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