Liquid drop micro-fluidic chip and biological detection system

Abstract

The invention relates to the technical field of biological detection, and discloses a droplet micro-fluidic chip and a biological detection system. The liquid drop micro-fluidic chip comprises a plurality of liquid drop micro-fluidic units, wherein each liquid drop micro-fluidic unit is provided with a flow channel structure, a main body structure and a cover plate structure; each flow channel structure comprises a micro-flow channel; the main body structure comprises a liquid drop generation module and a liquid drop collection module which are communicated with a micro-channel, the liquid drop collection module comprises a collection cavity with an opening in one end and a channel part, a first preset included angle is formed between the extending direction of the channel part and the plane where the micro-channel is located, the first end of the channel part is communicated with the micro-channel, and the second end of the channel part is communicated with the collection cavity and is flush with the opening end; the cover plate structure is arranged on the main body structure and seals the second end of the flow channel part and the open end of the collecting cavity under the action of external force. According to the invention, bubbles in a sample can be broken under the influence of surface tension when flowing through the top of the flow channel part, so that the bubbles in the sample are removed; the cover plate structure simultaneously seals the second end of the runner part and the opening end of the collecting cavity to ensure the sealing performance of the collecting cavity.

Description

Technical Field

[0001] The present invention relates to the field of biological detection technology, and in particular to a droplet microfluidic chip and a biological detection system. Background Art

[0002] Polymerase chain reaction (PCR) is a molecular biology technique used to amplify specific DNA fragments, which can be regarded as a special DNA replication outside the body. As the third-generation PCR technology, digital PCR (dPCR) can achieve absolute quantitative detection of nucleic acid molecules. Its basic principle is to divide the sample into a large number of reaction units, each unit contains one or more copies of the target molecule, and perform PCR amplification on the target molecule. After the amplification, the fluorescent signal of the reaction unit is statistically analyzed to finally obtain the copy number concentration of the original sample. Digital PCR technology has been widely used in many fields such as liquid biopsy, pathogen detection, single cell analysis, environmental monitoring and food safety.

[0003] In digital PCR technology, the droplet digital PCR (ddPCR for short) chip has the advantages of high sample throughput, large dynamic range, simple sample division and less sample loss. Its droplet generation, PCR amplification and fluorescence detection functional modules are independent of each other. The droplets of the liquid supply module need to be transferred to the PCR tube for amplification after generation. During this process, bubbles are easily generated in the sample and affect the droplet amplification. If the droplet generation, PCR amplification and fluorescence detection functional modules are integrated together, the difficulty of sealing the PCR tube will be greatly increased. Therefore, it is difficult to take into account the high integration of the existing droplet digital PCR chip and the strict sealing of the PCR tube. Bubbles are easily generated during the droplet delivery and amplification process, resulting in amplification failure, which ultimately affects the accuracy of biological molecule detection. Summary of the invention

[0004] In view of this, the present invention provides a droplet microfluidic chip and a biological detection system to solve the problem that the sealing of existing PCR tubes is difficult to ensure, bubbles are easily generated during droplet transportation and amplification, resulting in amplification failure, and ultimately affecting the accuracy of biological molecule detection.

[0005] In the first aspect, the present invention provides a droplet microfluidic chip, comprising a plurality of droplet microfluidic units, any droplet microfluidic unit comprising: a flow channel structure, a main body structure and a cover plate structure, the flow channel structure comprising a microfluidic channel for droplet generation and circulation; the main body structure comprising a liquid supply module and a droplet collection module, the liquid supply module being used to accommodate samples, the microfluidic channel connecting the liquid supply module and the droplet collection module, the droplet collection module comprising a collection chamber and a flow channel portion, one end of the collection chamber having an opening for receiving droplets, an extension direction of the flow channel portion having a first preset angle with a plane where the microfluidic channel is located, a first end of the flow channel portion connecting the microfluidic channel, a second end of the flow channel portion connecting the collection chamber, and the second end being flush with an end of the collection chamber having an opening; the cover plate structure being arranged on a side of the main body structure away from the flow channel structure, the cover plate structure being suitable for closing the second end of the flow channel portion and an end of the collection chamber having an opening under the action of an external force.

[0006] Beneficial effect: In the droplet microfluidic chip of the present invention, a horizontal microchannel for droplet formation and flow is arranged between the liquid supply module and the droplet collection module, and the droplet collection module is provided with a flow channel portion having a first preset angle with the plane where the microchannel is located. The sample gradually moves from the liquid supply module along the microchannel toward the flow channel portion. When the bubbles formed in the sample reach the top position of the flow channel portion, the bubbles come into contact with the external air. The bubbles are affected by gravity, injection pressure toward one end of the flow channel portion outlet, and surface tension toward one end of the flow channel portion inlet. The bubbles form a thin film dome at the outlet end of the flow channel portion, and the gas in the bubbles is gas. The bubbles provide support, and then as the injection pressure continues, the forces inside and outside the bubbles are unbalanced, the liquid film at the gas-liquid interface is disturbed and holes are generated, and after the holes are formed, the bubbles begin to burst. The bubbles accelerate their collapse under the action of surface tension and injection pressure, thereby achieving the purpose of removing bubbles in the sample. At the same time, the droplets in the sample maintain their shape under the net buoyancy, viscous resistance and injection pressure and will not burst; the cover plate structure simultaneously seals the second end of the flush flow channel portion and the opening end of the collection chamber to ensure the sealing of the collection chamber, thereby avoiding affecting the droplet amplification in the collection chamber, improving the droplet amplification quality and the accuracy of subsequent biological detection.

[0007] In an optional embodiment, a first preset angle between an extension direction of the flow channel portion and a surface of the flow channel structure is in a range of 30° to 150°.

[0008] Beneficial effect: The extension direction of the flow channel portion is not parallel to the surface of the flow channel structure, so that when the bubble reaches the top of the flow channel portion, a thin film dome can be formed at the outlet end of the top of the flow channel portion, and the liquid film at the gas-liquid interface is easily disturbed to produce holes, thereby breaking the force balance of the bubble and causing the bubble to burst; at the same time, within this angle range, the horizontal size of the flow channel portion can be limited, which is conducive to the further integration of the chip.

[0009] In an optional embodiment, the extension direction of the flow channel portion is perpendicular to the surface of the flow channel structure.

[0010] Beneficial effect: The first preset angle between the extension direction of the flow channel and the surface of the flow channel structure is 90°. At this time, the flow channel forms a fountain-like structure. When the sample flows in the flow channel, the bubbles in the sample are subject to the upward net buoyancy, the upward injection pressure and the downward viscous resistance. As the sample gradually flows upward until it reaches the gas-liquid interface; when the bubbles in the sample reach the gas-liquid interface at one end of the flow channel outlet, the bubbles are subject to the downward gravity, the upward injection pressure and the downward surface tension, and a thin film dome is formed. At this time, the gas in the bubble provides support for the bubble. Then, as the injection pressure continues, the force inside and outside the bubble is unbalanced, and the liquid film at the gas-liquid interface is disturbed to produce holes. After the holes are formed, the bubble begins to rupture, and collapses faster under the action of surface tension and injection pressure, so as to achieve the purpose of removing bubbles in the sample. The vertical flow channel surface is very easy to form a thin film dome at the end, which is convenient for destroying the equilibrium state of the bubble surface tension, and finally realizing the efficient removal of bubbles.

[0011] In an optional embodiment, one end of the opening of the collecting chamber has a buffer slope on a side close to the flow channel portion, and the buffer slope gradually decreases in height from the flow channel portion side to the collecting chamber side.

[0012] Beneficial effect: A downward sloping buffer slope is formed from one side of the flow channel portion to the other side of the collecting chamber to facilitate the droplets to smoothly enter the collecting chamber, so as to avoid that when the droplets fall directly downward into the collecting chamber from the second end of the flow channel portion, due to the large height difference, when they fall on the sample surface on the surface of the collecting chamber, bubbles are formed again due to the wrapped gas due to the instantaneous collision, thereby ultimately avoiding the influence of bubbles on the amplification of droplets in the collecting chamber, ensuring the purity of droplets in the sample entering the collecting chamber, and thereby improving the amplification quality.

[0013] In an optional embodiment, the flow channel structure includes: The microfluidic layer comprises a flow channel groove formed on one side surface of the microfluidic layer, and an inlet end, an outlet end and a first mounting hole penetrating the microfluidic layer, and a side surface of the microfluidic layer where the flow channel groove is not formed is used to connect with the main structure; the liquid supply module is connected to the inlet end of the microfluidic layer, the flow channel portion of the droplet collection module is connected to the outlet end of the microfluidic layer, and the collection cavity of the droplet collection module passes through the first mounting hole; The bottom plate layer is arranged on one side of the microchannel layer having the channel groove to form a microchannel with the channel groove; the bottom plate layer has a second mounting hole for the collection cavity to penetrate.

[0014] Beneficial effects: The flow channel structure of the present invention includes two detachable parts, which is helpful for the preparation and assembly of the flow channel structure and convenient for the maintenance of the micro-channel.

[0015] In an optional embodiment, the inlet end of the microfluidic layer includes a first liquid inlet and a second liquid inlet; the liquid supply module includes: A first accommodating chamber, the first accommodating chamber is suitable for accommodating a dispersed phase and is communicated with a first liquid inlet of the microfluidic channel layer so that the dispersed phase in the first accommodating chamber flows into the microfluidic channel; The second accommodating chamber is suitable for accommodating the continuous phase and is communicated with the second liquid inlet of the microfluidic layer so that the continuous phase in the second accommodating chamber flows into the microfluidic channel, and the continuous phase is suitable for wrapping the dispersed phase to form droplets.

[0016] Beneficial effect: The inlet end of the microfluidic layer includes a first liquid inlet for receiving a dispersed phase (aqueous phase sample) and a second liquid inlet for receiving a continuous phase (generated oil); the first liquid inlet is connected to the first liquid outlet of the liquid supply module, and then connects the first receiving chamber of the liquid supply module for loading the dispersed phase and the microfluidic channel, and the second liquid inlet is connected to the second liquid outlet of the liquid supply module, and then connects the second receiving chamber of the liquid supply module for loading the continuous phase and the microfluidic channel, ensuring that the dispersed phase can reduce gas encapsulation to form bubbles as much as possible, and at the same time allows the continuous phase to fully flow into the microfluidic channel to encapsulate the dispersed phase to form droplets.

[0017] In an optional embodiment, the cover plate structure is a flexible structure; the cover plate structure includes: a first cover plate arranged on the liquid supply module and a second cover plate arranged on the droplet collection module.

[0018] Beneficial effect: The first cover plate and the second cover plate are made of flexible materials, so the sealing of the cover plate structure to the main structure can be achieved by mechanical means. The first cover plate and the second cover plate are respectively arranged on the liquid supply module and the droplet collection module, which facilitates the injection of sample raw materials on the liquid supply module and realizes efficient sealing and fluorescence detection on the droplet collection module.

[0019] In an optional embodiment, it also includes: a connecting layer, which is arranged between the flow channel structure and the main structure to fix the flow channel structure and the main structure; the connecting layer has a third mounting hole corresponding to the collecting cavity of the main structure, and the collecting cavity is suitable for passing through the third mounting hole.

[0020] Beneficial effect: One side of the connecting layer is adhered to the upper surface of the flow channel structure, and the other side faces the lower surface of the main structure. After the collecting chamber passes through the third mounting hole, it is adhered to the plane part of the lower surface of the main structure, thereby realizing reliable assembly and fixation of the main structure and the microfluidic layer.

[0021] In the second aspect, the present invention also provides a biological detection system, including: a gene amplification instrument, the above-mentioned droplet microfluidic chip and a fluorescence detection device, the gene amplification instrument includes a base and a heating module, the base includes a plurality of holes arranged in an array; the droplet microfluidic chip is suitable for being placed on the base, and the collecting chamber is arranged in the hole; the heating module is suitable for heating the collecting chamber; the collecting end of the fluorescence detection device is suitable for extending into the collecting chamber so that the amplified droplets in the collecting chamber flow through the detection area of ​​the fluorescence detection device.

[0022] Beneficial effects: The shape of the collection chamber of the droplet microfluidic chip is the same as the shape of the PCR tube of the gene amplifier, so the droplet microfluidic chip can be compatible with the gene amplifier (PCR instrument) and can realize batch sample processing. The droplet collection module of the droplet microfluidic chip adopts a fountain-like flow channel, which can stably transport the droplets from the liquid supply module to the collection chamber of the droplet collection module, and effectively remove the bubbles in the sample to avoid the influence of bubbles on PCR amplification. After injecting the dispersed phase and continuous phase materials into the liquid supply module, the hot cover of the PCR instrument is closed, and the hot cover squeezes the second cover plate on the droplet collection module downward, which can achieve complete sealing of the droplet collection module, and then the heating structure heats the collection chamber to realize PCR amplification of biological molecules; after the amplification is completed, the sampling tube of the fluorescence detection device is inserted into the sample in the collection chamber through the sealing hole, and the droplets flow into the detection area along the sampling tube. The detection module emits and reads the fluorescence signal to realize the analysis of the droplets and distinguish between positive droplets and negative droplets.

[0023] In an optional embodiment, the liquid supply module and the droplet collection module are arranged along the first direction; the droplet microfluidic chip includes 8 droplet microfluidic units, and the 8 droplet microfluidic units are arranged along the second direction, and the first direction and the second direction form a second preset angle; the size of the droplet microfluidic chip in the first direction is 17 mm, and the plurality of droplet microfluidic chips are suitable for being arranged on the base along the first direction; The base is a 96-hole base with 12 holes in the first direction and 8 holes in the second direction. The center distance between adjacent holes in the first direction is 9 mm, and the holes in the second direction correspond to the collection chambers of 8 droplet microfluidic units.

[0024] Beneficial effect: In the present invention, in the first direction, the width dimension of the droplet microfluidic unit of the droplet microfluidic chip is smaller than the two columns of hole sizes of the gene amplification instrument; in the second direction, the number of holes in the base is equal to the number of droplet microfluidic units of the droplet microfluidic chip, that is, multiple droplet microfluidic chips can be arranged on the base along the first direction, then the two columns of holes of the base in the first direction can just accommodate one droplet microfluidic chip, and a total of 6 droplet microfluidic chips can be accommodated; a droplet microfluidic chip has 8 droplet microfluidic units, that is, it has 8 detection quantities, and the sample throughput of a single amplification of the biological detection system of the present invention can reach 48, which meets the needs of high-throughput applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0026] Figure 1 is an exploded schematic diagram of a droplet microfluidic chip according to an embodiment of the present invention; Figure 2 is a three-dimensional schematic diagram of the main structure of an embodiment of the present invention; Figure 3 is a schematic top view of the main structure of an embodiment of the present invention; Figure 4 is a schematic structural diagram of a droplet microfluidic unit according to an embodiment of the present invention; Figure 5 is a schematic structural diagram of a liquid supply module of the main structure of an embodiment of the present invention after loading a sample; Figure 6 is a schematic structural diagram of a droplet generation process according to an embodiment of the present invention; Figure 7 is a schematic structural diagram of a droplet transport process according to an embodiment of the present invention; Figure 8 is a schematic structural diagram of a droplet collection module in an embodiment of the present invention collecting droplets for amplification; Fig. 9 It is a schematic diagram of the structure of the fluorescence detection device of an embodiment of the present invention for detecting droplets in a collection chamber.

[0027] Description of reference numerals: 100, droplet microfluidic chip; 101, droplet microfluidic unit; 200, dispersed phase; 300, continuous phase; 400, fluorescence detection device; 401, sampling tube; 402, detection module; 1. channel structure; 11. microchannel layer; 111. first liquid inlet; 112. second liquid inlet; 113. outlet; 114. first mounting hole; 12. bottom plate layer; 121. second mounting hole; 13. microchannel; 2. Main structure; 21. Liquid supply module; 211. First accommodating chamber; 212. Second accommodating chamber; 213. First liquid outlet; 214. Second liquid outlet; 22. Droplet collecting module; 221. Collecting chamber; 222. Flow channel; 223. Buffer slope; 3. Cover plate structure; 31. First cover plate; 311. First loading port; 312. Second loading port; 32. Second cover plate; 321. Sealing hole; 4. Connection layer; 41. Third mounting hole. DETAILED DESCRIPTION

[0028] The present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. It is understood that the specific embodiments described herein are only used to explain the present invention, not to limit the present invention. It should also be noted that, for the convenience of description, only some structures related to the present invention are shown in the accompanying drawings, not all structures. In the following description, the description of known structures and technologies is omitted to avoid unnecessary confusion of the concept of the present invention. Various structural schematic diagrams according to embodiments of the present invention are shown in the accompanying drawings. These figures are not drawn to scale, and some details are magnified for the purpose of clear expression, and some details may be omitted. The shapes of various regions and layers shown in the figures and the relative sizes and positional relationships between them are only exemplary, and may be deviated due to manufacturing tolerances or technical limitations in practice, and those skilled in the art may additionally design regions / layers with different shapes, sizes, and relative positions according to actual needs. In the context of the present invention, when a layer / element is referred to as being "on" another layer / element, the layer / element may be directly on the other layer / element, or there may be an intermediate layer / element between them. In addition, if one layer / element is "on" another layer / element in one orientation, then when the orientation is reversed, the layer / element may be "below" the other layer / element.

[0029] The first generation of PCR technology is the original PCR, which uses an ordinary PCR amplifier to amplify the target gene, and then uses agarose gel electrophoresis to analyze the product. It can only perform qualitative analysis; the second generation of PCR is fluorescent quantitative PCR technology (Real-Time PCR), also called qPCR. Fluorescent quantitative PCR adds fluorescent probes that can indicate the reaction process to the reaction system, and monitors the accumulation of amplification products by the accumulation of fluorescent signals to achieve quantitative analysis; the third generation of PCR technology is called digital PCR (Digital PCR, abbreviated as dPCR), which can detect and quantify nucleic acids. It uses direct counting of target molecules without relying on any calibrants or appearances, and can determine the absolute number of target molecules to be tested as low as a single copy.

[0030] According to the sample division method, digital PCR technology platforms can be divided into two categories: droplet digital PCR and micro-chamber digital PCR. Compared with micro-chamber digital PCR, droplet digital PCR (ddPCR) has the advantages of high sample throughput, large dynamic range, simple sample division and less sample loss. For conventional ddPCR technology platforms, its droplet generation, PCR amplification and fluorescence detection functional modules are independent of each other and require droplet transfer, which will lead to droplet loss and cross contamination. Based on microfluidics technology, the above modules can be integrated into an integrated ddPCR chip, and the integrated ddPCR chip can be divided into three categories: (1) integrated ddPCR chip based on PCR tube; (2) integrated ddPCR chip based on flat cavity; (3) integrated ddPCR chip based on capillary.

[0031] The first type is an integrated ddPCR chip based on PCR tubes: after the droplets are generated, they enter the PCR tubes, where they are amplified and then detected using flow fluorescence detection or fluorescence imaging methods. The integrated ddPCR chip based on PCR tubes in the related art has the problem that the sample throughput is difficult to adjust on demand, and the sample throughput for a single run is at least 16, which cannot meet the needs of a small number of samples, and requires the use of complex peripheral equipment to seal the transport flow channel and the exhaust flow channel. Alternatively, droplets are transported using gas-liquid pipelines. In order to achieve on-chip droplet detection, the chip needs to be rotated 180° during the amplification process, and a low-density, low-volatile sealing oil needs to be used to seal the collection module to prevent sample volatilization. These operations also increase the requirements for peripheral equipment, and droplet loss is also likely to occur during the process of flipping the chip, and the collection module is not compatible with conventional PCR instruments.

[0032] The second type is an integrated ddPCR chip based on a flat cavity: after the droplets are generated, they enter a flat thin cavity, where they are spread out in a single layer. After amplification, they are detected using flow fluorescence detection or fluorescence imaging methods. The most difficult problem to solve in this type of ddPCR chip is the bubble problem. The airtightness of the flat thin cavity is difficult to ensure, and bubbles are easily generated during the plane transportation of the droplets. At the same time, due to the need for high-temperature heating during the amplification process, bubbles are also easily generated inside the chip. During the amplification process, the bubbles continue to expand and shrink, causing the droplets to merge, and ultimately leading to amplification failure. In order to suppress the bubble problem, the chip bonding strength needs to be improved, which greatly increases the difficulty and cost of chip processing, and has high requirements for peripheral equipment. At the same time, this integrated ddPCR chip based on a flat cavity occupies a large space, which limits the expansion of sample throughput.

[0033] The third type is the capillary-based integrated ddPCR chip: after the droplets are generated, they directly enter the capillary and are amplified in the capillary. After the amplification, they are detected by flow fluorescence detection. The three modules of droplet generation, transportation and collection all need to be connected by capillaries. The increase in the length of the capillary leads to an increase in its internal pressure, which increases the requirements for the system. The connection and assembly process between the capillary and other components is very cumbersome, which greatly reduces the practicality of the method. The serial streaming method is used to achieve the sequential generation and amplification of multiple samples, which is prone to cross-contamination problems, which makes it difficult to increase the sample throughput.

[0034] Based on the above situation, there is an urgent need to develop a droplet microfluidic chip to solve the problems of the existing integrated ddPCR chip, such as complex structure, cumbersome operation, easy existence of bubbles, and low compatibility, so as to realize the rapid and smooth transport and collection of droplets, and ultimately achieve efficient and reliable target molecule amplification.

[0035] refer to Figures 1 to 9 The present embodiment provides a droplet microfluidic chip 100, comprising a plurality of droplet microfluidic units 101; the droplet microfluidic unit 101 comprises: a flow channel structure 1, a main body structure 2 and a cover plate structure 3, the flow channel structure 1 comprises a microfluidic channel 13 for droplet generation and circulation; the main body structure 2 comprises a liquid supply module 21 and a droplet collection module 22, the liquid supply module 21 is suitable for accommodating samples, the microfluidic channel 13 connects the liquid supply module 21 and the droplet collection module 22, the droplet collection module 22 comprises a collection chamber 221 and a flow channel portion 222, One end of the collecting chamber 221 has an opening to receive droplets, the extension direction of the flow channel portion 222 has a first preset angle with the plane where the microchannel 13 is located, the first end of the flow channel portion 222 is connected to the microchannel 13, the second end of the flow channel portion 222 is connected to the collecting chamber 221, and the second end is flush with the end of the collecting chamber 221 with an opening; the cover structure 3 is arranged on a side of the main structure 2 away from the flow channel structure 1, and the cover structure 3 is suitable for closing the second end of the flow channel portion 222 and the end of the collecting chamber 221 with an opening under the action of external force.

[0036] Specifically, the above-mentioned droplet microfluidic chip 100 includes a plurality of droplet microfluidic units 101 arranged along the second direction and integrally formed; any droplet microfluidic unit 101 includes a flow channel structure 1, a main structure 2 and a cover structure 3 from bottom to top, the flow channel structure 1 has a horizontally extending microfluidic channel 13, the main structure 2 includes a liquid supply module 21 and a droplet collection module 22 arranged on the same plane, the liquid supply module 21 is used to load samples, and the collection chamber 221 is configured as a hollow cylinder. The shape of the collection chamber 221 in this embodiment is configured to be the same as that of a PCR tube to be compatible with a conventional gene amplification instrument (PCR instrument). The sample in this embodiment includes a dispersed phase 200 (aqueous phase sample) and a continuous phase 300 (generated oil). The sample is loaded into the liquid supply module 21, and then the cover structure 3 covers the upper end of the liquid supply module 21, such as Figure 5 As shown; then, injection pressure is applied to the liquid supply module 21, and the sample is driven by the injection pressure to enter the microchannel 13 of the flow channel structure 1 and form monodisperse oil-in-water droplets, and then the droplets continue to flow forward in the microchannel 13 to the first end of the flow channel portion 222 of the droplet collection module 22, that is, Figure 6 Then the droplets gradually flow upward along the flow channel portion 222, and then flow out from the second end of the flow channel portion 222, that is, the upper end outlet, and gradually flow into the collection chamber 221 along the side wall of the collection chamber 221, such as Figure 7As shown, in the process of the droplet gradually flowing upward along the flow channel portion 222, since the flow channel portion 222 and the surface of the flow channel structure 1 have a first preset angle, that is, the flow channel portion 222 and the plane where the horizontal microchannel 13 is located have an angle, the bubbles in the sample are subject to the net buoyancy component toward the outlet end of the flow channel portion 222, the injection pressure toward the outlet end of the flow channel portion 222 and the viscous resistance toward the inlet end of the flow channel portion 222, and the bubbles gradually flow upward with the sample until they reach the gas-liquid interface. When the bubbles in the sample reach the gas-liquid interface at the outlet end of the flow channel portion 222, the bubbles are subject to the gravity, the injection pressure toward the outlet end of the flow channel portion 222 and the surface tension toward the inlet end of the flow channel portion 222, and the bubbles form a thin film dome. At this time, the gas in the bubble provides support for the bubble. Then, as the injection pressure continues, the forces inside and outside the bubble are unbalanced, and the gas-liquid interface The liquid film is disturbed and holes are generated. After the holes are formed, the bubbles begin to burst and collapse faster under the action of surface tension and injection pressure, thereby achieving the purpose of removing bubbles in the sample. After the droplets enter the flow channel portion 222, they move upward due to the combined action of net buoyancy, viscous resistance and injection pressure. They will not burst after moving to the top outlet of the flow channel portion 222. Surfactants are added to the aqueous sample and the generated oil to maintain the stability of the oil-water interface. Therefore, the droplets will not burst under the action of interfacial tension. Finally, as more and more droplets are on the top of the flow channel portion 222, the force balance is broken, and the droplets will flow downward along the outer wall of the flow channel portion 222 to the collection chamber 221. After all the samples flow from the liquid supply module 21 to the droplet collection module 22, the droplets are collected in the collection chamber 221, and the sample in the collection chamber 221 does not have defects such as bubbles, such as Figure 8 As shown, at this time, the cover structure 3 is pressed down, and the lower end of the cover structure 3 abuts against the upper end of the flow channel portion 222 and the upper end of the collecting chamber 221 at the same time, so as to achieve complete sealing of the droplet collection module 22, that is, the flow channel portion 222 is blocked and the opening of the collecting chamber 221 is sealed, so as to perform PCR amplification of the target molecules carried by the droplets in the collecting chamber 221, thereby ensuring the sealing of the collecting chamber 221 during the amplification process.

[0037] The droplet microfluidic chip 100 of this embodiment has a main structure 2 that integrates a liquid supply module 21 and a droplet collection module 22. The droplet collection module 22 also includes a flow channel portion 222 with a conveying function, thereby achieving a high degree of integration of chip functions. A horizontal microchannel 13 for droplet formation and flow is provided between the liquid supply module 21 and the droplet collection module 22. The droplet collection module 22 is provided with a flow channel portion 222 having a first preset angle with the plane where the microchannel 13 is located. The sample gradually moves from the liquid supply module 21 along the microchannel 13 toward the flow channel portion 222. When the bubbles formed in the sample reach the top position of the flow channel portion 222, the bubble surface contacts the external air. The bubbles are affected by gravity, the injection pressure toward the outlet end of the flow channel portion 222, and the surface tension toward the inlet end of the flow channel portion 222. The bubbles will form a thin film dome at the outlet end of the flow channel portion 222. At this time, the gas in the bubble provides support for the bubble. Then, as the injection pressure continues, the forces inside and outside the bubble are unbalanced, and the liquid film at the gas-liquid interface is disturbed. The bubbles begin to burst after the holes are formed, and collapse faster under the action of surface tension and injection pressure, so as to achieve the purpose of removing bubbles in the sample. At the same time, the droplets in the sample maintain their shape under the net buoyancy, viscous resistance and injection pressure and will not burst. That is, the bubbles in the microchannel 13 can be quickly removed by adopting the fountain-like structure, and the droplets can be stably transported to the droplet collection module 22, so as to completely eliminate the influence of bubbles on droplet generation and PCR amplification. Mechanical pressurization is adopted to make the cover structure 3 seal the second end of the flush flow channel portion 222 and the open end of the collection chamber 221 at the same time, so as to ensure the sealing of the collection chamber 221, thereby avoiding affecting the droplet amplification in the collection chamber 221, and improving the droplet amplification quality and the accuracy of subsequent biological detection.

[0038] In addition, in the process of using the droplet microfluidic chip 100 of this embodiment, it is only necessary to add aqueous samples and generate oil, and there is no need to use equipment to transfer droplets. The operation is simple and convenient, which reduces the requirements for operators and greatly improves the user experience. In addition, the droplet microfluidic chip has low processing cost, less sample loss, high integration, closed process, no cross contamination, and is closer to clinical testing needs, so it can accelerate the market development and clinical application promotion of digital PCR technology.

[0039] As an optional embodiment, the first preset angle between the extension direction of the flow channel portion 222 and the surface of the flow channel structure 1 is in the range of 30° to 150°, such as 30°, 45°, 60°, 90°, 120°, 150°, etc. That is, the extension direction of the flow channel portion 222 is not parallel to the surface of the flow channel structure 1, so that when the bubble reaches the top of the flow channel portion 222, a film dome can be formed at the outlet end of the top of the flow channel portion 222, and the liquid film at the gas-liquid interface is easily disturbed to generate holes, thereby breaking the force balance of the bubble and causing the bubble to burst. At the same time, within this angle range, the horizontal size of the flow channel portion 222 can be limited, which is conducive to further integration of the chip.

[0040] Preferably, in this embodiment, the extension direction of the flow channel portion 222 is perpendicular to the surface of the flow channel structure 1. That is, the first preset angle between the extension direction of the flow channel portion 222 and the surface of the flow channel structure 1 is 90°. At this time, the flow channel portion 222 forms a fountain-like structure. When the sample flows in the flow channel portion 222, the bubbles in the sample are subjected to the upward net buoyancy, the upward injection pressure and the downward viscous resistance, and gradually flow upward with the sample until it reaches the gas-liquid interface; when the bubbles in the sample reach the gas-liquid interface at one end of the outlet of the flow channel portion 222, the bubbles are subjected to the downward gravity, the upward injection pressure and the downward surface tension, and form a film dome. At this time, the gas in the bubble provides support for the bubble. Then, as the injection pressure continues, the force inside and outside the bubble is unbalanced, and the liquid film at the gas-liquid interface is disturbed to generate holes. After the holes are formed, the bubbles begin to rupture. The bubbles accelerate collapse and rupture quickly under the action of surface tension and injection pressure, so as to achieve the purpose of efficiently removing bubbles in the sample. The vertical flow channel 222 can easily form a film dome at the end, which is convenient for destroying the equilibrium state of the bubbles and ultimately removing the bubbles efficiently. The flow channel 222 in this embodiment has a hole diameter of 0.8 mm and a height of 6.5 mm.

[0041] In one embodiment, the opening end of the collecting chamber 221 has a buffer slope 223 on a side close to the flow channel 222 , and the buffer slope 223 gradually decreases in height from the flow channel 222 side to the collecting chamber 221 side.

[0042] Specifically, Figures 2 to 9 As shown, a downward sloping buffer slope 223 is formed from one side of the flow channel portion 222 to the side of the collecting chamber 221 to facilitate the droplets to smoothly enter the collecting chamber 221, so as to avoid that when the droplets fall directly downward from the second end of the flow channel portion 222 into the collecting chamber 221, due to the large height difference, when they fall on the sample surface on the surface of the collecting chamber 221, bubbles are formed again due to the wrapped gas due to the instantaneous collision, thereby avoiding the influence of bubbles on the amplification of the droplets in the collecting chamber 221, ensuring the purity of the droplets in the sample entering the collecting chamber 221, and thereby improving the amplification quality.

[0043] refer to Figure 1 , Figures 5 to 9 The above-mentioned flow channel structure 1 includes: a microfluidic layer 11 and a bottom plate layer 12, the microfluidic layer 11 includes a flow channel groove formed on one side surface of the microfluidic layer 11 and an inlet end, an outlet end 113 and a first mounting hole 114 penetrating the microfluidic layer 11, and the side surface of the microfluidic layer 11 without the flow channel groove is used to connect with the main structure 2; the liquid supply module 21 is connected to the inlet end of the microfluidic layer 11, the flow channel portion 222 of the droplet collection module 22 is connected to the outlet end 113 of the microfluidic layer 11, and the collection cavity 221 of the droplet collection module 22 penetrates the first mounting hole 114; the bottom plate layer 12 is arranged on the side of the microfluidic layer 11 having the flow channel groove to form a microfluidic channel 13 with the flow channel groove, and the bottom plate layer 12 has a second mounting hole 121 for the collection cavity 221 to penetrate.

[0044] Specifically, the bottom plate layer 12 is a single-sided adhesive layer with a thickness of 0.1 mm, and the microfluidic layer 11 is made of olefin polymer (COP) with excellent physical properties, chemical properties, biocompatibility and optical properties. The microfluidic layer 11 is formed by a hot pressing molding process, and the maximum thickness of the microfluidic layer 11 is 0.8 mm. The specific process of forming the microfluidic layer 11 by hot pressing is as follows: first, the olefin polymer sheet and the metal mold are heated to above the glass transition temperature of the olefin polymer by a hot press; secondly, a pressure of 2MPa and 4MPa are applied to the whole respectively, and the holding time is 10min and 30min respectively; thirdly, the pressure is continued to be increased to 6MPa, and the whole is cooled at the same time. When the temperature of the metal mold drops below the demolding temperature (such as 80°C), the olefin polymer sheet is separated and demolded from the metal mold, thereby completing the transfer of the microfluidic layer 11; finally, the olefin polymer sheet is cut and punched by a precision engraving machine, and the hole diameter is 1mm, corresponding to the inlet and outlet ends 113 of the microfluidic layer 11. At this point, the microfluidic layer 11 is processed, and then the bottom plate layer 12 and the side of the microfluidic layer 11 with the flow channel groove are bonded together to form a closed microfluidic channel 13. That is, a flow channel groove with a depth of 75 μm is formed by hot pressing on one side surface of the olefin polymer sheet, and then the inlet end hole and the outlet end hole 113 that penetrate the olefin polymer sheet are formed by punching. In this way, the flow channel structure 1 with the microchannel 13 is formed in two parts, which is helpful for the preparation and assembly of the flow channel structure 1 and convenient for the maintenance of the microchannel 13. In addition, the flow channel structure 1 can be replaced according to the requirements of different droplet sizes, so the droplet size can be adjusted in a wide range to meet different application scenarios.

[0045] refer to Figures 5 to 9The inlet end of the above-mentioned microfluidic layer 11 includes a first liquid inlet 111 and a second liquid inlet 112; the liquid supply module 21 includes a first accommodating chamber 211 and a second accommodating chamber 212, the first accommodating chamber 211 is suitable for accommodating a dispersed phase 200, and is communicated with the first liquid inlet 111 of the microfluidic layer 11, so that the dispersed phase 200 in the first accommodating chamber 211 flows into the microfluidic channel 13; the second accommodating chamber 212 is suitable for accommodating a continuous phase 300, and is communicated with the second liquid inlet 112 of the microfluidic layer 11, so that the continuous phase 300 in the second accommodating chamber 212 flows into the microfluidic channel 13, and the continuous phase 300 is suitable for wrapping the dispersed phase 200 to form droplets.

[0046] Specifically, the inlet end of the microfluidic layer 11 includes a first liquid inlet 111 for receiving the dispersed phase 200 (aqueous phase sample) and a second liquid inlet 112 for receiving the continuous phase 300 (generated oil); the first liquid inlet 111 is connected to the first liquid outlet 213 of the liquid supply module 21, thereby connecting the first receiving chamber 211 of the liquid supply module 21 loaded with the dispersed phase 200 and the microfluidic channel 13, and the second liquid inlet 112 is connected to the second liquid outlet 214 of the liquid supply module 21, thereby connecting the second receiving chamber 212 of the liquid supply module 21 loaded with the continuous phase 300 and the microfluidic channel 13. In this embodiment, the width of the connection between the first liquid inlet 111 and the first liquid outlet 213 is 40 μm, and the width of the second liquid inlet 112 and the second liquid outlet 214 is 80 μm, which ensures that the dispersed phase 200 can reduce gas encapsulation to form bubbles as much as possible, and at the same time allows the continuous phase 300 to fully flow into the microfluidic channel 13 to encapsulate the dispersed phase 200 to form droplets.

[0047] The main structure 2 of this embodiment is obtained by injection molding process. Specifically, the molten cycloolefin polymer pellets are transported to the metal mold cavity by a screw to fill the entire metal mold; then the pressure is continuously increased and maintained for a period of time, and then the metal mold is cooled. When the temperature drops to the demolding temperature, the mold is demolded, thereby completing the injection molding of the main structure 2.

[0048] refer to Figure 1 , Figures 4 to 9 The cover plate structure 3 of this embodiment is a flexible structure; the cover plate structure 3 includes: a first cover plate 31 arranged on the droplet collecting module 22 and a second cover plate 32 arranged on the droplet collecting module 22 .

[0049] Exemplarily, the first cover plate 31 and the second cover plate 32 can both be made of silicone material and are made by molding, for example, pouring liquid silicone (such as DRSGJ02) into an aluminum alloy mold, heating and curing at 60°C for 30 minutes, and the cured silicone material is translucent; the mold is demoulded after cooling to room temperature, thereby completing the preparation of the silicone cover plate structure 3. The first cover plate 31 is provided with a first loading port 311 and a second loading port 312. The first loading port 311 is located above the first accommodating chamber 211 and is used for the injection of dispersed phase 200 materials. The second loading port 312 is located above the second accommodating chamber 212 and is used for the injection of continuous phase 300 materials. The second cover plate 32 is provided with a sealing hole 321, which is located above the collecting chamber 221 and is used for the subsequent installation of the fluorescence detection device 400 for post-amplification detection.

[0050] Based on the above scheme, refer to Figure 1 The droplet microfluidic chip 100 of this embodiment also includes a connecting layer 4, which is arranged between the flow channel structure 1 and the main structure 2 to fix the flow channel structure 1 and the main structure 2. The connecting layer 4 has a third mounting hole 41 corresponding to the collecting cavity 221 of the main structure 2, and the collecting cavity 221 is suitable for passing through the third mounting hole 41.

[0051] Specifically, the connection layer 4 of this embodiment is a double-sided tape with a thickness of 0.17 mm. One side of the connection layer 4 is pasted to the upper surface of the flow channel structure 1, and the other side faces the lower surface of the main structure 2. After the collection chamber 221 passes through the third mounting hole 41, it is pasted to the lower surface plane part of the main structure 2 to achieve the assembly and fixation of the main structure 2 and the microchannel layer 11. The bottom plate layer 12 and the connection layer 4 of this embodiment are both cut into the required shape using a carving die.

[0052] Therefore, the droplet microfluidic chip 100 of this embodiment first bonds the microchannel layer 11 and the bottom plate layer 12 to form a channel structure 1 with a closed microchannel 13; then, the connecting layer 4 is pasted on the microchannel layer 11, and the lower surface of the main structure 2 is bonded to the connecting layer 4; finally, the first cover plate 31 is covered on the liquid supply module 21 of the main structure 2, and the second cover plate 32 is covered on the droplet collection module 22 to complete the assembly of the droplet microfluidic chip 100.

[0053] In another embodiment, the flow channel structure 1 and the main structure 2 can be integrally formed, and the flow channel structure 1 is directly formed in the horizontal plate area of ​​the main structure 2 connecting the liquid supply module 21 and the droplet collection module 22, that is, no additional Figure 1 The flow channel structure 1 includes a micro-channel layer 11 and a bottom plate layer 12. Accordingly, there is no need to set a connecting layer 4 to fix the main structure 2 and the flow channel structure 1, so as to further improve the integration of the chip.

[0054] refer to Figures 1 to 9 The present embodiment further provides a biological detection system, comprising: a gene amplification instrument, the above-mentioned droplet microfluidic chip 100 and a fluorescence detection device 400, the gene amplification instrument comprising a base and a heating module, the base comprising a plurality of holes arranged in an array; the droplet microfluidic chip 100 is suitable for being placed on the base, and the collecting chamber 221 is arranged in the hole, and the heating module is suitable for heating the collecting chamber 221; the collecting end of the fluorescence detection device 400 is suitable for extending into the collecting chamber 221, so that the amplified droplets in the collecting chamber 221 flow through the detection area of ​​the fluorescence detection device 400.

[0055] In this embodiment, the shape of the collection chamber 221 of the droplet microfluidic chip 100 is the same as the shape of the PCR tube of the gene amplifier, so the droplet microfluidic chip 100 can be compatible with the gene amplifier (PCR instrument) and can realize batch sample processing. The droplet collection module 22 of the droplet microfluidic chip 100 adopts a fountain-like flow channel 222, which can stably transport droplets from the liquid supply module 21 to the collection chamber 221 of the droplet collection module 22, and effectively remove bubbles in the sample to avoid the influence of bubbles on PCR amplification. After the dispersed phase 200 and the continuous phase 300 materials are injected into the liquid supply module 21, the thermal cover of the PCR instrument is closed, and the thermal cover presses the second cover plate 32 on the droplet collection module 22 downward to achieve complete sealing of the droplet collection module 22. Then, the heating structure heats the collection chamber 221 to achieve PCR amplification of biological molecules. After the amplification is completed, the droplet microfluidic chip 100 is moved to the fluorescence detection device 400, and the sampling tube 401 of the fluorescence detection device 400 is inserted into the sample in the collection chamber 221 through the sealing hole 321. The droplets flow into the detection area along the sampling tube 401, and the detection module 402 emits and reads the fluorescence signal of the passing droplets. Fig. 9 As shown, the analysis of the droplets is realized to distinguish positive droplets from negative droplets. In this embodiment, the droplet microfluidic chip 100 is moved to the fluorescence detection device 400 for detection, so that another droplet microfluidic chip 100 can be amplified in the gene amplifier, thereby improving the overall detection efficiency and reducing the impact of the amplification time on the detection efficiency. Of course, in other embodiments, it is not excluded to move the fluorescence detection device 400 so that it can be directly detected in situ at the location of the gene amplifier.

[0056] In one embodiment, reference Figures 1 to 3, the liquid supply module 21 and the droplet collection module 22 are arranged along the first direction; the droplet microfluidic chip 100 includes 8 droplet microfluidic units 101, and the 8 droplet microfluidic units 101 are arranged along the second direction, and the first direction and the second direction form a second preset angle; the size of the droplet microfluidic chip 100 in the first direction is 17mm, and a plurality of droplet microfluidic chips 100 are suitable for being arranged on the base along the first direction; the base is a 96-hole base with 12 holes in the first direction and 8 holes in the second direction, and the center distance between adjacent holes in the first direction is 9mm, and the holes in the second direction correspond to the collection chambers 221 of the 8 droplet microfluidic units 101.

[0057] Specifically, the second preset angle of the present embodiment is set to 90 degrees, that is, the base of the gene thermal cycler is a base with 96 holes distributed in a rectangular array, with 12 holes arranged along the first direction and 8 holes arranged along the second direction; in the second direction, the number of holes in the base is equal to the number of droplet microfluidic units 101 of the droplet microfluidic chip 100, that is, multiple droplet microfluidic chips 100 can be arranged on the base along the first direction. Based on this, the width of the droplet microfluidic chip 100 in the first direction of this embodiment is 17 mm, which is smaller than the width of the two columns of holes in the first direction. Therefore, the two columns of holes in the first direction of the base can just accommodate one droplet microfluidic chip 100, and a total of 6 droplet microfluidic chips 100 can be accommodated; a droplet microfluidic chip 100 has 8 droplet microfluidic units 101, that is, it has 8 detection quantities. Therefore, the sample throughput range of a single amplification of the biological detection system of this embodiment is 1-48, which can meet the needs of high-throughput applications while allowing the sample throughput to be adjusted at will according to the detection requirements.

[0058] The further functional description of each of the above structures is the same as that of the above corresponding embodiments and will not be repeated here.

[0059] In the above description, the technical details of the patterning and etching of each layer are not described in detail. However, those skilled in the art should understand that various technical means can be used to form layers, regions, etc. of desired shapes. In addition, in order to form the same structure, those skilled in the art can also design methods that are not completely the same as the methods described above. In addition, although the various embodiments are described above separately, this does not mean that the measures in the various embodiments cannot be used in combination to advantage.

[0060] Although the embodiments of the present invention have been described in conjunction with the accompanying drawings, those skilled in the art may make various modifications and variations without departing from the spirit and scope of the present invention, and such modifications and variations are all within the scope defined by the appended claims.

Claims

1. A droplet microfluidic chip, characterized in that: The invention comprises a plurality of droplet microfluidic units; the droplet microfluidic unit comprises: A flow channel structure, the flow channel structure comprising a micro-channel for droplet generation and circulation; The main structure comprises a liquid supply module and a droplet collection module, wherein the liquid supply module is suitable for accommodating samples, and the microchannel connects the liquid supply module and the droplet collection module; the droplet collection module comprises a collection chamber and a flow channel portion, and one end of the collection chamber has an opening to receive droplets; the extension direction of the flow channel portion has a first preset angle with the plane where the microchannel is located, the first end of the flow channel portion connects with the microchannel, the second end of the flow channel portion connects with the collection chamber, and the second end is flush with the end of the collection chamber having an opening; The cover plate structure is arranged on a side of the main structure away from the flow channel structure; the cover plate structure is suitable for closing the second end of the flow channel part and the end of the collection chamber with an opening under the action of external force.

2. The droplet microfluidic chip according to claim 1, characterized in that: A first preset angle between an extending direction of the flow channel portion and a surface of the flow channel structure is in a range of 30° to 150°.

3. The droplet microfluidic chip according to claim 2, characterized in that: An extending direction of the flow channel portion is perpendicular to a surface of the flow channel structure.

4. The droplet microfluidic chip according to claim 1, characterized in that: One end of the opening of the collecting chamber has a buffer slope on a side close to the flow channel portion, and the buffer slope gradually decreases in height from one side of the flow channel portion to one side of the collecting chamber.

5. The droplet microfluidic chip according to claim 1, characterized in that: The flow channel structure comprises: The microfluidic layer comprises a flow channel groove formed on one side surface of the microfluidic layer, and an inlet end, an outlet end and a first mounting hole penetrating the microfluidic layer, and a side surface of the microfluidic layer where the flow channel groove is not formed is used to connect with the main structure; the liquid supply module is connected to the inlet end of the microfluidic layer, the flow channel portion of the droplet collection module is connected to the outlet end of the microfluidic layer, and the collection chamber of the droplet collection module passes through the first mounting hole; The bottom plate layer is arranged on one side of the microchannel layer having the channel groove to form a microchannel with the channel groove; the bottom plate layer has a second mounting hole for the collection cavity to pass through.

6. The droplet microfluidic chip according to claim 5, characterized in that: The inlet end of the microfluidic layer includes a first liquid inlet and a second liquid inlet; the liquid supply module includes: A first accommodating chamber, the first accommodating chamber is suitable for accommodating a dispersed phase and is communicated with a first liquid inlet of the microfluidic channel layer so that the dispersed phase in the first accommodating chamber flows into the microfluidic channel; The second accommodating chamber is suitable for accommodating a continuous phase and is communicated with a second liquid inlet of the microfluidic channel layer so that the continuous phase in the second accommodating chamber flows into the microfluidic channel, and the continuous phase is suitable for wrapping the dispersed phase to form droplets.

7. The droplet microfluidic chip according to claim 1, characterized in that: The cover plate structure is a flexible structure; the cover plate structure comprises: a first cover plate arranged on the liquid supply module and a second cover plate arranged on the droplet collection module.

8. The droplet microfluidic chip according to claim 1, characterized in that: Also includes: A connecting layer, wherein the connecting layer is arranged between the flow channel structure and the main body structure to fix the flow channel structure and the main body structure; The connection layer has a third mounting hole corresponding to the collecting cavity of the main structure, and the collecting cavity is suitable for passing through the third mounting hole.

9. A biological detection system, characterized in that: include: A gene amplification instrument, comprising a base and a heating module, wherein the base comprises a plurality of wells arranged in an array; The droplet microfluidic chip according to any one of claims 1 to 8, wherein the droplet microfluidic chip is suitable for being placed on the base, and the collecting chamber is arranged in the hole; and the heating module is suitable for heating the collecting chamber; and a fluorescence detection device, wherein the collecting end of the fluorescence detection device is suitable for extending into the collecting cavity, so that the amplified droplets in the collecting cavity flow through the detection area of ​​the fluorescence detection device.

10. The biological detection system according to claim 9, characterized in that: The liquid supply module and the droplet collection module are arranged along a first direction; the droplet microfluidic chip includes 8 droplet microfluidic units, and the 8 droplet microfluidic units are arranged along a second direction, and the first direction and the second direction form a second preset angle; the size of the droplet microfluidic chip in the first direction is 17 mm, and a plurality of the droplet microfluidic chips are suitable for being arranged on the base along the first direction; The base is a 96-hole base having 12 holes along the first direction and 8 holes along the second direction, and the center distance between adjacent holes in the first direction is 9 mm, and the holes in the second direction correspond to the collection chambers of the 8 droplet microfluidic units.

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