Liquid storage type micro-fluidic chip and working method thereof

Abstract

The invention discloses a liquid storage type micro-fluidic chip and a working method thereof. The chip comprises a chip body, a sealing film and gum, the sealing film and the back adhesive are respectively bonded on the front surface and the back surface of the chip body; a plurality of solution storage cavities are formed in the chip body, outflow runners at the bottoms of the solution storage cavities are connected with corresponding first runners, sealing partition pieces are arranged on the first runners, the outflow runners and the corresponding first runners are blocked through the sealing partition pieces, and the first runners are communicated with second runners through corresponding gum runners. The whole experiment process does not need to be externally connected with a reagent bottle, the purposes of instant detection, preparation and sample separation of the micro-fluidic chip can be achieved, the detection time is greatly shortened, and the experiment steps are simplified. Waste liquid generated in the experiment process flows into the waste liquid cavity, and cross contamination of a laboratory is avoided. The chip provided by the invention can be used for identifying the species of pathogenic microorganisms.

Description

Technical Field

[0001] The present invention belongs to the technical field of microfluidic chips, and in particular, relates to a liquid storage type microfluidic chip and a working method thereof. Background Art

[0002] Microfluidic chips, also known as chip laboratories, can integrate basic operating units such as sample preparation, reaction, separation, and detection in the fields of biology, chemistry, and medicine on a single chip. They have the characteristics of high integration and strong automation. In recent years, microfluidic chips have been applied to fully automated, integrated biological testing POCT (full name: point-of-care testing, Chinese name: instant testing), which has broad application prospects and important application value.

[0003] Currently, most functions of microfluidic chips, such as reagent storage, require connecting the chip to different reagent bottles, and using a driving pump to provide power to transport the external reagents into the microfluidic chip.

[0004] Its operation is cumbersome and complicated, requiring quantitative devices (such as pipettes) and external pipes and reagent bottles. The structural design is complex and the repeated use of external pipes will cause contamination, which may lead to erroneous experimental results. It is not conducive to transportation and storage, and it is not conducive to simplifying the process. It requires a high level of operation of the experimenter, which is not conducive to the development of full automation of microfluidic chips. At the same time, for microfluidic chip full automation and integrated biological detection equipment, sample in and result out are important goals. To achieve the goal of sample in and result out, various reagents need to be pre-filled. The external reagent bottle method limits the promotion and application of microfluidic chip instant testing methods; in addition, it is also necessary to ensure that the liquid reagents pre-filled in the microfluidic chip can be effectively released when needed, so the pre-storage and release of liquid reagents are crucial in microfluidic chips. Therefore, how to realize reagent storage and sealing on microfluidic chips is an urgent problem to be solved.

[0005] Chinese patent application 201810957492.1 provides a microfluidic chip, in which the storage module and the reaction module are designed to be separated. When in use, the storage module and the reaction module need to be docked, which is complicated and cumbersome to operate, and requires additional complex connecting structures and valves to connect the storage module and the reaction chamber module, which requires a high technical level of the operator, is not conducive to the promotion and application of microfluidic chip instant detection, and has a high cost of use.

[0006] Chinese patent application 202310632430.4 provides a rotary valve microfluidic chip, which requires an additional rotary valve drive device to drive the rotary valve to rotate. The assembly and operation are cumbersome, which is not conducive to the promotion and application of microfluidic chip instant testing. Summary of the invention

[0007] In view of the above-mentioned deficiencies in the prior art, an object of the present invention is to provide a liquid storage type microfluidic chip for solving the problem of reagent storage and sealing in the microfluidic chip.

[0008] The technical solution of the present invention is specifically described as follows.

[0009] The present invention provides a liquid storage type microfluidic chip, which comprises a chip body, a sealing film and a backing glue; the sealing film and the backing glue are bonded to the front and back of the chip body respectively; a solution storage cavity, an outflow channel, a first channel and a second channel are arranged inside the chip body, and a backing glue channel is arranged on the backing glue; wherein: There are several solution storage chambers, the openings of which are arranged on the top surface of the microfluidic chip, and the top surface of the solution storage chamber is isolated and sealed from the outside by a sealant; the bottom of each solution storage chamber is connected to an outflow channel, and the outflow channel is connected to a corresponding first channel, a sealing partition is arranged on the first channel, and the outflow channel and the corresponding first channel are blocked by the sealing partition, and the first channel is connected to the second channel through the corresponding adhesive channel; when working, the top surface of the solution storage chamber is connected to the atmosphere, the adhesive and the sealing partition are pierced so that the chamber is connected to the corresponding adhesive channel, and under the action of negative pressure, the liquid pre-stored in the chamber is released and flows in a direction in the chip through the outflow channel, the first channel, the adhesive channel, and the second channel in sequence.

[0010] In the present invention, a back glue flow channel blind hole is arranged on the back glue, and the back glue flow channel blind hole corresponds to the outlet position of the first flow channel.

[0011] In the present invention, a premixing chamber, a reaction chamber and a waste liquid chamber are also provided on the chip body; the output end of the second flow channel is connected to the mixing flow channel, the output end of the mixing flow channel is connected to the premixing chamber, and the premixing chamber is connected to the reaction chamber through the flow channel; the waste liquid chamber is used to collect waste liquid generated during the experiment.

[0012] In the present invention, there are two waste liquid chambers on the chip body: a first waste liquid chamber and a second waste liquid chamber, which are respectively connected to the premixing chamber and the reaction chamber through flow channels; the first waste liquid chamber, the premixing chamber, and the second waste liquid chamber are respectively connected to one end of the negative pressure extraction channel, and the other end of the negative pressure extraction channel is a closed end. A blind hole is arranged on the closed end on the backing glue. When extracting liquid, the blind hole is punctured to form a first negative pressure extraction port, a second negative pressure extraction port, and a third negative pressure extraction port, respectively.

[0013] In the present invention, the solution storage chamber is used to store sample solution, cleaning solution, elution solution and reaction solution of pathogenic microorganisms to be tested that have been lysed by lysate; a nucleic acid adsorption component is arranged in the mixing flow channel, and there are several reaction chambers in which different pathogenic microorganism nucleic acid amplification primers are pre-buried.

[0014] In the present invention, the solution storage chamber for storing the reaction solution is omitted, and the reaction solution freeze-dried microspheres are pre-embedded in the premixing chamber to replace the reaction solution.

[0015] In the present invention, the sealing member on the top surface of the solution storage chamber is designed with a sealing film or a sealing cover; the sealing film is selected from a pressure-sensitive film, an adhesive-backed film, a PET film or a PC film; the adhesive-backed film is made of an elastic material; the bonding method is one or more of hot pressing bonding, ultrasonic bonding, laser bonding, adhesive bonding, and solvent bonding; the core body and the sealing spacer are designed as an integrated unit and are manufactured by injection molding, machining or 3D printing; the bottom surface of the solution storage chamber is a funnel-shaped inclined surface.

[0016] The present invention also includes a puncture component used in conjunction with the chip; the puncture component includes a needle and a needle seat, the needle is used to puncture the backing glue and the sealing spacer, and the needle seat is used to fix the needle.

[0017] The present invention also provides a working method of the above-mentioned liquid storage type microfluidic chip, which includes the steps of driving the pre-stored liquid in the solution storage cavity to directional flow and release, which are specifically as follows: (1) Connecting the top surface of the solution storage chamber to the atmosphere; external force drives the needle seat to press the adhesive backing, and the needle pierces the adhesive backing and the sealing spacer. At this time, the adhesive backing blocks the channel where the sealing spacer is located, and each solution storage chamber and its corresponding adhesive backing flow channel are not connected to each other; (2) After puncturing the sealing spacer, the needle is pulled outward, driving the needle seat away from the adhesive backing, and each solution storage chamber is connected to its corresponding adhesive backing flow channel. At this time, liquid is extracted, and the pre-stored liquid in the chamber passes through the outflow flow channel, the first flow channel, the adhesive backing flow channel, and the second flow channel in sequence and is released and flows in the chip in a direction; (3) After the liquid stored in the solution storage chamber is exhausted, the external force drives the needle seat to press the adhesive backing, and the adhesive backing blocks the channel where the sealing spacer is located, and the solution storage chamber is not connected to the corresponding adhesive backing flow channel.

[0018] After each of the above-mentioned liquid extraction steps is completed, the corresponding flow channel must be blocked to ensure that the liquid can be extracted smoothly in other solution storage cavities that are connected and share the flow channel in the subsequent steps.

[0019] Furthermore, the working method of the liquid storage type microfluidic chip of the present invention specifically comprises the following steps: First, the top surface of the solution storage chamber storing the sample solution of the pathogenic microorganism to be detected that has been lysed by the lysing solution is connected to the atmosphere, the sealing partition in the corresponding first flow channel is punctured, and negative pressure is drawn to the first negative pressure port. While the nucleic acid in the sample of the pathogenic microorganism to be detected is adsorbed on the nucleic acid adsorption component, the lysate in the lysing solution storage chamber is drawn to the first waste liquid chamber; after the drawing is completed, the needle seat is driven to press the back glue, and the needle head presses the corresponding back glue flow channel blind hole to block the back glue flow channel; Secondly, the top surface of the solution storage chamber storing the cleaning solution is connected to the atmosphere, the sealing partition in the corresponding first flow channel is punctured, and negative pressure is drawn to the first negative pressure port. The cleaning solution cleans the impurities of the nucleic acid, and the cleaning solution and impurities are drawn to the first waste liquid chamber. After the drawing is completed, the needle seat is driven to press the back glue, and the needle head presses the corresponding back glue flow channel blind hole to block the back glue flow channel, and the first negative pressure port is closed; Next, the top surface of the solution storage chamber storing the eluent is connected to the atmosphere, the sealing partition in the corresponding first flow channel is punctured, and negative pressure is drawn to the second negative pressure pumping port, and the eluent elutes the nucleic acid on the nucleic acid adsorption component into the premixing chamber. After the pumping is completed, the needle seat is driven to press the back glue, and the needle head presses the corresponding back glue flow channel blind hole to block the back glue flow channel; Then, the top surface of the solution storage chamber storing the reaction solution is connected to the atmosphere, the sealing spacer in the corresponding first flow channel is punctured, and negative pressure is drawn to the second negative pressure drawing port, so that the reaction solution is drawn into the premixing chamber and mixed evenly with the nucleic acid; Finally, negative pressure is drawn at the third negative pressure port, and the nucleic acid and reaction solution evenly mixed in the premixing chamber flow into each reaction chamber under the action of negative pressure, and the excess solution flows into the second waste liquid chamber; by observing the color changes of the liquids in multiple reaction chambers, the positive and negative nature of the nucleic acid reaction of the pathogenic microorganisms in the sample is judged.

[0020] Compared with the prior art, the present invention has the following beneficial effects: The present invention provides a sealing spacer inside each chamber and the corresponding flow channel, and the sealing spacer is integrally formed with the microfluidic chip body. Compared with the additional sealing spacer, this sealing method has a better sealing effect. The solution stored in each chamber will not leak or seep out through the integrally formed sealing spacer, so that the solution in the chamber can be stably sealed and stored in each chamber. The opening and closing of each blind hole is controlled by pressing on and off (pressing the needle with external force to keep the flow channel sealed). There is no need to use additional complex valve structures and transfer devices. The solution is pre-stored in the microfluidic chip body. Through reasonable structure and flow channel design, the solution is transferred and released in different experimental steps. It is easy to operate and is very suitable for instant experiments on microfluidic chips. The disposable, ready-to-use design avoids cross-contamination problems.

[0021] Through the reasonable structural design of the flow channel, each chamber and the sealing method of each chamber of the microfluidic chip, various reagents can be stably and long-term sealed and stored inside the microfluidic chip. The waste liquid generated during the experiment flows into the first waste liquid chamber and the second waste liquid chamber in the microfluidic chip without contacting the external environment of the laboratory, thus avoiding cross contamination of the laboratory.

[0022] Before the experiment, each chamber of the present invention is separated from the solution by a sealing partition, and one solution is released in each step; the present invention can achieve the purpose of instant preparation, reaction, separation and detection of samples on the microfluidic chip without the need for external reagent bottles, greatly shortening the operation time and simplifying the operation steps.

[0023] The present invention is aimed at the situation of liquid reaction liquid. The liquid reaction liquid has an independent storage chamber and is released into the premixing chamber only after the experiment starts, thereby ensuring the stability of the reaction liquid.

[0024] In view of the situation that the reaction liquid freeze-dried microspheres are pre-embedded in the premixing chamber, the present invention physically isolates each liquid storage device from the freeze-dried microspheres, so that the freeze-dried microspheres are not affected by moisture from the reagent, thereby ensuring the activity of the enzyme and the accuracy and precision of the experimental results.

[0025] The chip of the present invention can realize that the reaction liquid flows into the premixing chamber and is evenly mixed with the nucleic acid after lysis, cleaning, adsorption and elution of the pathogenic microorganism sample, and then enters the reaction chamber for reaction to realize the identification of the pathogenic microorganism type. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1A It is a schematic diagram of the state of puncturing the sealing spacer and pressing the on-off switch.

[0027] Figure 1B It is a schematic diagram of the state of puncturing the sealing spacer and pressing the on-off state.

[0028] Figure 1C It is a schematic diagram of the state of puncturing the sealing spacer and pressing the on-off state.

[0029] Figure 1D It is a schematic diagram of the state of puncturing the sealing spacer and pressing the on-off state.

[0030] Figure 2 It is a schematic diagram of the main structure of the liquid storage microfluidic chip.

[0031] Figure 3 It is a schematic diagram of the adhesive structure of the liquid storage microfluidic chip.

[0032] Figure 4 is a schematic diagram of a nucleic acid adsorption component.

[0033] Figure 5 This is a three-layer structure diagram of the chip.

[0034] Markings in the figure: 1-chip body, 101-lysate storage chamber, 102-cleaning liquid storage chamber, 103-elution liquid storage chamber, 104-reaction liquid storage chamber, 105-premixing chamber, 106-reaction chamber, 107-first waste liquid chamber, 108-second waste liquid chamber, 109-first lysate flow channel, 111-first cleaning liquid flow channel, 113-first eluent flow channel, 115-first reaction liquid flow channel, 117-second lysate Lysis liquid flow channel, 118-second cleaning liquid flow channel, 119-second elution liquid flow channel, 120-second reaction liquid flow channel, 121-mixing flow channel, 122-waste liquid chamber flow channel, 123-premixing chamber inflow flow channel, 124-reaction chamber flow channel, 125-nucleic acid adsorption component, 126-sealing spacer, 127-first negative pressure pumping channel, 128-second negative pressure pumping channel, 129-third negative pressure pumping channel, 130-outflow flow channel; 2-sealing film, 3-back glue, 301-first back glue flow channel, 302-first back glue flow channel blind hole, 303-second back glue flow channel, 304-second back glue flow channel blind hole, 305-third back glue flow channel, 306-third back glue flow channel blind hole, 307-fourth back glue flow channel, 308-fourth back glue flow channel blind hole, 309-first negative pressure pumping channel blind hole, 310-second negative pressure pumping channel blind hole, 311-third negative pressure pumping channel blind hole. DETAILED DESCRIPTION

[0035] The technical solution of the present invention is described in detail below in conjunction with the accompanying drawings and embodiments.

[0036] The present invention provides a liquid storage microfluidic cavity chip, which includes a chip body 1, a sealing film 2 and a back glue 3, wherein the sealing film 2 is bonded to the front of the chip body 1, and the back glue 3 is bonded to the back of the chip body 1; the back glue 3 is a back glue material with elasticity (rubber, back glue, and other materials are acceptable), and the bonding method is one or a combination of heat pressing bonding, ultrasonic bonding, laser bonding, adhesive bonding, and solvent bonding. The sealing film 2 is used to seal the flow channel and the chamber arranged on the front of the chip, and the sealing film 2 is selected from one of a pressure-sensitive film, a back glue film, a PET film, and a PC film, and preferably, it is a transparent film.

[0037] The chip body 1 is provided with: Lysis solution storage chamber 101, cleaning solution storage chamber 102, eluent storage chamber 103, reaction solution storage chamber 104, premixing chamber 105, reaction chamber 106, first waste liquid chamber 107, second waste liquid chamber 108; A first lysate flow channel 109, a first cleaning solution flow channel 111, a first eluent flow channel 113, and a first reaction solution flow channel 115; A second lysate flow channel 117, a second cleaning solution flow channel 118, a second eluent flow channel 119, and a second reaction solution flow channel 120; Mixing channel 121 and waste liquid chamber channel 122 .

[0038] The lysis solution storage chamber 101 is used for filling and storing samples of pathogenic microorganisms to be tested that have been lysed by the lysis solution; the cleaning solution storage chamber 102 is used for storing cleaning solution; the eluent storage chamber 103 is used for storing eluent; and the reaction solution storage chamber 104 is used for storing reaction solution; the lysis solution storage chamber 101, the cleaning solution storage chamber 102, the eluent storage chamber 103 and the reaction solution storage chamber 104 are all independently arranged inside the same chip body 1, so that the solution stored in each solution storage chamber only contacts the chip body, but does not contact the sealing film 2 of the chip body 1 and the adhesive backing 3 on the back of the chip, thereby preventing the sealing film 2 and the adhesive backing material from releasing interfering substances into the solution, so that the solutions stored in each solution storage chamber can be stably stored for a long time without deterioration, that is, they do not interfere with each other, and at the same time prevent the sealing film 2 and the adhesive backing material from releasing interfering substances to inhibit the reaction solution and nucleic acid reaction, resulting in false negatives, thereby ensuring the accuracy of the experimental results.

[0039] Each solution storage cavity is connected to the corresponding outflow channel 130 arranged at the bottom thereof, and the outflow channel 130 is arranged inside the microfluidic chip body, and the corresponding outflow channel 130 is respectively connected to the corresponding first flow channel (the first lysate flow channel 109, the first cleaning liquid flow channel 111, the first eluent flow channel 113 and the first reaction liquid flow channel 115), and the outflow channel 130 and the corresponding first flow channel are blocked by a sealing spacer 126 (the sealing spacer 126 is arranged at any place of the first flow channel. In order to facilitate processing and production, the sealing spacer 126 can be arranged at the junction of the outflow channel 130 and the corresponding first flow channel. The sealing spacer 126 and the chip body 1 are integrally formed. , the first flow channel is connected to the adhesive flow channel opened on the corresponding adhesive 3. With such a design, the solution pre-stored in each solution storage cavity will neither contact the sealing film 2 on the front side of the chip nor the adhesive 3 on the back side, thus preventing the substances in the sealing film 2 and the adhesive 3 from being released into the solution in each solution storage cavity, thereby inhibiting the subsequent reaction solution from reacting with the nucleic acid, thus ensuring the accuracy and reliability of the experimental results; at the same time, it further ensures that the solution pre-stored in each solution storage cavity does not contact the sealing film 2 and the adhesive 3, thus preventing the solution in each solution storage cavity from deteriorating, allowing the solution pre-stored in each solution storage cavity to be stably stored for a long time, further ensuring the accuracy of the experimental results.

[0040] The bottom surface of each solution storage chamber is a funnel-shaped slope. With this design, the sample solution after lysis can be completely drained under the action of negative pressure, so that there is no loss or residue of the sample solution after lysis, and the nucleic acid in the sample is prevented from remaining on the bottom surface of the lysis solution storage chamber, thereby preventing the loss of nucleic acid samples and ensuring the accuracy of experimental results during quantitative detection.

[0041] In each solution storage cavity, after various solutions are canned in each cavity, a sealing film is provided on the top surface or a sealing cover with an elasticity is provided on the cover to prevent the solution pre-canned in each solution storage cavity from volatilizing; when driving the liquid in each cavity to flow, the sealing film on the top surface of each cavity is punctured or the sealing cover is opened.

[0042] The first flow channel (the first lysate flow channel 109, the first cleaning liquid flow channel 111, the first eluent flow channel 113, and the first reaction liquid flow channel 115) is designed not to penetrate the microfluidic chip body. The lysate storage chamber 101 is connected to the corresponding lysate outflow channel, and the lysate outflow channel is connected to the first lysate flow channel 109. For the convenience of processing and production, the first lysate flow channel 109 is provided with a sealing spacer 126 at the junction of the first lysate flow channel 109 and the lysate outflow channel. The sealing spacer 126 is integrally formed with the chip body 1. With such a design, the solution pre-stored in the lysate storage chamber 101 is The liquid only contacts the chip body 1 and does not contact other materials, ensuring that the pre-stored solution is stably stored and sealed without leakage; and avoiding the solution from contacting other materials, preventing other materials from releasing uncertain substances to inhibit the reaction between nucleic acid and reaction solution, resulting in false negatives, and ensuring the accuracy and reliability of experimental results; the sealing spacer 126 is made by injection molding, machining or 3D printing, and the first lysate flow channel 109 and the lysate storage chamber 101 are separated so that the lysate is sealed and stored in the lysate storage chamber 101; the sealing spacer 126 is punctured, and the lysate storage chamber 101 is connected with the first lysate flow channel 109; A sealing spacer 126 is provided in the first cleaning liquid flow channel 111. The sealing spacer 126 is integrally formed with the chip body 1. The sealing spacer 126 separates the first cleaning liquid flow channel 111 from the cleaning liquid storage chamber 102 so that the cleaning liquid is sealed and stored in the cleaning liquid storage chamber 102. The sealing spacer 126 is punctured, and the cleaning liquid storage chamber 102 is connected to the first cleaning liquid flow channel 111. A sealing spacer 126 is provided in the first eluent flow channel 113, and the sealing spacer 126 is integrally formed with the chip body 1. The sealing spacer 126 separates the first eluent flow channel 113 from the eluent storage chamber 103 so that the eluent is sealed and stored in the eluent storage chamber 103; the sealing spacer 126 is punctured, and the eluent storage chamber 103 is connected to the first eluent flow channel 113; A sealing spacer 126 is provided in the first reaction liquid flow channel 115. The sealing spacer 126 is integrally formed with the chip body 1. The sealing spacer 126 separates the first reaction liquid flow channel 115 from the reaction liquid storage chamber 104 so that the reaction liquid is sealed and stored in the reaction liquid storage chamber 104; the sealing spacer 126 is punctured, and the reaction liquid storage chamber 104 is connected to the first reaction liquid flow channel 115.

[0043] The second flow channels (second lysate flow channel 117, second cleaning solution flow channel 118, second eluent flow channel 119 and second reaction solution flow channel 120) and the first flow channels of the corresponding solution storage chambers are interconnected through the corresponding adhesive flow channels opened on the adhesive backing 3. The second flow channels are all designed to penetrate the microfluidic chip body, and are all connected to the mixing flow channel 121 and penetrate the front and back sides of the microfluidic chip body 1; the mixing flow channel 121 is arranged on the front side of the chip body 1, and the mixing flow channel 121 is connected to the premixing chamber 105; The waste liquid chamber flow channel 122 (arranged on the front side of the chip body 1) is connected to the first waste liquid chamber 107 and the mixing flow channel 121; the waste liquid chamber flow channel outlet of the first waste liquid chamber 107 is arranged in the middle and upper part of the first waste liquid chamber 107, and the volume of the waste liquid chamber is greater than the volume of the lysis solution plus the cleaning solution.

[0044] Nucleic acid adsorption component 125: preset in the mixing channel 121, preset between the second eluent channel 119 and the waste liquid chamber channel 122, for adsorbing nucleic acids. The nucleic acid adsorption component 125 is an existing silicone mold, the width of the silicone mold matches the width of the mixing channel 121, the microfluidic chip body 1 is a hard material, such as PP or PC, the mixing channel 121 is arranged on the front of the chip body 1, and after the silicone mold is arranged in the mixing channel 121, the sealing film 2 is covered on the front of the microfluidic chip, so that the silicone mold can be stably and firmly fixed in the mixing channel 121, and is not affected by the elastic adhesive material on the back, avoiding the elastic adhesive material from squeezing the silicone mold, resulting in the solution being unable to pass through the silicone mold smoothly, causing the experiment to fail; at the same time, it can also avoid the mixing channel 121 being squeezed and deformed by the adhesive 3 during assembly, affecting the smooth passage of the solution.

[0045] Before the reaction, the lysate storage chamber 101, the cleaning solution storage chamber 102, the eluent storage chamber 103, the reaction solution storage chamber 104, the premixing chamber 105, the reaction chamber 106 and the first waste liquid chamber 107 are not connected to each other; the volumes of the multiple reaction chambers 106 are the same, and a second waste liquid chamber 108 is further provided at the end of the reaction chamber 106 for collecting waste liquid. The waste liquid after the reaction is not discharged, so that the laboratory environment will not be polluted.

[0046] For experiments using freeze-dried microspheres as reaction materials, the freeze-dried microspheres can be pre-placed in the premixing chamber 105 , and the reaction liquid storage chamber 104 and the corresponding reaction liquid flow channel are not provided.

[0047] The adhesive backing 3 is provided with: The first adhesive channel 301 and the first adhesive channel blind hole 302, the second adhesive channel 303 and the second adhesive channel blind hole 304, the third adhesive channel 305 and the third adhesive channel blind hole 306, the fourth adhesive channel 307 and the fourth adhesive channel blind hole 308, the first negative pressure channel blind hole 309, the second negative pressure channel blind hole 310 and the third negative pressure channel blind hole 311.

[0048] The first lysate flow channel 109 and the second lysate flow channel 117 are connected through the first adhesive flow channel 301; The first cleaning liquid flow channel 111 and the second cleaning liquid flow channel 118 are connected through the second adhesive flow channel 303; The first eluent flow channel 113 and the second eluent flow channel 119 are connected via the third adhesive flow channel 305; The first reaction liquid flow channel 115 and the second reaction liquid flow channel 120 are connected through the fourth adhesive flow channel 307; The first lysis liquid flow channel outlet corresponds to the first adhesive flow channel blind hole 302, the first cleaning liquid flow channel outlet corresponds to the second adhesive flow channel blind hole 304, the first eluent flow channel outlet corresponds to the third adhesive flow channel blind hole 306, the first reaction liquid flow channel outlet corresponds to the fourth adhesive flow channel blind hole 308, the position where the liquid passes through for the first time after flowing out of the first flow channel is called the adhesive flow channel blind hole, and the size of the adhesive flow channel blind hole is larger than the corresponding adhesive flow channel. This design can correspond to the position of the first flow channel outlet, improve the reliability of the docking position of the first flow channel outlet and the blind hole, and even if there is a slight deviation during docking, it can ensure that the solution coming out of the first flow channel outlet smoothly enters the adhesive flow channel; the first negative pressure channel blind hole 309 corresponds to the first negative pressure channel 127, the second negative pressure channel blind hole 310 corresponds to the second negative pressure channel 128, and the third negative pressure channel blind hole 311 corresponds to the third negative pressure channel 129.

[0049] When using the liquid storage microfluidic chip of the present invention, first, various solutions are respectively filled in each chamber, and a sealing film is attached to the top surface or an elastic sealing cover is added to the top surface of each chamber. When external force is used to extract the liquid, a hollow needle is used to pierce the sealing film or open the sealing cover. Through the hollow structure of the needle, each chamber is connected to the atmosphere, and the sealing partition 126 separating different chambers is pierced to drive the liquid in each chamber to flow under the action of negative pressure. After the liquid is extracted, the needle is pulled out, and the sealing can be achieved again because the back glue 3 is elastic.

[0050] As shown in FIG1 , the state of puncturing the sealing spacer includes: State 1 ( Figure 1A ): Sealed Initially, the solution storage cavity is isolated from the corresponding adhesive flow channel by the sealing spacer 126, and the solution storage cavity and the corresponding adhesive flow channel are not connected to each other; State 2 ( Figure 1B ): Insert the needle to break the membrane The external force drives the needle to press the adhesive backing 3, and the needle pierces the adhesive backing 3 and the sealing spacer 126. At this time, the adhesive backing 3 blocks the channel where the sealing spacer 126 is located, and each solution storage cavity and its corresponding adhesive flow channel are not connected to each other; State 3 ( Figure 1C ): Connect the needle After puncturing the sealing spacer 126, the needle is pulled outward to drive the needle seat away from the adhesive backing, and each solution storage cavity is connected to its corresponding adhesive backing flow channel, and liquid can be drawn at this time; State 4 ( Figure 1D ): Insert the pin to the bottom and seal it After the liquid stored in the solution storage chamber is exhausted, the external force further drives the needle seat to press the adhesive backing 3, and the adhesive backing 3 blocks the channel where the sealing spacer 126 is located, and the solution storage chamber is not connected with the corresponding adhesive backing channel.

[0051] After each liquid extraction step, the corresponding flow channel must be blocked to ensure the smooth extraction. The liquid flow resistance is greater than the gas flow resistance. If the corresponding flow channel is not blocked after the liquid is extracted, the external negative pressure will draw away the air in the previous step, which may cause the liquid in the corresponding chamber to be unable to be extracted in the next step.

[0052] In the present invention, the outlet of the waste liquid chamber flow channel of the first waste liquid chamber 107 is arranged in the middle and upper part of the first waste liquid chamber 107, which ensures that the waste liquid in the first waste liquid chamber 107 will not flow back. The volume of the waste liquid chamber is larger than the volume of the lysate plus the cleaning liquid. After the lysate and the cleaning liquid are pumped into the first waste liquid chamber 107, the liquid level is lower than the outlet of the waste liquid chamber flow channel. The second waste liquid chamber 108 flows into the excess reaction liquid and nucleic acid. In the last step, the third negative pressure channel blind hole 311 is negatively pressured, and the second waste liquid chamber 108 and each reaction chamber are always in a negative pressure state, and the liquid in the second waste liquid chamber 108 will not flow back. After the negative pressure is pumped, there is no external force, the microfluidic chip is placed vertically, and the liquid in the second waste liquid chamber 108 will not flow back. Therefore, the isolation of the waste liquid and the channel can be achieved.

[0053] In the present invention, the volumes of the multiple reaction chambers 106 are the same, so the volumes of the nucleic acids and reaction liquids in the multiple reaction chambers 106 are also the same. Different pathogenic microorganism nucleic acid amplification primers are pre-buried in different reaction chambers 106. After the nucleic acids and reaction liquids in the premixing chamber 105 are evenly diverted into the multiple different reaction chambers 106, the nucleic acids and pathogenic microorganism nucleic acid amplification primers react under the action of the reaction liquid. By observing the color changes of the liquids in the multiple reaction chambers 106, it is possible to determine which types of pathogenic microorganisms are present in the sample. After the experiment is completed, the microfluidic chip can be discarded without cross infection or contamination of the laboratory.

[0054] In the present invention, nucleic acids exist in samples of pathogenic microorganisms. By adding the sample containing pathogenic microorganisms into the lysis solution storage chamber 101, the lysis solution lyses the pathogenic microorganisms, and the nucleic acids in the pathogenic microorganisms are released in the lysis solution. By pumping negative pressure, the liquid in each storage chamber is driven to perform nucleic acid adsorption, nucleic acid cleaning and nucleic acid elution, and finally flows into the premixing chamber 105, thereby realizing the identification of the sample of pathogenic microorganisms based on the reaction between nucleic acid and reaction solution.

[0055] In a specific embodiment, the workflow of the liquid storage microfluidic chip is carried out in the following steps: 1. First, add a sample containing pathogenic microorganisms into the lysis solution storage chamber 101, the lysis solution lyses the pathogenic microorganisms, and the nucleic acid of the pathogenic microorganism is released into the lysis solution under the action of the lysis solution, puncturing the top surface of the lysis solution storage chamber 101 (the purpose is to allow the lysis solution storage chamber 101 to be ventilated so that the solution can be pumped), and the external force drives the needle to pierce the first adhesive flow channel blind hole 302 and the sealing spacer 126 in the first lysis solution flow channel 109, puncture the first negative pressure channel blind hole 309 and draw negative pressure here, the nucleic acid in the sample is adsorbed on the nucleic acid adsorption component 125, and the lysis solution flows from the lysis solution storage chamber 101 through the outflow channel 130, the first lysis solution flow channel 109, the first adhesive flow channel 301, the second lysis solution flow channel 117, the mixing flow channel 121, and the waste liquid chamber flow channel 122 in sequence, and finally flows into the first waste liquid chamber 107. (The purpose of step 1 is to adsorb the nucleic acid on the nucleic acid adsorption component 125. After the extraction, the needle is driven to press the first adhesive flow channel blind hole 302 to block the first adhesive flow channel 301. At this time, the lysis solution storage chamber 101 is not connected to the first adhesive flow channel 301).

[0056] 2. Puncture the top surface of the cleaning liquid storage chamber 102, and drive the needle with external force to pierce the blind hole 304 of the second adhesive flow channel and the sealing spacer in the first cleaning liquid flow channel 111, and draw negative pressure on the blind hole 309 of the first negative pressure channel. The cleaning liquid flows from the cleaning liquid storage chamber 102 through the outflow channel 130, the first cleaning liquid flow channel 111, the second adhesive flow channel 303, the second cleaning liquid flow channel 118, the mixing flow channel 121, and the waste liquid chamber flow channel 122 in sequence, and finally flows into the first waste liquid chamber 107. (The purpose of step 2 is to purify the nucleic acid. After the extraction, the needle is driven to press the blind hole 304 of the second adhesive flow channel, block the second adhesive flow channel 303, and close the first negative pressure channel 127). The following methods can be used to close the first negative pressure channel 127: 1) pull out the needle; 2) directly turn off the negative pressure pump; 3. Add a pinch valve to the tube connecting the negative pressure pump and the negative pressure channel.

[0057] 3. Puncture the top surface of the eluent storage chamber 103, and drive the needle with external force to pierce the third adhesive flow channel blind hole 306 and the sealing spacer 126 in the first eluent flow channel 113, puncture the second negative pressure channel blind hole 310 and draw negative pressure here, and the eluent flows from the eluent storage chamber 103 through the outflow channel 130, the first eluent flow channel 113, the third adhesive flow channel 305, the second eluent flow channel 119, the mixing flow channel 121, the premixing chamber inflow channel 123, and finally flows into the premixing chamber 105 (step 3 elutes the nucleic acid and allows the nucleic acid to flow into the premixing chamber 105. After the extraction, the needle is driven to press the third adhesive flow channel blind hole 306 to block the third adhesive flow channel 305).

[0058] 4. Puncture the top surface of the reaction liquid storage chamber 104, and drive the needle with external force to puncture the fourth adhesive flow channel blind hole 308 and the sealing spacer 126 in the first reaction liquid flow channel 115, and draw negative pressure at the second negative pressure channel blind hole 310. The reaction liquid flows from the reaction liquid storage chamber 104 through the outflow channel 130, the first reaction liquid flow channel 115, the fourth adhesive flow channel 307, the second reaction liquid flow channel 120, and the premixing chamber inflow channel 123 in sequence, and finally the reaction liquid flows into the premixing chamber 105 and is evenly mixed with the nucleic acid.

[0059] 5. Puncture the blind hole 311 of the third negative pressure channel and draw negative pressure here. The nucleic acid and reaction liquid mixed evenly in the premixing chamber 105 pass through the reaction chamber flow channel 124 under the action of negative pressure and finally flow into each reaction chamber 106 for reaction, and the excess liquid finally flows into the second waste liquid chamber 108.

[0060] In the above-mentioned liquid storage microfluidic chip workflow, for experiments using freeze-dried microspheres as reaction raw materials, the above-mentioned step 4 is omitted.

[0061] As described above, the present invention pre-cans a variety of different solutions in each storage cavity in the microfluidic chip, and the sealing spacer 126 is integrally formed and arranged in each flow channel during processing. No additional quantitative device and reagent bottle are required, which is convenient for transportation and storage and easy to operate. Only one microfluidic chip is needed each time to simultaneously detect multiple pathogenic microorganisms in one sample and one experiment, which is quick and convenient, and realizes the instant detection experiment of the microfluidic chip.

Claims

1. A liquid storage microfluidic chip, characterized in that: It includes a chip body, a sealing film and a back glue; the sealing film and the back glue are bonded to the front and back of the chip body respectively; a solution storage cavity, an outflow channel, a first flow channel and a second flow channel are arranged inside the chip body, and a back glue flow channel is arranged on the back glue; wherein: There are several solution storage chambers, the openings of which are arranged on the top surface of the microfluidic chip, and the top surface of the solution storage chamber is isolated and sealed from the outside by a sealant; the bottom of each solution storage chamber is connected to an outflow channel, and the outflow channel is connected to a corresponding first channel, a sealing partition is arranged on the first channel, and the outflow channel and the corresponding first channel are blocked by the sealing partition, and the first channel is connected to the second channel through the corresponding adhesive channel; when working, the top surface of the solution storage chamber is connected to the atmosphere, the adhesive and the sealing partition are pierced so that the chamber is connected to the corresponding adhesive channel, and under the action of negative pressure, the liquid pre-stored in the chamber is released and flows in a direction in the chip through the outflow channel, the first channel, the adhesive channel, and the second channel in sequence.

2. The liquid storage microfluidic chip according to claim 1, characterized in that: The back glue is provided with a back glue flow channel blind hole, and the back glue flow channel blind hole corresponds to the outlet position of the first flow channel.

3. The liquid storage microfluidic chip according to claim 1, characterized in that: A premixing chamber, a reaction chamber and a waste liquid chamber are also provided on the chip body. The output end of the second flow channel is connected to the mixing flow channel, the output end of the mixing flow channel is connected to the premixing chamber, and the premixing chamber is connected to the reaction chamber through the flow channel; the waste liquid chamber is used to collect waste liquid generated during the experiment.

4. The liquid storage microfluidic chip according to claim 3, characterized in that: There are two waste liquid chambers on the chip body: the first waste liquid chamber and the second waste liquid chamber, which are connected to the premixing chamber and the reaction chamber through flow channels respectively; the first waste liquid chamber, the premixing chamber and the second waste liquid chamber are respectively connected to one end of the negative pressure extraction channel, and the other end of the negative pressure extraction channel is a closed end. The blind hole arranged on the closed end is on the backing glue. When extracting liquid, the blind hole is punctured to form the first negative pressure extraction port, the second negative pressure extraction port and the third negative pressure extraction port, respectively.

5. The liquid storage microfluidic chip according to claim 3, characterized in that: The solution storage chamber is used to store sample solutions, cleaning solutions, elution solutions and reaction solutions of pathogenic microorganisms to be tested that have been lysed by lysing solutions; a nucleic acid adsorption component is arranged in the mixing flow channel, and there are several reaction chambers in which different pathogenic microorganism nucleic acid amplification primers are pre-buried.

6. The liquid storage microfluidic chip according to claim 5, characterized in that: The solution storage chamber for storing the reaction solution is omitted, and the reaction solution freeze-dried microspheres are pre-embedded in the premixing chamber to replace the reaction solution.

7. The liquid storage microfluidic chip according to claim 1, characterized in that: The seal on the top surface of the solution storage chamber is designed with a sealing film or a sealing cover; the sealing film is selected from one of a pressure-sensitive film, a back-adhesive film, a PET film or a PC film; the back-adhesive is an elastic material; the bonding method is one or more of hot pressing bonding, ultrasonic bonding, laser bonding, adhesive bonding, and solvent bonding; the core body and the sealing spacer are designed as an integrated whole and are manufactured by injection molding, machining or 3D printing; the bottom surface of the solution storage chamber is a funnel-shaped slope.

8. The liquid storage microfluidic chip according to claim 1, characterized in that: It also includes a puncture component used in conjunction with the chip; the puncture component includes a needle and a needle seat, the needle is used to puncture the backing glue and the sealing spacer, and the needle seat is used to fix the needle.

9. A method for operating the liquid storage microfluidic chip according to any one of claims 1 to 8, characterized in that: The method comprises the steps of driving the liquid in the solution storage chamber to release and flow in a directional manner, which are specifically as follows: (1) Connecting the top surface of the solution storage chamber to the atmosphere; external force drives the needle seat to press the adhesive backing, and the needle pierces the adhesive backing and the sealing spacer. At this time, the adhesive backing blocks the channel where the sealing spacer is located, and each solution storage chamber and its corresponding adhesive backing flow channel are not connected to each other; (2) After puncturing the sealing spacer, the needle is pulled outward, driving the needle seat away from the adhesive backing, and each solution storage chamber is connected to its corresponding adhesive backing flow channel. At this time, liquid is extracted, and the pre-stored liquid in the chamber passes through the outflow flow channel, the first flow channel, the adhesive backing flow channel, and the second flow channel in sequence and is released and flows in the chip in a direction; (3) After the liquid stored in the solution storage chamber is exhausted, the external force drives the needle seat to press the adhesive backing, and the adhesive backing blocks the channel where the sealing spacer is located, and the solution storage chamber is not connected to the corresponding adhesive backing flow channel.

10. The working method of the liquid storage type microfluidic chip according to claim 9, characterized in that: The following steps are involved: First, the top surface of the solution storage chamber storing the sample solution of the pathogenic microorganism to be detected that has been lysed by the lysing solution is connected to the atmosphere, the sealing partition in the corresponding first flow channel is punctured, and negative pressure is drawn to the first negative pressure port. At the same time, the nucleic acid in the sample of the pathogenic microorganism to be detected is adsorbed on the nucleic acid adsorption component, and the lysate in the lysing solution storage chamber is drawn to the first waste liquid chamber; After the extraction, the needle seat is driven to press the adhesive, and the needle presses the corresponding adhesive flow channel blind hole to block the adhesive flow channel; Secondly, the top surface of the solution storage chamber storing the cleaning solution is connected to the atmosphere, the sealing partition in the corresponding first flow channel is punctured, and negative pressure is drawn to the first negative pressure port. The cleaning solution cleans the impurities on the nucleic acid adsorption component, and the cleaning solution and impurities are drawn to the first waste liquid chamber. After the drawing is completed, the needle seat is driven to press the back glue, and the needle head presses the corresponding back glue flow channel blind hole to block the back glue flow channel, and the first negative pressure port is closed; Next, the top surface of the solution storage chamber storing the eluent is connected to the atmosphere, the sealing partition in the corresponding first flow channel is punctured, and negative pressure is drawn to the second negative pressure pumping port, and the eluent elutes the nucleic acid on the nucleic acid adsorption component into the premixing chamber. After the pumping is completed, the needle seat is driven to press the back glue, and the needle head presses the corresponding back glue flow channel blind hole to block the back glue flow channel; Then, the top surface of the solution storage chamber storing the reaction solution is connected to the atmosphere, the sealing spacer in the corresponding first flow channel is punctured, and negative pressure is drawn to the second negative pressure drawing port, so that the reaction solution is drawn into the premixing chamber and mixed evenly with the nucleic acid; Finally, negative pressure is drawn at the third negative pressure port, and the nucleic acid and reaction solution evenly mixed in the premixing chamber flow into each reaction chamber under the action of negative pressure, and the excess solution flows into the second waste liquid chamber; by observing the color changes of the liquids in multiple reaction chambers, the positive and negative nature of the nucleic acid reaction of the pathogenic microorganisms in the sample is judged.

Images