A liquid storage type microfluidic chip and its working method

Abstract

The present invention discloses a liquid storage type microfluidic chip and its working method. The chip includes a chip body, a sealing film and an adhesive; the sealing film and the adhesive are respectively bonded to the front and back of the chip body; a number of solution storage cavities are arranged in the chip body, the outflow channels at the bottom of the solution storage cavities are connected to the corresponding first channels, a sealing spacer is arranged on the first channels, the outflow channels and the corresponding first channels are blocked by the sealing spacer, and the first channels are communicated with the corresponding second channels through the adhesive channels. In the whole experimental process of the present invention, no external reagent bottle is required, and the purposes of instant detection, preparation and separation of samples by the microfluidic chip can be achieved, greatly shortening the detection time and simplifying the experimental steps. The waste liquid generated during the experimental process of the present invention flows into the waste liquid cavity, avoiding cross-contamination in the laboratory. The chip of the present invention can be used to identify the types of pathogenic microorganisms.

Description

Technical Field

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

[0002] A microfluidic chip, also known as a lab-on-a-chip, can integrate basic operation units such as sample preparation, reaction, separation, and detection in the fields of biology, chemistry, medicine, etc. on a single chip, with the characteristics of high integration and strong automation. In recent years, there has emerged the application of microfluidic chips in fully automated and integrated biological detection POCT (Full name: point-of-care testing, Chinese name: instant testing), which has broad application prospects and important application values.

[0003] Currently, for functions such as reagent storage in microfluidic chips, it is mostly necessary to externally connect different reagent bottles to the chip and use a driving pump to provide power to transport the external reagents into the microfluidic chip.

[0004] Its operation is cumbersome and complex, requiring the use of quantitative devices (such as pipettes) and external pipelines and reagent bottles. The structural design is complex, and repeated use of the external pipelines may cause contamination, which may lead to incorrect experimental results, is not conducive to transportation and storage, is not conducive to simplifying the process, has high requirements for the operation level of experimental personnel, and is not conducive to the development of the full automation of microfluidic chips. At the same time, for fully automated and integrated biological detection devices of microfluidic chips, the goal of sample in and result out is important. To achieve the goal of sample in and result out, it is necessary to pre-fill various reagents. The method of externally connecting reagent bottles limits the popularization and application of the instant testing method of microfluidic chips; in addition, it is also necessary to ensure that the liquid reagents pre-filled in the microfluidic chip can be effectively released when needed. Therefore, the pre-storage and release of liquid reagents in microfluidic chips are crucial. Therefore, how to achieve reagent storage and sealing on a microfluidic chip is an urgent problem to be solved currently.

[0005] Chinese Patent Application No. 201810957492.1 provides a microfluidic chip with a separated design of the storage module and the reaction module. During use, it is necessary to dock the storage module and the reaction module, and the operation is complex and cumbersome. Moreover, it requires additional complex connection structures and valves to connect the storage module and the reaction chamber module, has high requirements for the technical level of operators, is not conducive to the popularization and application of instant detection of microfluidic chips, and has a high use cost.

[0006] Chinese Patent Application No. 202310632430.4 provides a rotary valve type microfluidic chip, which requires an additional rotary valve driving device to drive the rotary valve to rotate, and the assembly and operation are cumbersome, and it is not conducive to the popularization and application of instant testing of microfluidic chips. 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:

[0010] 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.

[0011] 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.

[0012] 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.

[0013] 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.

[0014] 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.

[0015] In the present invention, the solution storage chamber for storing the reaction solution is omitted, and the reaction solution freeze-dried microspheres are embedded in the premixing chamber to replace the reaction solution.

[0016] In the present invention, 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, an adhesive film, a PET film, or a PC film; the adhesive is made of an elastic material; the bonding method is one or several of thermocompression bonding, ultrasonic bonding, laser bonding, adhesive bonding, and solvent bonding; the core body and the sealing spacer are integrally designed and manufactured by injection molding, machining, or 3D printing; the bottom surface of the solution storage chamber is an inclined surface in the shape of a funnel.

[0017] In the present invention, it further includes a puncturing assembly used in cooperation with the chip; the puncturing assembly includes a needle and a needle seat, the needle is used to puncture the adhesive and the sealing spacer, and the needle seat is used to fix the needle.

[0018] The present invention also provides a working method for the above-mentioned liquid storage microfluidic chip, which includes the step of driving the pre-stored liquid in the solution storage chamber to flow out directionally, specifically as follows:

[0019] (1) Connect the top surface of the solution storage chamber to the atmosphere; an external force drives the needle seat to press the adhesive tightly, and the needle pierces the adhesive and the sealing spacer. At this time, the adhesive blocks the channel where the sealing spacer is located, and each solution storage chamber and its corresponding adhesive flow channel are not connected to each other;

[0020] (2) After piercing the sealing spacer, pull out the needle outward, drive the needle seat away from the adhesive, and each solution storage chamber is connected to its corresponding adhesive flow channel. At this time, liquid extraction is carried out, and the pre-stored liquid in the chamber sequentially passes through the outflow channel, the first channel, the adhesive flow channel, and the second channel and is released and flows directionally in the chip;

[0021] (3) After the liquid stored in the solution storage chamber is pumped out, an external force drives the needle seat to press the adhesive tightly, and the adhesive blocks the channel where the sealing spacer is located, and the solution storage chamber is not connected to its corresponding adhesive flow channel.

[0022] After each liquid extraction step described above, the corresponding flow channel needs to be blocked to ensure that the liquid extraction in other solution storage chambers with a connection relationship and a shared flow channel can proceed smoothly in the subsequent steps.

[0023] Further, the working method of the liquid storage microfluidic chip of the present invention specifically includes the following steps:

[0024] First, the top surface of the solution storage chamber storing the sample solution of the pathogenic microorganism to be detected lysed by the lysis solution is communicated with the atmosphere. The sealing partition in the corresponding first flow channel is punctured, and negative pressure is applied to the first negative pressure extraction port. While the nucleic acid in the sample of the pathogenic microorganism to be detected is adsorbed on the nucleic acid adsorption component, the lysis solution in the lysis solution storage chamber is pumped into the first waste liquid chamber; after pumping is completed, the needle seat is driven to press the back glue, and the needle presses the corresponding blind hole of the back glue flow channel to block the back glue flow channel;

[0025] Secondly, the top surface of the solution storage chamber storing the cleaning solution is communicated with the atmosphere. The sealing partition in the corresponding first flow channel is punctured, and negative pressure is applied to the first negative pressure extraction port. The cleaning solution cleans the impurities of the nucleic acid, and the cleaning solution and the impurities are pumped into the first waste liquid chamber. After pumping is completed, the needle seat is driven to press the back glue, and the needle presses the corresponding blind hole of the back glue flow channel to block the back glue flow channel, and the first negative pressure extraction port is closed;

[0026] Next, the top surface of the solution storage chamber storing the elution solution is communicated with the atmosphere. The sealing partition in the corresponding first flow channel is punctured, and negative pressure is applied to the second negative pressure extraction port. The elution solution elutes the nucleic acid on the nucleic acid adsorption component into the premixing chamber. After pumping is completed, the needle seat is driven to press the back glue, and the needle presses the corresponding blind hole of the back glue flow channel to block the back glue flow channel;

[0027] Then, the top surface of the solution storage chamber storing the reaction solution is communicated with the atmosphere. The sealing partition in the corresponding first flow channel is punctured, and negative pressure is applied to the second negative pressure extraction port. The reaction solution is pumped into the premixing chamber and mixed evenly with the nucleic acid;

[0028] Finally, negative pressure is applied to the third negative pressure extraction port. The nucleic acid and the reaction solution mixed evenly 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 change of the liquid in multiple reaction chambers, the positive or negative of the nucleic acid reaction of the pathogenic microorganism in the sample is judged.

[0029] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0030] By arranging sealing partitions inside each chamber and the corresponding flow channels, and the sealing partitions are integrally formed with the microfluidic chip body. This sealing method has a better sealing effect compared with the additional arrangement of sealing partitions. The solutions stored in each chamber will not leak or ooze out through the integrally formed sealing partitions, enabling the solutions in the chambers to be stably sealed and stored inside each chamber;

[0031] By means of pressing for on / off (pressing the needle tip with external force to keep the flow channel sealed), the opening and closing of each blind hole are controlled. Without the need to additionally use complex valve structures and transfer devices, the solution is pre-stored in the microfluidic chip body. Through reasonable structure and flow channel design, the transfer and release of the solution in different experimental steps are realized. The operation is simple and convenient, which is very suitable for the on-site experiments of microfluidic chips. The disposable design of use-and-throw avoids the problem of cross-contamination.

[0032] Through the reasonable structural design of the flow channels, each chamber and the sealing method of each chamber in the microfluidic chip, the stable and long-term sealed storage of various reagents inside the microfluidic chip is realized. Moreover, the waste liquid generated during the experiment flows into the first waste liquid chamber and the second waste liquid chamber in the microfluidic chip and will not come into contact with the external environment of the laboratory, avoiding cross-contamination in the laboratory.

[0033] Before the experiment of the present invention, the solutions in each chamber are separated 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 of the microfluidic chip without connecting an external reagent bottle, greatly shortening the operation time and simplifying the operation steps.

[0034] For the case of liquid reaction solutions, the liquid reaction solutions have an independent storage chamber, and will only be released into the premixing chamber after the experiment starts, ensuring the stability of the reaction solutions.

[0035] For the case of pre-embedded lyophilized microspheres of reaction solutions in the premixing chamber, each liquid storage device is physically isolated from the lyophilized microspheres, so that the lyophilized microspheres are not affected by the moisture of the reagent, ensuring the activity of the enzyme and the accuracy and precision of the experimental results.

[0036] The chip of the present invention can enable the reaction solution to flow into the premixing chamber, be mixed evenly with the nucleic acid after the pathogenic microorganism sample is lysed, washed, adsorbed and eluted, and then enter the reaction chamber for reaction to realize the identification of the types of pathogenic microorganisms. Description of the Drawings

[0037] Figure 1A It is a schematic diagram of the first state of piercing the sealing partition and pressing for on / off.

[0038] Figure 1B It is a schematic diagram of the second state of piercing the sealing partition and pressing for on / off.

[0039] Figure 1C It is a schematic diagram of the second state of piercing the sealing partition and pressing for on / off.

[0040] Figure 1D It is a schematic diagram of the second state of piercing the sealing partition and pressing for on / off.

[0041] Figure 2It is a schematic structural diagram of the liquid storage type microfluidic chip body.

[0042] Figure 3 It is a schematic structural diagram of the adhesive layer of the liquid storage type microfluidic chip.

[0043] Figure 4 It is a schematic diagram of a nucleic acid adsorption component.

[0044] Figure 5 It is a three-layer structure diagram of the chip.

[0045] Markings in the figure: 1-chip body, 101-lysis solution storage chamber, 102-washing solution storage chamber, 103-elution solution storage chamber, 104-reaction solution storage chamber, 105-premixing chamber, 106-reaction chamber, 107-first waste liquid chamber, 108-second waste liquid chamber, 109-first lysis solution flow channel, 111-first washing solution flow channel, 113-first elution solution flow channel, 115-first reaction solution flow channel, 117-second lysis solution flow channel, 118-second washing solution flow channel, 119-second elution solution flow channel, 120-second reaction solution flow channel, 121-mixing flow channel, 122-waste liquid chamber flow channel, 123-premixing chamber inflow 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 channel;

[0046] 2-sealing film, 3-adhesive layer, 301-first adhesive layer flow channel, 302-first adhesive layer flow channel blind hole, 303-second adhesive layer flow channel, 304-second adhesive layer flow channel blind hole, 305-third adhesive layer flow channel, 306-third adhesive layer flow channel blind hole, 307-fourth adhesive layer flow channel, 308-fourth adhesive layer 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. Specific embodiments

[0047] The technical solutions of the present invention will be introduced in detail below in conjunction with the accompanying drawings and embodiments.

[0048] The present invention provides a liquid storage type microfluidic cavity chip, which includes a chip body 1, a sealing film 2 and an adhesive layer 3. The sealing film 2 is bonded to the front surface of the chip body 1, and the adhesive layer 3 is bonded to the back surface of the chip body 1; the adhesive layer 3 is made of an elastic adhesive material (rubber, adhesive layer, or other materials can be used), and the bonding method is one or a combination of hot pressing bonding, ultrasonic bonding, laser bonding, adhesive bonding, and solvent bonding. The sealing film 2 is used to seal the flow channels and chambers provided on the front surface of the chip. The sealing film 2 is selected from one of a pressure-sensitive film, an adhesive film, a PET film, and a PC film. Preferably, it is a transparent film.

[0049] The chip body 1 is provided with:

[0050] 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;

[0051] First lysis solution flow channel 109, first cleaning solution flow channel 111, first eluent flow channel 113, first reaction solution flow channel 115;

[0052] Second lysis solution flow channel 117, second cleaning solution flow channel 118, second eluent flow channel 119, second reaction solution flow channel 120;

[0053] Mixing flow channel 121, waste liquid chamber flow channel 122.

[0054] The lysis solution storage chamber 101 is used for filling and storing samples of pathogenic microorganisms to be detected lysed by the lysis solution; the cleaning solution storage chamber 102 is used for storing the cleaning solution; the eluent storage chamber 103 is used for storing the eluent; the reaction solution storage chamber 104 is used for storing the 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 solutions stored in each solution storage chamber only contact the chip body, and do not contact the sealing film 2 of the chip body 1 and the adhesive 3 on the back of the chip, avoiding the release of interfering substances from the sealing film 2 and the adhesive material into the solution, enabling the solutions stored in each solution storage chamber to be stored stably for a long time without deterioration, that is, not interfering with each other or mixing, and at the same time preventing the release of interfering substances from the sealing film 2 and the adhesive material from inhibiting the reaction between the reaction solution and nucleic acid, resulting in false negatives, ensuring the accuracy of the experimental results.

[0055] Each solution storage chamber communicates with a corresponding outflow channel 130 provided at its bottom. The outflow channels 130 are provided inside the microfluidic chip body. The corresponding outflow channels 130 are respectively connected to corresponding first channels (the first lysis solution channel 109, the first cleaning solution channel 111, the first elution solution channel 113, and the first reaction solution channel 115). The outflow channels 130 and the corresponding first channels are blocked by a sealing spacer 126 (the sealing spacer 126 is provided at any position of the first channel. For the convenience of processing and production, the sealing spacer 126 can be provided at the junction of the outflow channel 130 and the corresponding first channel. The sealing spacer 126 is integrally formed with the chip body 1, and the first channel communicates with the adhesive channel provided on the corresponding adhesive 3). With such a design, the solutions pre-stored in each solution storage chamber neither contact the sealing film 2 on the front side of the chip nor contact the adhesive 3 on the back side, avoiding the release of substances in the materials of the sealing film 2 and the adhesive 3 into the solutions in each solution storage chamber, thereby inhibiting the reaction between the subsequent reaction solution and nucleic acid, and ensuring the accuracy and reliability of the experimental results; at the same time, it further ensures that the solutions pre-stored in each solution storage chamber do not contact the sealing film 2 and the adhesive 3, preventing the solutions in each solution storage chamber from deteriorating, enabling the solutions pre-stored in each solution storage chamber to be stored stably for a long time, and further ensuring the accuracy of the experimental results.

[0056] The bottom surface of each solution storage chamber is an inclined surface in the shape of a funnel. With such a design, the lysed sample solution can be completely drained under the action of negative pressure, so that there is no loss and residue of the lysed sample solution, avoiding the nucleic acid in the sample remaining on the bottom surface of the lysis solution storage chamber, preventing the loss of nucleic acid samples, and ensuring the accuracy of the experimental results during quantitative detection.

[0057] In each solution storage chamber, after filling various solutions into each chamber, a sealing film is covered on the top surface or an elastic sealing cover is covered to prevent the solutions pre-filled in each solution storage chamber from volatilizing; when driving the liquid in each chamber to flow, the sealing film on the top surface of each chamber is punctured or the sealing cover is opened.

[0058] The first flow channels (the first lysis solution flow channel 109, the first cleaning solution flow channel 111, the first elution solution flow channel 113, the first reaction solution flow channel 115) are designed not to penetrate through the microfluidic chip body. The lysis solution storage cavity 101 is communicated with the corresponding lysis solution outflow channel, and the lysis solution outflow channel is communicated with the first lysis solution flow channel 109. For the convenience of processing and production, a sealing spacer 126 is provided at the junction of the first lysis solution flow channel 109 and the lysis solution outflow channel. The sealing spacer 126 is integrally formed with the chip body 1. With such a design, the solution pre-stored in the lysis solution storage cavity 101 only contacts the chip body 1 and does not contact other materials, ensuring the stable storage and sealed non-leakage of the pre-stored solution; and it avoids the solution contacting other materials, preventing other materials from releasing uncertain substances to inhibit the reaction between nucleic acid and the reaction solution, resulting in false negatives, and ensuring the accuracy and reliability of the experimental results; it is fabricated by injection molding, machining or 3D printing. The sealing spacer 126 separates the first lysis solution flow channel 109 from the lysis solution storage cavity 101 to keep the lysis solution sealed in the lysis solution storage cavity 101; piercing the sealing spacer 126, the lysis solution storage cavity 101 is communicated with the first lysis solution flow channel 109;

[0059] A sealing spacer 126 is provided in the first cleaning solution flow channel 111. The sealing spacer 126 is integrally formed with the chip body 1. The sealing spacer 126 separates the first cleaning solution flow channel 111 from the cleaning solution storage cavity 102 to keep the cleaning solution sealed in the cleaning solution storage cavity 102; piercing the sealing spacer 126, the cleaning solution storage cavity 102 is communicated with the first cleaning solution flow channel 111;

[0060] A sealing spacer 126 is provided in the first elution solution flow channel 113. The sealing spacer 126 is integrally formed with the chip body 1. The sealing spacer 126 separates the first elution solution flow channel 113 from the elution solution storage cavity 103 to keep the elution solution sealed in the elution solution storage cavity 103; piercing the sealing spacer 126, the elution solution storage cavity 103 is communicated with the first elution solution flow channel 113;

[0061] A sealing spacer 126 is provided in the first reaction solution flow channel 115. The sealing spacer 126 is integrally formed with the chip body 1. The sealing spacer 126 separates the first reaction solution flow channel 115 from the reaction solution storage cavity 104 to keep the reaction solution sealed in the reaction solution storage cavity 104; piercing the sealing spacer 126, the reaction solution storage cavity 104 is communicated with the first reaction solution flow channel 115.

[0062] 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;

[0063] 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.

[0064] 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.

[0065] 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.

[0066] 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.

[0067] The adhesive backing 3 is provided with:

[0068] The first back glue runner 301 and the first back glue runner blind hole 302, the second back glue runner 303 and the second back glue runner blind hole 304, the third back glue runner 305 and the third back glue runner blind hole 306, the fourth back glue runner 307 and the fourth back glue runner blind hole 308, the first negative pressure extraction channel blind hole 309, the second negative pressure extraction channel blind hole 310 and the third negative pressure extraction channel blind hole 311.

[0069] The first lysis solution runner 109 and the second lysis solution runner 117 are connected and communicated through the first back glue runner 301;

[0070] The first cleaning solution runner 111 and the second cleaning solution runner 118 are connected and communicated through the second back glue runner 303;

[0071] The first elution solution runner 113 and the second elution solution runner 119 are connected and communicated through the third back glue runner 305;

[0072] The first reaction solution runner 115 and the second reaction solution runner 120 are connected and communicated through the fourth back glue runner 307;

[0073] The outlet of the first lysis solution runner corresponds to the first back glue runner blind hole 302, the outlet of the first cleaning solution runner corresponds to the second back glue runner blind hole 304, the outlet of the first elution solution runner corresponds to the third back glue runner blind hole 306, the outlet of the first reaction solution runner corresponds to the fourth back glue runner blind hole 308. The position where the liquid passes through for the first time after flowing out of the first runner is called the back glue runner blind hole. The size of the back glue runner blind hole is larger than that of the corresponding back glue runner. Such a design can correspond to the position of the first runner outlet, improving the reliability of the docking position between the first runner outlet and the blind hole. Even if there is a slight deviation during docking, it can ensure that the solution flowing out of the first runner outlet smoothly enters the back glue runner; the first negative pressure extraction channel blind hole 309 corresponds to the first negative pressure extraction channel 127, the second negative pressure extraction channel blind hole 310 corresponds to the second negative pressure extraction channel 128, and the third negative pressure extraction channel blind hole 311 corresponds to the third negative pressure extraction channel 129.

[0074] When using the liquid storage type microfluidic chip of the present invention, various solutions are first filled into each chamber respectively, 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 extracting liquid by external force, the sealing film is punctured with a hollow needle or the sealing cover is opened, and each chamber is connected to the atmosphere through the hollow structure of the needle. The sealing partition 126 separating different chambers is punctured, and the liquid in each chamber is driven to flow under the action of negative pressure. After the liquid is extracted, when the needle is pulled out, due to the elasticity of the back glue 3, sealing can be achieved again.

[0075] As shown in Figure 1, the states of puncturing the sealing partition include:

[0076] State 1 ( Figure 1A ) : Sealed

[0077] Initially, the solution storage chamber is isolated from its corresponding back glue flow channel by the sealing spacer 126, and the solution storage chamber and its corresponding back glue flow channel are not connected to each other.

[0078] State 2 ( Figure 1B ): Insert the needle to break the film

[0079] An external force drives the needle to press against the back glue 3, and the needle pierces through the back glue 3 and the sealing spacer 126. At this time, the back glue 3 blocks the channel where the sealing spacer 126 is located, and each solution storage chamber and its corresponding back glue flow channel are not connected to each other.

[0080] State 3 ( Figure 1C ): Connect and pull out the needle

[0081] After piercing the sealing spacer 126, pull out the needle outward, drive the needle seat away from the back glue, and each solution storage chamber is connected to its corresponding back glue flow channel. At this time, liquid can be pumped.

[0082] State 4 ( Figure 1D ): Insert the needle to the bottom and then seal

[0083] After the liquid stored in the solution storage chamber is pumped out, further, an external force drives the needle seat to press against the back glue 3, and the back glue 3 blocks the channel where the sealing spacer 126 is located, and the solution storage chamber is not connected to its corresponding back glue flow channel.

[0084] After each liquid pumping step, the corresponding flow channel should be blocked, which ensures the smooth progress of liquid pumping. The liquid flow resistance is greater than the gas flow resistance. If the corresponding flow channel is not blocked after pumping the liquid, the external negative pressure will suck away the air in the previous step, which may cause the liquid in the corresponding chamber in the next step to be unable to be pumped.

[0085] In the present invention, the waste liquid chamber flow channel outlet of the first waste liquid chamber 107 is arranged in the upper middle 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 greater than the volume of the lysis solution plus the cleaning solution. After the lysis solution and the cleaning solution are pumped into the first waste liquid chamber 107, the liquid level is lower than the waste liquid chamber flow channel opening. What flows into the second waste liquid chamber 108 are the excess reaction liquid and nucleic acid. In the last step, a negative pressure is applied to the blind hole 311 of the third negative pressure pumping channel. 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 pumping is completed, without an external force and with the microfluidic chip placed vertically, 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.

[0086] In the present invention, the volumes of the multiple reaction chambers 106 are the same. Therefore, the volumes of nucleic acids and reaction solutions in the multiple reaction chambers 106 are also the same. Different pathogenic microorganism nucleic acid amplification primers are pre-embedded in different reaction chambers 106. When the nucleic acids and reaction solutions in the premixing chamber 105 are respectively 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 solution. By observing the color change of the liquid 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 causing cross-infection and without contaminating the laboratory.

[0087] In the present invention, nucleic acids are present in a sample 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 into the lysis solution. By applying negative pressure, the liquids in each storage chamber are driven for nucleic acid adsorption, nucleic acid washing, and nucleic acid elution, and finally flow into the premixing chamber 105, thereby realizing the identification of the sample of pathogenic microorganisms based on the reaction of nucleic acids and the reaction solution.

[0088] In a specific embodiment, the working process of the liquid storage type microfluidic chip is carried out in the following steps in sequence:

[0089] 1. First, add the sample containing pathogenic microorganisms into the lysis solution storage chamber 101. The lysis solution lyses the pathogenic microorganisms, and the nucleic acids of the pathogenic microorganisms are released into the lysis solution under the action of the lysis solution. Pierce the top surface of the lysis solution storage chamber 101 (the purpose is to make the lysis solution storage chamber 101 communicate with the atmosphere so that the solution can be pumped). Drive a needle to pierce the sealing spacer 126 in the first back glue flow channel blind hole 302 and the first lysis solution flow channel 109, pierce the first negative pressure pumping channel blind hole 309 and apply negative pressure here. The nucleic acids in the sample are adsorbed on the nucleic acid adsorption assembly 125. 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 back glue flow channel 301, the second lysis solution flow channel 117, the mixing flow channel 121, and the waste liquid chamber flow channel 122, and finally flows into the first waste liquid chamber 107. (The purpose of step 1 is to adsorb the nucleic acids on the nucleic acid adsorption assembly 125. After pumping, drive the needle to press tightly against the first back glue flow channel blind hole 302 to block the first back glue flow channel 301. At this time, the lysis solution storage chamber 101 is not connected to the first back glue flow channel 301).

[0090] 2. Pierce the top surface of the cleaning liquid storage chamber 102. Drive the needle through the sealing partitions in the second back glue flow channel blind hole 304 and the first cleaning liquid flow channel 111 by external force, and apply negative pressure to the first negative pressure extraction channel blind hole 309. The cleaning liquid flows through the outflow channel 130, the first cleaning liquid flow channel 111, the second back glue flow channel 303, the second cleaning liquid flow channel 118, the mixing channel 121, and the waste liquid chamber flow channel 122 in sequence from the cleaning liquid storage chamber 102, and finally flows into the first waste liquid chamber 107. (The purpose of step 2 is to purify nucleic acid. After extraction, drive the needle to press tightly against the second back glue flow channel blind hole 304 to block the second back glue flow channel 303, and close the first negative pressure extraction channel 127); The first negative pressure extraction channel 127 can be closed by the following methods: 1) Pull out the needle; 2) Directly close the negative pressure pump; 3. Install a pinch valve on the tube connecting the negative pressure pump and the negative pressure extraction channel.

[0091] 3. Pierce the top surface of the eluent storage chamber 103. Drive the needle through the sealing partitions 126 in the third back glue flow channel blind hole 306 and the first eluent flow channel 113 by external force, pierce the second negative pressure extraction channel blind hole 310 and apply negative pressure here. The eluent flows through the outflow channel 130, the first eluent flow channel 113, the third back glue flow channel 305, the second eluent flow channel 119, the mixing channel 121, and the pre-mixing chamber inflow channel 123 in sequence from the eluent storage chamber 103, and finally flows into the pre-mixing chamber 105 (step 3 elutes the nucleic acid and allows the nucleic acid to flow into the pre-mixing chamber 105. After extraction, drive the needle to press tightly against the third back glue flow channel blind hole 306 to block the third back glue flow channel 305).

[0092] 4. Pierce the top surface of the reaction liquid storage chamber 104. Drive the needle through the sealing partitions 126 in the fourth back glue flow channel blind hole 308 and the first reaction liquid flow channel 115 by external force, and apply negative pressure at the second negative pressure extraction channel blind hole 310. The reaction liquid flows through the outflow channel 130, the first reaction liquid flow channel 115, the fourth back glue flow channel 307, the second reaction liquid flow channel 120, and the pre-mixing chamber inflow channel 123 in sequence from the reaction liquid storage chamber 104, and finally the reaction liquid flows into the pre-mixing chamber 105 to be mixed evenly with the nucleic acid.

[0093] 5. Pierce the third negative pressure extraction channel blind hole 311 and apply negative pressure here. The evenly mixed nucleic acid and reaction liquid in the pre-mixing chamber 105 flow 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.

[0094] In the working process of the above liquid storage type microfluidic chip, when conducting experiments with freeze-dried microspheres as reaction raw materials, omit step 4 above.

[0095] In summary, in the present invention, a variety of different solutions are pre-filled in each storage cavity within the microfluidic chip, and the sealing spacer 126 is integrally formed in each flow channel during processing, eliminating the need for additional metering devices and reagent bottles, facilitating transportation and storage, with simple operation. Each time, only one microfluidic chip is required to simultaneously detect multiple pathogenic microorganisms in one sample in one experiment, which is fast and convenient, realizing 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 opening of the solution storage chamber is 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 sealing member; the bottom of each solution storage chamber is connected to the outflow channel, the outflow channel is connected to the corresponding first channel, a sealing spacer is arranged on the first channel, the outflow channel and the corresponding first channel are blocked by the sealing spacer, and the first channel is connected to the second channel through the corresponding adhesive backing channel; 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 adhesive backing and the sealing spacer, and the needle seat is used to fix the needle; When working, the top surface of the solution storage cavity is connected to the atmosphere, and the adhesive and the sealing spacer are pierced to connect the cavity with its corresponding adhesive flow channel. Under the action of negative pressure, the liquid pre-stored in the cavity is released and flows in the chip in a direction through the outflow flow channel, the first flow channel, the adhesive flow channel, and the second flow channel in sequence; the state of the piercing component piercing the sealing spacer includes: State 1: Closed Initially, the solution storage cavity is isolated from the corresponding adhesive flow channel by a sealing spacer, and the solution storage cavity and the corresponding adhesive flow channel are not connected to each other; State 2: The membrane is broken by the needle The external force drives the needle to compress 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 cavity and its corresponding adhesive backing flow channel are not connected to each other; State 3: Connecting and removing needle After puncturing the sealing spacer, 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 the liquid can be drawn at this time; State 4: Insert the pin to the bottom and then seal it After the liquid stored in the solution storage chamber is exhausted, the external force further drives the needle seat to compress the adhesive backing, and the adhesive backing blocks the channel where the sealing spacer is located, and the solution storage chamber is disconnected from the adhesive backing flow channel corresponding thereto.

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 chip 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. A method for operating the liquid storage microfluidic chip according to any one of claims 1 to 7, characterized in that: It is used for non-disease diagnosis and treatment purposes; it includes the steps of driving the directional release flow of liquid in the solution storage chamber, which are 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.

9. The working method of the liquid storage type microfluidic chip according to claim 8, 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.

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