Disclosure of Invention
Aiming at the defects of the prior art, the invention aims to provide a liquid storage type micro-fluidic chip which is used for solving the problems of reagent storage and sealing of the micro-fluidic chip.
The technical scheme of the invention is specifically introduced as follows.
The invention provides a liquid storage type micro-fluidic chip, which comprises a chip body, a sealing film and a back adhesive, wherein the sealing film and the back adhesive are respectively bonded on the front surface and the back surface of the chip body, a solution storage cavity, an outflow runner, a first runner and a second runner are arranged in the chip body, and the back adhesive runner is arranged on the back adhesive, wherein:
The liquid storage device comprises a microfluidic chip, a plurality of solution storage cavities, sealing elements, sealing spacers, a plurality of back glue channels and a plurality of sealing spacers, wherein the openings of the solution storage cavities are arranged on the top surface of the microfluidic chip, the top surface of the solution storage cavities is isolated and sealed with the outside through the sealing elements, the bottoms of the solution storage cavities are communicated with outflow channels, the outflow channels are connected with corresponding first channels, the sealing spacers are arranged on the first channels, the outflow channels are blocked by the sealing spacers, the first channels are communicated with corresponding back glue channels through the second channels, the top surface of the solution storage cavities are communicated with the atmosphere during operation, the back glue and the sealing spacers are pierced to enable the cavities to be communicated with the corresponding back glue channels, and liquid pre-stored in the cavities sequentially passes through the outflow channels, the first channels, the back glue channels and the second channels under the action of negative pressure to directionally release and flow in the chip.
In the invention, the back adhesive is provided with a back adhesive flow passage blind hole, and the back adhesive flow passage blind hole corresponds to the outlet position of the first flow passage.
In the invention, the chip body is also provided with a premixing cavity, a reaction cavity and a waste liquid cavity, the output end of the second flow channel is communicated with the mixing flow channel, the output end of the mixing flow channel is communicated with the premixing cavity, the premixing cavity is connected with the reaction cavity through the flow channel, and the waste liquid cavity is used for collecting waste liquid generated in the experimental process.
The chip comprises a chip body, a first waste liquid cavity, a second waste liquid cavity, a pre-mixing cavity, a reaction cavity, a negative pressure pumping channel, a blind hole, a first negative pressure pumping port, a second negative pressure pumping port and a third negative pressure pumping port, wherein the first waste liquid cavity and the second waste liquid cavity are respectively connected with the pre-mixing cavity and the reaction cavity through a flow channel, the first waste liquid cavity, the pre-mixing cavity and the second waste liquid cavity are respectively connected with one end of the negative pressure pumping channel, the other end of the negative pressure pumping channel is a closed end, the blind hole is formed in the closed end and is on a back adhesive, and the blind hole is pierced during liquid pumping.
In the invention, the solution storage cavity is used for storing sample solution, cleaning solution, eluent and reaction solution of pathogenic microorganisms to be detected, which are cracked by the cracking solution, the nucleic acid adsorption components are arranged in the mixing flow channel, the reaction cavities are provided with a plurality of reaction cavities, and different pathogenic microorganism nucleic acid amplification primers are pre-embedded in the reaction cavities.
In the invention, a solution storage cavity for storing the reaction solution is omitted, and the reaction solution freeze-dried microspheres are pre-buried in the pre-mixing cavity to replace the reaction solution.
The sealing piece on the top surface of the solution storage cavity is designed by adopting a sealing film or a sealing cover, the sealing film is one of a pressure sensitive film, a back adhesive film, a PET film or a PC film, the back adhesive is made of an elastic material, the bonding mode is one or more of hot press bonding, ultrasonic bonding, laser bonding, adhesive bonding and solvent bonding, the core body and the sealing partition piece are integrally designed and manufactured in an injection molding or machining or 3D printing mode, and the bottom surface of the solution storage cavity is a funnel-shaped inclined plane.
The invention also comprises a puncture assembly matched with the chip, wherein the puncture assembly comprises a needle head and a needle seat, the needle head is used for puncturing the back glue and sealing the partition, and the needle seat is used for fixing the needle head.
The invention also provides a working method of the liquid storage type micro-fluidic chip, which comprises the steps of driving the pre-stored liquid in the solution storage cavity to directionally flow and release, and specifically comprises the following steps:
(1) The needle head pierces the back glue and the sealing spacer, at the moment, the back glue blocks the channel where the sealing spacer is positioned, and each solution storage cavity is not communicated with the corresponding back glue flow channel;
(2) After the sealing partition piece is pierced, the needle is pulled outwards, the needle seat is driven to be far away from the back glue, each solution storage cavity is communicated with the corresponding back glue flow channel, liquid is pumped at the moment, and the liquid pre-stored in the cavity passes through the outflow flow channel, the first flow channel, the back glue flow channel and the second flow channel in sequence to release and flow in the chip in a directional manner;
(3) After the liquid stored in the solution storage cavity is pumped out, the external force drives the needle seat to compress the back glue, the back glue blocks the channel where the sealing partition piece is located, and the solution storage cavity is not communicated with the corresponding back glue flow channel.
After each liquid pumping step is finished, the corresponding flow channel is blocked, so that liquid pumping in other solution storage cavities with communication relation and shared flow channels in the subsequent steps can be smoothly performed.
Further, the working method of the liquid storage type micro-fluidic chip specifically comprises the following steps:
Firstly, the top surface of a solution storage cavity for storing a sample solution of pathogenic microorganisms to be detected, which is cracked by a cracking solution, is communicated with the atmosphere, a sealing spacer in a corresponding first runner is pierced, negative pressure is pumped to a first negative pressure pumping port, and simultaneously, the nucleic acid in the sample of the pathogenic microorganisms to be detected is adsorbed on a nucleic acid adsorption component, the cracking solution in the cracking solution storage cavity is pumped to a first waste liquid cavity;
Secondly, the top surface of a solution storage cavity for storing the cleaning solution is communicated with the atmosphere, a sealing partition piece in a corresponding first runner is pierced, negative pressure is pumped to a first negative pressure pumping port, the cleaning solution cleans impurities of nucleic acid, the cleaning solution and the impurities are pumped to a first waste liquid cavity, a needle seat is driven to press back glue after the cleaning solution and the impurities are pumped, a needle head presses a blind hole of the corresponding back glue runner, the back glue runner is blocked, and the first negative pressure pumping port is closed;
Then, the top surface of a solution storage cavity for storing the eluent is communicated with the atmosphere, a sealing partition piece in a corresponding first runner is pierced, negative pressure is pumped to a second negative pressure pumping port, the eluent elutes nucleic acid on the nucleic acid adsorption component into a premixing cavity, a needle seat is driven to press gum after the elution is completed, and a needle head presses a blind hole of a corresponding gum runner to block the gum runner;
Then, the top surface of a solution storage cavity for storing the reaction solution is communicated with the atmosphere, a sealing partition piece in a corresponding first flow channel is pierced, negative pressure is pumped to a second negative pressure pumping port, and the reaction solution is pumped into a premixing cavity and is uniformly mixed with nucleic acid;
And finally, pumping negative pressure at a third negative pressure pumping port, leading the nucleic acid and the reaction liquid which are uniformly mixed in the premixing chamber to flow into each reaction cavity under the action of the negative pressure, leading the redundant solution to flow into a second waste liquid cavity, and judging the negative and positive of the nucleic acid reaction of pathogenic microorganisms in the sample by observing the color change of the liquid in the reaction cavities.
Compared with the prior art, the invention has the beneficial effects that:
According to the invention, the sealing partition pieces are arranged in each cavity and the corresponding flow channel, and the sealing partition pieces and the microfluidic chip body are integrally formed, so that compared with the sealing mode, the sealing mode is additionally provided with the sealing partition pieces, the sealing effect is better, and the solution stored in each cavity cannot leak and leak through the integrally formed sealing partition pieces, so that the solution in the cavity can be stably sealed and stored in each cavity;
The method has the advantages that the opening and closing of each blind hole are controlled by pressing on-off (the needle is pressed by external force to keep the runner sealed), the complex valve structure and the transfer device are not required to be additionally used, the solution is pre-stored in the microfluidic chip body, the solution is transferred and released in different experimental steps through reasonable structure and runner design, the operation is simple and convenient, the method is very suitable for the instant experiment of the microfluidic chip, the disposable instant-use instant-casting design is adopted, and the cross contamination problem is avoided.
Through the reasonable structural design to the runner, each cavity and the sealing mode of each cavity of micro-fluidic chip, realized at the inside stable long-term sealed various reagents of preserving of micro-fluidic chip, and the waste liquid that produces in the experimentation flows into the first waste liquid chamber and the second waste liquid intracavity in the micro-fluidic chip, can not with the outside environment contact of laboratory, avoided the cross contamination in laboratory.
Prior to the experiments of the invention, the solutions are separated by the sealing partition in each chamber, and each step releases one solution; the invention can realize the purposes of instantly preparing, reacting, separating and detecting samples of the microfluidic chip without externally connecting a reagent bottle, thereby greatly shortening the operation time and simplifying the operation steps.
According to the invention, aiming at the condition of liquid reaction liquid, the liquid reaction liquid is provided with an independent storage chamber, and can be released into the premixing cavity only after the experiment starts, so that the stability of the reaction liquid is ensured.
According to the invention, aiming at the condition that the reaction liquid freeze-dried microspheres are pre-buried in the pre-mixing cavity, each liquid storage device is physically isolated from the freeze-dried microspheres, so that the freeze-dried microspheres are not influenced by moisture from the reagent, the activity of enzyme is ensured, and the accuracy and precision of experimental results are ensured.
The chip can realize that the reaction liquid flows into the premix chamber to be uniformly mixed with the pathogenic microorganism sample for cracking, cleaning, adsorbing and eluting the nucleic acid, and then enters the reaction chamber for reaction so as to realize the identification of the pathogenic microorganism species.
Detailed Description
The technical scheme of the invention is described in detail below with reference to the accompanying drawings and examples.
The invention provides a liquid storage type micro-fluidic cavity chip which comprises a chip body 1, a sealing film 2 and a back adhesive 3, wherein the sealing film 2 is bonded on the front surface of the chip body 1, the back adhesive 3 is bonded on the back surface of the chip body 1, the back adhesive 3 is made of elastic back adhesive materials (rubber, back adhesive, and other materials can be used), and the bonding mode is one or the combination of a plurality of hot-press bonding, ultrasonic bonding, laser bonding, adhesive bonding and solvent bonding. The sealing film 2 is used for sealing a flow channel and a cavity arranged on the front surface of the chip, and the sealing film 2 is one of a pressure sensitive film, a back adhesive film, a PET film and a PC film, and is preferably a transparent film.
The chip body 1 is provided with:
A lysate storage chamber 101, a cleaning liquid storage chamber 102, an eluent storage chamber 103, a reaction liquid storage chamber 104, a premixing chamber 105, a reaction chamber 106, a first waste liquid chamber 107, a second waste liquid chamber 108;
a first cleavage liquid flow path 109, a first cleaning liquid flow path 111, a first eluent flow path 113, a first reaction liquid flow path 115;
A second lysate flow path 117, a second cleaning solution flow path 118, a second eluent flow path 119, and a second reactant flow path 120;
Mixing channel 121, waste chamber channel 122.
The chip comprises a chip body 1, a chip body 2, a chip back adhesive 3, a sealing film 2, a chip back adhesive material, a reaction solution storage cavity 101, a reaction solution storage cavity 104, a cleaning solution storage cavity 102, an eluent storage cavity 103, a reaction solution storage cavity 101 and a reaction solution storage cavity 104, wherein the chip body 1 is filled with a sample of pathogenic microorganisms to be detected, the sample is subjected to pyrolysis by the pyrolysis solution, the chip back adhesive 3 is arranged on the back surface of the chip body, the chip back adhesive is arranged on the back surface of the chip body, and the chip back adhesive is arranged on the back surface of the chip body.
The method is characterized in that the outflow flow channels 130 are respectively connected with corresponding first flow channels (a first cracking liquid flow channel 109, a first cleaning liquid flow channel 111, a first eluent flow channel 113 and a first reaction liquid flow channel 115), the outflow flow channels 130 and the corresponding first flow channels are blocked by a sealing partition 126 (the sealing partition 126 is arranged at any position of the first flow channels, and in order to facilitate processing and production, the sealing partition 126 can be arranged at the junction of the outflow flow channels 130 and the corresponding first flow channels, the sealing partition 126 and the chip body 1 are integrally formed, and the first flow channels and the corresponding back glue flow channels arranged on the back glue 3 are communicated.
The bottom surface of each solution storage cavity is a funnel-shaped inclined surface, so that the cracked sample solution can be completely pumped out under the action of negative pressure, so that the cracked sample solution has no loss and residues, the nucleic acid in the sample is prevented from being remained on the bottom surface of the cracking solution storage cavity, the loss of the nucleic acid sample is prevented, and the accuracy of an experimental result is ensured during quantitative detection.
After various solutions are canned in each cavity, a layer of sealing film is covered on the top surface or an elastic sealing cover is covered on the top surface so as to prevent the solution pre-canned in each solution storage cavity from volatilizing, and when the liquid in each cavity is driven to flow, the sealing film on the top surface of each cavity is pierced or the sealing cover is opened.
The first flow channel (the first lysate flow channel 109, the first cleaning solution flow channel 111, the first eluent flow channel 113 and the first reaction liquid flow channel 115) is designed to be a non-penetrating microfluidic chip body, the lysate storage cavity 101 is communicated with a corresponding lysate flow channel, the lysate flow channel is communicated with the first lysate flow channel 109, and a sealing separation piece 126 is arranged at the junction of the first lysate flow channel 109 and the lysate flow channel for convenient processing and production, the sealing separation piece 126 is integrally formed with the chip body 1, so that the solution pre-stored in the lysate storage cavity 101 is only contacted with the chip body 1, and does not contact with other materials, thereby ensuring that the pre-stored solution is stably stored and sealed and does not leak;
the first cleaning solution flow channel 111 is internally provided with a sealing separation member 126, the sealing separation member 126 and the chip body 1 are integrally formed, and the sealing separation member 126 separates the first cleaning solution flow channel 111 and the cleaning solution storage cavity 102 so as to enable the cleaning solution to be stored in the cleaning solution storage cavity 102 in a sealing manner;
The first eluent flow channel 113 is internally provided with a sealing separation piece 126, the sealing separation piece 126 and the chip body 1 are integrally formed, and the sealing separation piece 126 separates the first eluent flow channel 113 and the eluent storage cavity 103 so as to enable eluent to be stored in the eluent storage cavity 103 in a sealing manner;
The first reaction liquid flow channel 115 is internally provided with a sealing spacer 126, the sealing spacer 126 and the chip body 1 are integrally formed, the sealing spacer 126 separates the first reaction liquid flow channel 115 and the reaction liquid storage cavity 104 so that the reaction liquid is stored in the reaction liquid storage cavity 104 in a sealing manner, and the sealing spacer 126 is pierced, so that the reaction liquid storage cavity 104 is communicated with the first reaction liquid flow channel 115.
The second flow channels (a second cracking liquid flow channel 117, a second cleaning liquid flow channel 118, a second eluent flow channel 119 and a second reaction liquid flow channel 120) are communicated with the first flow channels of the corresponding solution storage cavities through the corresponding gum flow channels arranged on the gum 3, the second flow channels are all designed to penetrate through the microfluidic chip body, are all communicated with the mixing flow channels 121 and penetrate through the front and back surfaces of the microfluidic chip body 1, the mixing flow channels 121 are arranged on the front surface of the chip body 1, and the mixing flow channels 121 are communicated with the premixing cavity 105;
the waste liquid cavity flow channel 122 (arranged on the front surface of the chip body 1), the waste liquid cavity flow channel 122 is communicated with the first waste liquid cavity 107 and the mixing flow channel 121, and the waste liquid cavity flow channel outlet of the first waste liquid cavity 107 is arranged at the middle upper part of the first waste liquid cavity 107, and the volume of the waste liquid cavity is larger than the volume of the pyrolysis liquid and the cleaning liquid.
The nucleic acid adsorbing module 125 is provided in the mixing channel 121 between the second eluent channel 119 and the waste liquid chamber channel 122, and is used for adsorbing nucleic acids. The nucleic acid adsorption component 125 is an existing silica gel mold, the width of the silica gel mold is matched with the width of the mixing flow channel 121, the microfluidic chip body 1 is made of hard materials, such as PP or PC, the mixing flow channel 121 is arranged on the front surface of the chip body 1, after the silica gel mold is arranged on the mixing flow channel 121, the front surface of the microfluidic chip is covered with the sealing film 2, so that the silica gel mold can be stably and firmly fixed in the mixing flow channel 121 without being influenced by elastic gum materials on the back surface, the problem that the elastic gum materials extrude the silica gel mold, so that a solution cannot smoothly pass through the silica gel mold to cause experimental failure is avoided, and meanwhile, the problem that the mixing flow channel 121 is deformed due to extrusion of the gum 3 during assembly, so that smooth passing of the solution is influenced can be avoided.
Before the reaction, the pyrolysis liquid storage cavity 101, the cleaning liquid storage cavity 102, the eluent storage cavity 103, the reaction liquid storage cavity 104, the premixing cavity 105, the reaction cavity 106 and the first waste liquid cavity 107 are not communicated with each other, the volumes of the reaction cavities 106 are the same, the tail end of the reaction cavity 106 is also provided with a second waste liquid cavity 108 for collecting waste liquid, and the reacted waste liquid is not discharged outwards, so that the laboratory environment is not polluted.
For experiments using freeze-dried microspheres as reaction raw materials, freeze-dried microspheres can be preset in the premix chamber 105, and the reaction liquid storage chamber 104 and the corresponding reaction liquid flow channel are not arranged.
The back adhesive 3 is provided with:
The first adhesive flow path 301 and the first adhesive flow path blind hole 302, the second adhesive flow path 303 and the second adhesive flow path blind hole 304, the third adhesive flow path 305 and the third adhesive flow path blind hole 306, the fourth adhesive flow path 307 and the fourth adhesive flow path blind hole 308, the first negative pressure pumping channel blind hole 309, the second negative pressure pumping channel blind hole 310 and the third negative pressure pumping channel blind hole 311.
The first lysate flow channel 109 and the second lysate flow channel 117 are communicated by a first gum flow channel 301;
the first cleaning solution flow channel 111 and the second cleaning solution flow channel 118 are communicated through a second gum flow channel 303;
The first eluent flow channel 113 and the second eluent flow channel 119 are in communication via a third gum flow channel 305;
the first reaction liquid flow channel 115 and the second reaction liquid flow channel 120 are communicated through a fourth back glue flow channel 307;
The first cracking liquid flow channel outlet corresponds to the first back glue flow channel blind hole 302, the first cleaning liquid flow channel outlet corresponds to the second back glue flow channel blind hole 304, the first eluent flow channel outlet corresponds to the third back glue flow channel blind hole 306, the first reaction liquid flow channel outlet corresponds to the fourth back glue flow channel blind hole 308, the position of the first time passing through after the liquid flows out of the first flow channel is called the back glue flow channel blind hole, the size of the back glue flow channel blind hole is larger than that of the corresponding back glue flow channel, the design can correspond to the position of the first flow channel outlet, the reliability of the butt joint position of the first flow channel outlet and the blind hole is improved, even if slight deviation exists in butt joint, the solution coming out of the first flow channel outlet can be ensured to smoothly enter the back glue flow channel, the first negative pressure pumping channel blind hole 309 corresponds to the first negative pressure pumping channel 127, the second negative pressure pumping channel blind hole 310 corresponds to the second negative pressure pumping channel 128, and the third negative pressure pumping channel blind hole 311 corresponds to the third negative pressure pumping channel 129.
When the liquid storage type microfluidic chip is used, various solutions are firstly respectively canned in each cavity, a layer of sealing film is attached to the top surface of each cavity or an elastic sealing cover is added to the top surface of each cavity, when external force is used for pumping liquid, the sealing film is punctured by a hollow needle or the sealing cover is opened, each cavity is communicated with the atmosphere through the hollow structure of the needle, the sealing partition 126 for isolating different cavities is punctured, the liquid in each cavity is driven to flow under the action of negative pressure, after the liquid is pumped out, the needle is pulled out, and sealing can be realized again due to the elasticity of the back glue 3.
As shown in fig. 1, the state of piercing the sealing barrier includes:
State one (FIG. 1A) sealing
Initially, the solution storage cavities are isolated from their corresponding gum flow channels by the sealing spacers 126, and the solution storage cavities are not in communication with their corresponding gum flow channels;
state two (FIG. 1B) contact pin rupture of membranes
The needle is driven by external force to press the back adhesive 3, the needle pierces the back adhesive 3 and the sealing partition 126, at the moment, the back adhesive 3 blocks the channel where the sealing partition 126 is positioned, and each solution storage cavity is not communicated with the corresponding back adhesive flow channel;
State III (FIG. 1C) connecting the needle
After the sealing partition 126 is pierced, the needle is pulled outwards, the needle seat is driven to be far away from the back glue, and each solution storage cavity is communicated with the corresponding back glue flow channel, so that liquid can be pumped out;
State four (FIG. 1D) the pins are resealed to the bottom
After the liquid stored in the solution storage cavity is pumped out, further, the external force drives the needle seat to compress the back glue 3, the back glue 3 blocks the channel where the sealing spacer 126 is located, and the solution storage cavity is not communicated with the corresponding back glue flow channel.
After each liquid pumping step is finished, the corresponding flow channel is blocked, so that the smooth liquid pumping is ensured, the liquid flow resistance is larger than the gas flow resistance, if the corresponding flow channel is not blocked after the liquid is pumped, the air in the previous step can be pumped away by external negative pressure, and the liquid in the corresponding cavity in the next step can not be pumped.
In the invention, the waste liquid cavity flow channel outlet of the first waste liquid cavity 107 is arranged at the middle upper part of the first waste liquid cavity 107, so that the waste liquid in the first waste liquid cavity 107 can not flow backwards, the volume of the waste liquid cavity is larger than the volume of the pyrolysis liquid and the cleaning liquid, and the liquid level is lower than the flow channel opening of the waste liquid cavity after the pyrolysis liquid and the cleaning liquid are pumped into the first waste liquid cavity 107. In the last step, negative pressure is pumped to the third negative pressure pumping channel blind hole 311, the second waste liquid cavity 108 and the inside of each reaction cavity are always in a negative pressure state, the liquid in the second waste liquid cavity 108 cannot flow backwards, no external force exists after negative pressure pumping is finished, the microfluidic chip is vertically placed, and the liquid in the second waste liquid cavity 108 cannot flow backwards, so that the isolation of waste liquid and channels can be realized.
In the invention, the volumes of the reaction chambers 106 are the same, so that the volumes of nucleic acid and reaction liquid in the reaction chambers 106 are the same, different pathogenic microorganism nucleic acid amplification primers are pre-embedded in different reaction chambers 106, and after the nucleic acid and the reaction liquid in the pre-mixing chamber 105 are evenly distributed into the different reaction chambers 106, the nucleic acid and the pathogenic microorganism nucleic acid amplification primers react under the action of the reaction liquid, and by observing the color change of the liquid in the reaction chambers 106, the pathogenic microorganisms of which types are in a sample can be judged, and after experiments are completed, a microfluidic chip is discarded, so that cross infection and no pollution to a laboratory are avoided.
In the invention, nucleic acid exists in a sample of pathogenic microorganisms, the pathogenic microorganisms are cracked by adding the sample containing the pathogenic microorganisms into the cracking liquid storage cavity 101, the nucleic acid in the pathogenic microorganisms is released in the cracking liquid, and the liquid in each storage cavity is driven to carry out nucleic acid adsorption, nucleic acid cleaning and nucleic acid elution by pumping negative pressure and finally flows into the premixing cavity 105, so that the identification of the sample of the pathogenic microorganisms is realized based on the reaction of the nucleic acid and the reaction liquid.
In a specific embodiment, the workflow of the liquid storage type microfluidic chip is sequentially performed according to the following steps:
1. Firstly, a sample containing pathogenic microorganisms is added into a lysis solution storage cavity 101, the lysis solution lyses the pathogenic microorganisms, nucleic acid of the pathogenic microorganisms is released into the lysis solution under the action of the lysis solution, the top surface of the lysis solution storage cavity 101 is pierced (the aim is to enable the lysis solution storage cavity 101 to be opened to the atmosphere, the solution can be pumped out), an external force driving needle pierces a first gum flow channel blind hole 302 and a sealing partition 126 in a first lysis solution flow channel 109, a first negative pumping pressure channel blind hole 309 is pierced, negative pressure is pumped out at the first negative pumping channel blind hole 309, the nucleic acid in the sample is adsorbed on a nucleic acid adsorption component 125, the lysis solution sequentially flows through an outflow flow channel 130 from the lysis solution storage cavity 101, and finally flows into a first waste liquid cavity 107, a first gum flow channel 109, a first gum flow channel 301, a second lysis solution flow channel 117, a mixing flow channel 121 and a waste liquid cavity flow channel 122. (the purpose of step 1 is to adsorb nucleic acid on the nucleic acid adsorbing component 125, after the nucleic acid adsorbing component is completely withdrawn, the needle is driven to press against the first gum flow channel blind hole 302 to block the first gum flow channel 301, and at this time, the lysate storage chamber 101 is not communicated with the first gum flow channel 301).
2. The top surface of the cleaning solution storage cavity 102 is pierced, the external force driving needle pierces the second gum flow passage blind hole 304 and the sealing partition in the first cleaning solution flow passage 111, negative pressure is pumped to the first negative pressure pumping passage blind hole 309, and cleaning solution flows from the cleaning solution storage cavity 102 through the outflow flow passage 130, the first cleaning solution flow passage 111, the second gum flow passage 303, the second cleaning solution flow passage 118, the mixing flow passage 121 and the waste liquid cavity flow passage 122 in sequence, and finally flows into the first waste liquid cavity 107. (the purpose of step 2 is to purify nucleic acid, after the extraction, the needle is driven to press the blind hole 304 of the second gum flow channel, the second gum flow channel 303 is blocked, and the first negative pressure extraction channel 127 is closed, and the method for closing the first negative pressure extraction channel 127 can be adopted, namely 1) needle extraction, 2) direct closing of the negative pressure pump, and 3, a pinch valve is additionally arranged on a pipe connected with the negative pressure pump and the negative pressure extraction channel.
3. Piercing the top surface of the eluent reservoir 103, the external force driving needle pierces the third gum flow channel blind hole 306 and the sealing spacer 126 in the first eluent flow channel 113, piercing the second negative pressure pumping channel blind hole 310, pumping negative pressure there, the eluent sequentially flows from the eluent reservoir 103 through the outflow flow channel 130, the first eluent flow channel 113, the third gum flow channel 305, the second eluent flow channel 119, the mixing flow channel 121, the premix chamber inflow flow channel 123 and finally flows into the premix chamber 105 (step 3 elutes nucleic acid, lets the nucleic acid flow into the premix chamber 105, and after pumping out, the driving needle compresses the third gum flow channel blind hole 306, and blocks the third gum flow channel 305).
4. The top surface of the reaction liquid storage cavity 104 is pierced, the external force drives the needle to pierce the fourth back glue runner blind hole 308 and the sealing partition 126 in the first reaction liquid runner 115, negative pressure is pumped at the second negative pressure pumping channel blind hole 310, the reaction liquid sequentially flows through the outflow runner 130, the first reaction liquid runner 115, the fourth back glue runner 307, the second reaction liquid runner 120 and the premix chamber inflow runner 123 from the reaction liquid storage cavity 104, and finally the reaction liquid flows into the premix chamber 105 to be uniformly mixed with nucleic acid.
5. The third negative pressure pumping channel blind hole 311 is pierced, negative pressure is pumped here, nucleic acid and reaction liquid which are uniformly mixed in the premixing cavity 105 finally flow into each reaction cavity 106 to react through the reaction cavity flow channel 124 under the action of the negative pressure, and redundant liquid finally flows into the second waste liquid cavity 108.
In the liquid storage type microfluidic chip workflow, the step 4 is omitted for experiments using freeze-dried microspheres as reaction raw materials.
In the invention, various different solutions are pre-canned in each storage cavity in the microfluidic chip, and the sealing partition 126 is integrally formed and arranged in each flow channel during processing, so that the microfluidic chip is convenient to transport and store, is easy and convenient to operate, can simultaneously detect various pathogenic microorganisms in one sample experiment only by one microfluidic chip at a time, is quick and convenient, and realizes the instant detection experiment of the microfluidic chip.