A microfluidic detection device and detection method for pathogenic microorganisms

Abstract

The present invention discloses a microfluidic detection device and detection method for pathogenic microorganisms, which relates to the field of food detection technology. The device comprises a microfluidic chip lower substrate and a microfluidic chip upper substrate connected by irreversible bonding, a sample liquid microfluidic channel structure, a particle capture structure, a bubble puncture structure and a bubble attraction component. The sample liquid microfluidic channel structure comprises a sample liquid inlet channel, the bottom end of the sample liquid inlet channel is connected to at least a pair of mutually parallel flow channel walls, and a microfluidic channel is formed between every two mutually parallel flow channel walls. The present invention can attract bubbles to approach the bubble puncture structure by providing a bubble attraction component, and improves the effect of eliminating bubbles in the sample liquid compared to the traditional exhaust hole exhaust method. By providing the bubble puncture structure, bubbles in the sample liquid can be actively punctured, thereby eliminating bubble interference in the sample liquid detection process and improving the accuracy of the detection results.

Description

Technical Field

[0001] The present invention relates to the technical field of food detection, and in particular to a microfluidic detection device and a detection method for pathogenic microorganisms. Background Art

[0002] Microfluidic chips utilize semiconductor-like microelectromechanical processing technology to construct microfluidic systems on a chip, offloading experimental and analytical processes onto a chip structure composed of interconnected pathways and liquid phase chambers. Compared to traditional pathogen detection technologies, microfluidic detection offers advantages such as reduced sample requirements, a high degree of automation, and minimal operator skill requirements. Microfluidic technology, combined with various detection techniques, can be used to detect a wide range of pathogens.

[0003] Microfluidic chips used for microbial detection are usually composed of two layers of glass substrates, including structural units such as microchannels, microstructures, sample inlets and detection windows. Peripheral equipment mainly includes peristaltic pumps, microinjection pumps, temperature control systems, as well as ultraviolet, fluorescence, electrochemical, chromatography and other detection components. Combined with multiple detection systems such as laser induced fluorescence, polymerase chain reaction, DNA hybridization reaction, electrochemical and chemical, as well as various detection methods combined with analytical methods such as mass spectrometry, they can perform rapid, accurate and high-throughput analysis of samples.

[0004] At present, one of the difficulties that hinders microfluidic detection technology from improving the accuracy of detection results is that the sample is blocked by interference factors when flowing through the microchannel. Although relevant personnel can complete the discharge of some bubbles by opening an exhaust hole structure on the surface of the microfluidic chip, the amount of bubbles discharged by the exhaust hole is limited, and it is impossible to take active exclusion measures for bubble and particulate interference factors. As a result, the interference exclusion ability of the microfluidic chip is limited, which is not conducive to improving the accuracy of sample detection results. To this end, we propose a microfluidic detection device and detection method for pathogenic microorganisms that can actively exclude interference factors. Summary of the Invention

[0005] The object of the present invention is to provide a microfluidic detection device and a detection method for pathogenic microorganisms to solve the problems raised in the above background technology.

[0006] To achieve the above object, the present invention provides the following technical solutions:

[0007] On the one hand, a microfluidic detection device for pathogenic microorganisms is provided, comprising a microfluidic chip lower substrate and a microfluidic chip upper substrate connected by irreversible bonding, the microfluidic chip lower substrate and the microfluidic chip upper substrate being made of cycloolefin polymer material or glass silicon respectively, and a closed microfluidic detection cavity is formed between the microfluidic chip lower substrate and the microfluidic chip upper substrate, the microfluidic detection device also comprises a sample liquid microfluidic channel structure, a particle capture structure, a bubble puncture structure and a bubble attraction component, the sample liquid microfluidic channel structure is used to pass the sample liquid to be detected, and the particle capture structure is used to capture and intercept the sample liquid. The bubble puncture structure can puncture the bubbles contained in the sample liquid flowing through the microfluidic channel to prevent the bubbles from blocking the smoothness of the sample liquid circulation in the microfluidic channel. The microfluidic channel structure serves as a circulation channel for the sample liquid and can limit the sample liquid flowing through it to maintain the singleness of the sample liquid circulation path. The sample liquid microfluidic channel structure is welded to the top surface of the structural integration area. The sample liquid microfluidic channel structure includes a sample liquid inlet channel. The bottom end of the sample liquid inlet channel is connected to at least a pair of mutually parallel flow channel walls. A microfluidic channel is formed between every two mutually parallel flow channel walls. One end of the microfluidic channel is connected to the sample liquid. The discharge channel is used to discharge the sample liquid that has been tested after the reaction; the particle capture structure includes a retention groove integrally formed with the flow channel wall, the retention groove is used to accommodate the trapped particles to prevent non-reactive particles in the sample liquid from clogging the microfluidic channel, and the opening of the retention groove is connected to a reboundable opening and closing door structure, the opening and closing door structure and the retention groove form a fully enclosed particle retention space, the opening and closing door structure prevents the trapped particles from escaping from the retention groove, and can prevent the trapped particles from re-entering the sample liquid; the bubble puncture structure is installed on the structural integration area opposite the opening and closing door structure, and the bubble puncture structure includes A telescopic component, one end of the telescopic end of the telescopic component is integrally formed with a stop pick, and a locking structure adapted to the telescopic movement of the stop pick is installed on the flow channel wall facing the opening and closing door structure; the bubble attraction component includes an adsorption channel running through the stop pick and the telescopic end, one end of the adsorption channel is connected with a bellows, a first valve is installed at the connection between the telescopic end and the stop pick, a second valve is installed at the connection between the bellows and the telescopic end, one end of the bellows is connected to an air storage bag through a micro negative pressure pump, a third valve is installed at the connection between the air storage bag and the bellows, and the third valve closes the opening of the air storage bag to prevent gas backflow.

[0008] As a further solution of the present invention: the structural integration area is integrally formed on the top of the lower substrate of the microfluidic chip, and an annular bonding area is formed on the edge of the lower substrate of the microfluidic chip. A plurality of bonding grooves are opened on one side of the bonding area, and a convex portion adapted to the bonding groove is formed on the side where the upper substrate of the microfluidic chip and the lower substrate of the microfluidic chip are bonded to each other, which is beneficial to strengthening the connection strength between the lower substrate of the microfluidic chip and the upper substrate of the microfluidic chip.

[0009] As a further solution of the present invention: the bottom ends of the sample liquid inlet channel and the sample liquid discharge channel are both provided with a notch connected to the microfluidic channel, and the retention groove is a U-shaped groove or a semicircular groove protruding from one side of the flow channel wall. The height of the retention groove is not less than the height of the flow channel wall, which can prevent the sample liquid in the microfluidic channel from flowing into the retention groove; the opening and closing door structure includes a positioning shaft passing through both ends of the retention groove opening, and the surfaces of the two positioning shafts are respectively passed through torsion springs, and one end of the torsion spring is passed through and fixed with a door panel. The door panel connected by the torsion spring can maintain the closed state of the two door panels by the elastic force of the torsion spring to restore the elastic deformation after being pushed open by the intercepting pick.

[0010] As a further solution of the present invention: the locking structure includes a through-opening, a mounting groove and a locking door, the through-opening is tangentially adapted to the cut-off pick and is opened through the flow channel wall; the mounting groove is opened on one side of the flow channel wall where the through-opening is located; the locking door is symmetrical about the center line of the through-opening, one end of the locking door is fixedly connected to a traction cylinder, one end of the traction cylinder is fixed on the inner wall of the mounting groove, and the traction cylinder can quickly open or close the locking door that closes the through-opening.

[0011] As a further solution of the present invention: a servo is fixed through the interior of the lower substrate of the microfluidic chip, and the output end of the servo is welded and fixed to the bottom of the telescopic component. The output end of the servo pulls the telescopic component to rotate, and the telescopic end at one end of the telescopic component can be adjusted to adapt to different through-holes to intercept different particles or eliminate bubbles.

[0012] As a further solution of the present invention: the telescopic component is a telescopic cylinder, the telescopic end is a telescopic rod running through the telescopic cylinder, the telescopic rod can telescopically move in the telescopic cylinder to adjust the distance between the telescopic cylinder and the through-hole.

[0013] As a further solution of the present invention: a crescent groove is provided at one end of the cutting pick, and a plurality of parallel cutting edges are provided on the inner wall of the crescent groove; a hollow tube is coaxially welded at the opening of the adsorption channel, and the length of the hollow tube is smaller than the radius of the crescent groove. The hollow tube can extend into the bubble to absorb the gas in the bubble, thereby facilitating the rapid collection of the gas in the bubble.

[0014] As a further solution of the present invention: a micro pressure gauge is installed through the top of the air storage bag to measure the air pressure in the air storage bag.

[0015] As a further solution of the present invention: a leaked gas drainage structure is installed on the surface of the bellows, and the leaked gas drainage structure includes a drainage channel and a micro air valve. The drainage channel runs through and is connected to the surface of the bellows. The drainage channel is a hollow cylindrical tube, and the diameter of the drainage channel is smaller than the inner diameter of the bellows; the micro air valve is installed at the connection between the drainage channel and the bellows. When the bubble bursts, the micro negative pressure pump is started and the micro air valve is opened, and the gas generated after the bubble is punctured is absorbed into the air storage bag through the drainage channel.

[0016] As a further solution of the present invention: the top of the substrate on the microfluidic chip is connected through a sampling tube adapted for the sample liquid entering the channel, and the top of the substrate on the microfluidic chip is connected through a fluorescent detection probe and a sample discharge tube adapted for the sample liquid discharge channel, and the sample discharge tube is used to discharge the sample liquid after detection into a waste liquid collection bottle.

[0017] On the other hand, a detection method of the above-mentioned microfluidic detection device for pathogenic microorganisms is also provided, and the detection method comprises the following steps:

[0018] Step 1: After making the mask, take the clean silicon wafer substrates of the microfluidic chip lower substrate and the microfluidic chip upper substrate and perform plasma cleaning. Spin-coat photoresist to make the microfluidic channel template. Silanize the silicon wafer with the channel after 3-5 minutes. Pour 1:10 polydimethylsiloxane potting glue and let it stand horizontally for 25-35 minutes.

[0019] Step 2: Place the silicon wafer substrate with the potting compound prepared in step 1 in a baking oven, adjust the baking temperature to 80°C, and dry it by electric heating for 30 minutes. Then, press the prepared potting compound silicon wafer substrate onto the cycloolefin polymer at a temperature of 120°C.

[0020] Step 3: Use the engraved optically transparent adhesive to irreversibly bond the two silicon wafer substrates made in step 2, so that the bonding area on the silicon wafer substrate and the bonding groove, the sample injection tube and the sample liquid inlet channel, and the sample liquid discharge channel and the sample discharge tube are adapted and connected one by one. Place them in an 80°C electric drying oven and bake for 10-15 minutes to complete the production of the detection chip;

[0021] Step 4: Connect the chip made in step 3 to an external control device. Specifically, use a PVC tube with a micro lift pump to connect it between the sample discharge tube and the waste liquid collection bottle;

[0022] Step 5: Air-tightly connect the sample injection tube to one end of the delivery channel, and connect one end of the delivery channel to the sample liquid injection pump. A syringe pump monitor is installed on the side of the sample liquid injection pump, and the syringe pump monitor is electrically connected to the computer using a data cable;

[0023] Step 6: Connect the fluorescence detection probe to the signal transmission line, which is connected to the input interface of the computer. The sample liquid to be tested is pumped into the sample liquid entry channel through the sample injection tube by the sample liquid injection pump for detection. When the sample liquid flows through the microfluidic channel corresponding to the fluorescence detection probe, the fluorescence detection probe catalyzes the addition reaction between 3-azido-7-hydroxycoumarin and 3-butyn-1-ol to generate a fluorescent product. The detection result signal is transmitted to the computer terminal through the signal transmission line to complete the detection of the pathogenic bacteria Escherichia coli.

[0024] As a further solution of the present invention: a flow detector is connected to the middle of the delivery channel in step 4 to detect the flow rate of the sample liquid.

[0025] Compared with the prior art, the present invention has the following beneficial effects:

[0026] 1. The present invention provides a particle capture structure that can actively intercept particles, and can actively capture the same particle multiple times while it flows through different positions of the microfluidic channel, thereby greatly improving the interception efficiency of particles in the sample liquid.

[0027] 2. The present invention is capable of attracting bubbles to the bubble puncture structure by setting a bubble attraction component, which improves the effect of eliminating bubbles in the sample liquid compared with the traditional exhaust hole exhaust method. By setting the bubble puncture structure, the bubbles in the sample liquid can be actively punctured, eliminating the bubble interference in the sample liquid detection process and improving the accuracy of the detection results.

[0028] 3. The present invention forms multiple microfluidic channels through multiple parallel flow channel walls, which can perform multi-channel detection on sample liquids. By adopting fluorescent detection probes adapted to different microfluidic channels, several pathogenic microorganisms can be detected simultaneously, saving detection time and improving detection efficiency.

[0029] 4. The present invention installs a servo that penetrates the lower substrate of the microfluidic chip. The output end of the servo can adjust the direction of the telescopic end of the telescopic component, thereby intercepting particles flowing through different openings. It has the advantages of intercepting different particles at multiple angles and intercepting the same particle at different angles.

[0030] 5. The present invention provides a door panel elastically connected to a torsion spring. After the particles enter the retention groove, the intercepting pick automatically withdraws from the retention groove, causing the door panel to rebound under the traction of the torsion spring to restore its elastic deformation, thereby locking the particles into the particle retention space and avoiding ineffective retention. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 It is the overall structural diagram of the present invention;

[0032] Figure 2 A top view of the lower substrate of the microfluidic chip of the present invention;

[0033] Figure 3 For the present invention Figure 2 A magnified view of middle A;

[0034] Figure 4 It is a partial exploded structural diagram of the present invention;

[0035] Figure 5 This is a flow channel wall connection structure diagram of the present invention;

[0036] Figure 6For the present invention Figure 5 Enlarged view of middle B;

[0037] Figure 7 For the present invention Figure 5 Enlarged view of middle C;

[0038] Figure 8 For the present invention Figure 5 Enlarged view of middle D;

[0039] Figure 9 For the present invention Figure 5 Enlarged view of E.

[0040] In the figure: 1. Microfluidic chip lower substrate; 101. Bonding area; 102. Bonding groove; 2. Structural integration area; 3. Sample liquid inlet channel; 301. Notch; 302. Sample liquid outlet channel; 4. Flow channel wall; 5. Retention groove; 6. Opening and closing door structure; 601. Positioning shaft; 602. Torsion spring; 603. Door panel; 7. Microfluidic channel; 8. Through-hole; 9. Mounting groove; 10. Traction cylinder; 11. Locking door; 12. Servo; 13. Telescopic component; 14. Telescopic end; 15. Stop pick; 16. Crescent groove; 17. Adsorption channel; 171. Hollow tube; 18 , first valve; 19, bellows; 20, second valve; 21, micro negative pressure pump; 22, drainage channel; 23, micro air valve; 24, air storage bag; 25, micro pressure gauge; 26, third valve; 27, microfluidic chip substrate; 28, sample injection tube; 29, sample discharge tube; 30, fluorescence detection probe; 31, sample liquid injection pump; 310, flow meter; 311, delivery channel; 32, injection pump monitor; 33, data cable; 34, computer; 35, signal transmission line; 36, PVC tube; 37, waste liquid collection bottle; 38, micro lifting pump. DETAILED DESCRIPTION

[0041] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0042] like Figure 1The figure shows the overall structure of the microfluidic detection device for pathogenic microorganisms of the present application. The device includes a microfluidic chip lower substrate 1 and a microfluidic chip upper substrate 27 connected by irreversible bonding. The microfluidic chip lower substrate 1 and the microfluidic chip upper substrate 27 are respectively made of cycloolefin polymer material or glass silicon. A closed microfluidic detection cavity is formed between the microfluidic chip lower substrate 1 and the microfluidic chip upper substrate 27. The microfluidic detection device also includes a sample liquid microfluidic channel structure, a particle capture structure, a bubble puncture structure and a bubble attraction component. The sample liquid microfluidic channel structure is used to pass through the detected The sample liquid and the particle capture structure are used to capture irrelevant particles in the sample liquid that hinder the flow of the sample liquid. The bubble puncture structure can puncture the bubbles contained in the sample liquid flowing through the microfluidic channel 7 to prevent the bubbles from blocking the smooth flow of the sample liquid in the microfluidic channel 7. The microfluidic channel structure serves as a flow channel for the sample liquid and can limit the flow of the sample liquid to maintain the singleness of the sample liquid flow path. The top of the substrate 27 on the microfluidic chip is connected through a sample injection tube 28 adapted for the sample liquid to enter the channel 3. The top of the substrate 27 on the microfluidic chip is connected through a fluorescent detection probe 30 and a sample liquid discharge channel 302. The adapted sample discharge tube 29 is used to discharge the sample liquid after detection into the waste liquid collection bottle 37. The interior of the lower substrate 1 of the microfluidic chip is fixed with a servo 12. The output end of the servo 12 is welded and fixed to the bottom of the telescopic component 13. The output end of the servo 12 pulls the telescopic component 13 to rotate, and the telescopic end 14 at one end of the telescopic component 13 can be adjusted to adapt to different through-holes 8 to intercept different particles or eliminate bubbles. The telescopic component 13 is a telescopic cylinder, and the telescopic end 14 is a telescopic rod running through the telescopic cylinder. The telescopic rod can telescope and move in the telescopic cylinder to adjust the telescopic air flow. The distance between the cylinder and the through-hole 8 is a gas drainage structure installed on the surface of the bellows 19. The gas drainage structure includes a drainage channel 22 and a micro air valve 23. The drainage channel 22 passes through and is connected to the surface of the bellows 19. The drainage channel 22 is a hollow cylindrical tube. The diameter of the drainage channel 22 is smaller than the inner diameter of the bellows 19; the micro air valve 23 is installed at the connection between the drainage channel 22 and the bellows 19. When the bubble bursts, the micro negative pressure pump 21 is started and the micro air valve 23 is opened, and the gas generated after the bubble is punctured is absorbed into the air storage bag 24 through the drainage channel 22.

[0043] like Figure 2 The figure shows a top view of the lower substrate of the microfluidic chip. The sample liquid microfluidic channel structure is welded to the top surface of the structural integration area 2. The sample liquid microfluidic channel structure includes a sample liquid inlet channel 3. The bottom end of the sample liquid inlet channel 3 is connected to at least a pair of mutually parallel flow channel walls 4. A microfluidic channel 7 is formed between every two mutually parallel flow channel walls 4. One end of the microfluidic channel 7 is connected to a sample liquid discharge channel 302. The sample liquid discharge channel 302 is used to discharge the sample liquid after reaction and detection.

[0044] like Figure 3 Shown Figure 2 In the enlarged view of A in the middle, the particle capture structure includes a retention groove 5 integrally formed with the flow channel wall 4, the retention groove 5 is used to accommodate the trapped particles to prevent non-reactive particles in the sample liquid from clogging the microfluidic channel 7, and the bubble attraction component includes an adsorption channel 17 running through the stop pick 15 and the telescopic end 14, one end of the adsorption channel 17 is connected to a bellows 19, a first valve 18 is installed at the connection between the telescopic end 14 and the stop pick 15, a second valve 20 is installed at the connection between the bellows 19 and the telescopic end 14, one end of the bellows 19 is connected to an air storage bag 24 through a micro negative pressure pump 21, and a third valve 26 is installed at the connection between the air storage bag 24 and the bellows 19. The third valve 26 closes the opening of the air storage bag 24 to prevent gas backflow.

[0045] like Figure 4 The figure shows a partial decomposition structure diagram of the present application. The structural integration area 2 is integrally formed on the top of the lower substrate 1 of the microfluidic chip. The edge of the lower substrate 1 of the microfluidic chip is formed with an annular bonding area 101. A plurality of bonding grooves 102 are opened on one side of the bonding area 101. The side where the upper substrate 27 of the microfluidic chip is bonded to the lower substrate 1 of the microfluidic chip is formed with a convex portion adapted to the bonding groove 102, which is beneficial to strengthen the connection strength between the lower substrate 1 of the microfluidic chip and the upper substrate 27 of the microfluidic chip.

[0046] like Figure 5 This is a flow channel wall connection structure diagram. A micro pressure gauge 25 is installed through the top of the air storage bag 24 to measure the air pressure in the air storage bag 24.

[0047] like Figure 6 for Figure 5 In the enlarged view of B, the bubble puncture structure is installed on the structural integration area 2 facing the opening and closing door structure 6, and the bubble puncture structure includes a telescopic component 13. One end of the telescopic end 14 of the telescopic component 13 is integrally formed with a stopping pick 15, and a locking structure adapted to the telescopic movement of the stopping pick 15 is installed on the flow channel wall 4 facing the opening and closing door structure 6; a crescent groove 16 is provided at one end of the stopping pick 15, and a plurality of cutting edges parallel to each other are provided on the inner wall of the crescent groove 16; a hollow tube 171 is coaxially welded at the opening of the adsorption channel 17, and the length of the hollow tube 171 is less than the radius of the crescent groove 16. The hollow tube 171 can be extended into the bubble to absorb the gas in the bubble, so as to facilitate the rapid collection of the gas in the bubble.

[0048] like Figure 7 for Figure 5In the enlarged view of middle C, the opening of the retention groove 5 is connected to a reboundable opening and closing door structure 6, and the opening and closing door structure 6 and the retention groove 5 constitute a fully enclosed particle retention space. The opening and closing door structure 6 prevents the trapped particles from escaping from the retention groove 5, and can prevent the trapped particles from entering the sample liquid again; the retention groove 5 is a U-shaped groove or a semicircular groove protruding from one side of the flow channel wall 4, and the height of the retention groove 5 is not less than the height of the flow channel wall 4, which can prevent the sample liquid in the microfluidic channel 7 from flowing into the retention groove 5; the opening and closing door structure 6 includes a positioning shaft 601 passing through both ends of the opening of the retention groove 5, and the surfaces of the two positioning shafts 601 are respectively penetrated by torsion springs 602, and one end of the torsion spring 602 is penetrated and fixed with a door plate 603. The door plate 603 connected by the torsion spring 602 can maintain the closed state of the two door plates 603 through the elastic force of the torsion spring 602 to restore the elastic deformation after being pushed open by the intercepting pick 15.

[0049] like Figure 8 for Figure 5 In the enlarged view of middle D, a notch 301 communicating with the microfluidic channel 7 is provided at the bottom of each of the sample liquid inlet channel 3 and the sample liquid outlet channel 302 .

[0050] like Figure 9 for Figure 5 In the enlarged view of E in the middle, the locking structure includes a through-opening 8, a mounting groove 9 and a locking door 11. The through-opening 8 is tangentially fitted with the cut-off pick 15 and is opened through the flow channel wall 4; the mounting groove 9 is opened on one side of the flow channel wall 4 where the through-opening 8 is located; the locking door 11 is symmetrical about the center line of the through-opening 8, and one end of the locking door 11 is fixedly connected to a traction cylinder 10, and one end of the traction cylinder 10 is fixed on the inner wall of the mounting groove 9. The traction cylinder 10 can quickly open or close the locking door 11 that closes the through-opening 8.

[0051] The detection method of the microfluidic detection device for pathogenic microorganisms in this application is as follows:

[0052] Step 1: After making the mask, take the clean silicon wafer substrates of the microfluidic chip lower substrate 1 and the microfluidic chip upper substrate 27 and plasma clean them. Spin-coat photoresist to make the microfluidic channel 7 template. Silanize the silicon wafer with the channel after 3-5 minutes. Pour 1:10 polydimethylsiloxane potting glue and let it stand horizontally for 25-35 minutes.

[0053] Step 2: Place the silicon wafer substrate with the potting compound prepared in step 1 in a baking oven, adjust the baking temperature to 80°C, and dry it by electric heating for 30 minutes. Then, press the prepared potting compound silicon wafer substrate onto the cycloolefin polymer at a temperature of 120°C.

[0054] Step 3: Use the engraved optically transparent adhesive to irreversibly bond the two silicon wafer substrates produced in step 2, so that the bonding area 101 on the silicon wafer substrate is matched with the bonding groove 102, the sample inlet tube 28 is matched with the sample liquid inlet channel 3, and the sample liquid discharge channel 302 is matched with the sample discharge tube 29. Then, bake them in an 80°C electric drying oven for 10-15 minutes to complete the production of the detection chip;

[0055] Step 4: Connect the chip made in step 3 to an external control device. Specifically, use a PVC tube 36 with a micro lift pump 38 to connect between the sample discharge tube 29 and the waste liquid collection bottle 37;

[0056] Step 5: Air-tightly connect the sample injection tube 28 to one end of the delivery channel 311. One end of the delivery channel 311 is connected to the sample liquid injection pump 31. A syringe pump monitor 32 is installed on the side of the sample liquid injection pump 31. The syringe pump monitor 32 is electrically connected to the computer 34 using a data cable 33.

[0057] Step 6: Connect the fluorescence detection probe 30 to the signal transmission line 35, and connect the signal transmission line 35 to the input interface of the computer 34. The sample liquid to be detected is pumped into the sample liquid through the sampling tube 28 by the sample liquid injection pump 31 into the sample liquid entering the channel 3 for detection. When the sample liquid flows through the microfluidic channel 7 corresponding to the fluorescence detection probe 30, the fluorescence detection probe 30 catalyzes the addition reaction between 3-azido-7-hydroxycoumarin and 3-butyn-1-ol to generate a fluorescent product, and transmits the detection result signal to the computer 34 terminal through the signal transmission line 35 to complete the detection of the pathogenic bacteria Escherichia coli.

[0058] In step 4, a flow detector 310 is connected to the middle of the delivery channel 311 to detect the flow rate of the sample liquid.

[0059] Principle of interception of particles or bubbles: After the sample liquid enters the sample liquid entry channel 3, it flows into the microfluidic channel 7 from the notch 301 at its bottom end. When the particles in the sample liquid flow through the retention groove 5, the servo 12 near the retention groove 5 is started. The output end of the servo 12 adjusts the telescopic end 14 of the telescopic component 13 to align with the through-hole 8 facing the particle. By starting the traction cylinder 10 at the opening of the through-hole 8, the traction cylinder 10 pulls the locking door 11 to move linearly, which can open the through-hole 8, so that the intercepting pick 15 extends out of the through-hole 8 and pushes the particles into the retention groove 5. By setting a door plate 603 elastically connected by a torsion spring 602, after the particles enter the retention groove 5, as the intercepting pick 15 automatically withdraws from the retention groove 5, the door plate 603 rebounds under the traction of the torsion spring 602 to restore the elastic deformation, thereby locking the particles into the particle retention space. The interception of microparticles is achieved, and the risk of microparticles clogging the microfluidic channel 7 is reduced. By pulling the telescopic component 13 to rotate through the output end of the servo 12, the telescopic end 14 can be adjusted to align with the through-holes 8 at different positions in the circumferential direction, so that on the premise that the interception of the microparticles fails once or twice, the microparticles can be intercepted a second or third time, which is beneficial to improving the interception success rate. When the bubble flows through the through-hole 8, the first valve 18 and the second valve 20 are opened, and by starting the micro negative pressure pump 21, a negative pressure is formed at the opening of the adsorption channel 17, attracting the bubble to approach the interception pick 15 and absorbing the gas in the bubble into the air storage bag 24. In the process of the bubble approaching the interception pick 15, the bubble can be punctured through the crescent groove 16 and the micro air valve 23 is opened, and the gas generated after the bubble is punctured is absorbed into the air storage bag 24 through the drainage channel 22.

[0060] It should be noted that, in this document, relational terms such as first and second, etc., are used only to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any actual relationship or order between these entities or operations. Moreover, the terms "comprises," "comprising," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that includes a list of elements includes not only those elements but also other elements not explicitly listed, or elements inherent to such process, method, article, or apparatus.

[0061] While embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions, and variations may be made to these embodiments without departing from the principles and spirit of the invention, and that the scope of the invention is defined by the appended claims and their equivalents.

Claims

1. A microfluidic detection device for pathogenic microorganisms, comprising a microfluidic chip lower substrate and a microfluidic chip upper substrate connected by irreversible bonding, characterized in that: A sealed microfluidic detection cavity is formed between the lower substrate of the microfluidic chip and the upper substrate of the microfluidic chip. The microfluidic detection device further includes: A sample liquid microfluidic channel structure, which is welded to the top surface of the structural integration area, and includes a sample liquid inlet channel, the bottom end of which is connected to at least a pair of mutually parallel flow channel walls, a microfluidic channel is formed between each pair of mutually parallel flow channel walls, and one end of the microfluidic channel is connected to a sample liquid discharge channel; A particle capture structure comprising a retention groove integrally formed with the flow channel wall, the opening of the retention groove being connected to a resilient opening and closing door structure, the opening and closing door structure and the retention groove forming a fully enclosed particle retention space; A bubble puncture structure is installed on the structural integration area facing the opening and closing door structure. The bubble puncture structure includes a telescopic component. A stop pick is integrally formed at one end of the telescopic end of the telescopic component. A locking structure adapted to the telescopic movement of the stop pick is installed on the flow channel wall facing the opening and closing door structure. The bubble suction component includes an adsorption channel that runs through the stop pick and the telescopic end. One end of the adsorption channel is connected to a bellows. A first valve is installed at the connection between the telescopic end and the stop pick. A second valve is installed at the connection between the bellows and the telescopic end. One end of the bellows is connected to an air storage bag through a micro negative pressure pump. A third valve is installed at the connection between the air storage bag and the bellows.

2. A microfluidic detection device for pathogenic microorganisms according to claim 1, characterized in that: The structural integration area is integrally formed on the top of the lower substrate of the microfluidic chip. The lower substrate of the microfluidic chip and the upper substrate of the microfluidic chip are respectively made of cycloolefin polymer material. An annular bonding area is formed on the edge of the lower substrate of the microfluidic chip. A plurality of bonding grooves are opened on one side of the bonding area. A convex portion adapted to the bonding groove is formed on the side where the upper substrate of the microfluidic chip and the lower substrate of the microfluidic chip are bonded.

3. The microfluidic detection device for pathogenic microorganisms according to claim 1, characterized in that: The bottom ends of the sample liquid inlet channel and the sample liquid discharge channel are both provided with notches connecting to the microfluidic channel. The retention groove is a U-shaped groove or a semicircular groove protruding from one side of the flow channel wall, and the height of the retention groove is not less than the height of the flow channel wall; the opening and closing door structure includes a positioning shaft passing through both ends of the retention groove opening, and the surfaces of the two positioning shafts are respectively passed through torsion springs, and one end of the torsion spring is passed through and fixed with a door panel.

4. The microfluidic detection device for pathogenic microorganisms according to claim 1, characterized in that: The locking structure includes: A through-hole, the through-hole being tangentially adapted to the cut-off pick and penetrating through the flow channel wall; An installation groove is provided on one side of the flow channel wall where the through opening is located; The locking door is symmetrical about the center line of the through opening, one end of the locking door is fixedly connected with a traction cylinder, and one end of the traction cylinder is fixed on the inner wall of the mounting groove.

5. The microfluidic detection device for pathogenic microorganisms according to claim 1, characterized in that: A servo is fixed through the interior of the lower substrate of the microfluidic chip. The output end of the servo is welded and fixed to the bottom of the telescopic component. The output end of the servo pulls the telescopic component to rotate.

6. The microfluidic detection device for pathogenic microorganisms according to claim 1, characterized in that: The telescopic component is a telescopic cylinder, and the telescopic end is a telescopic rod running through the telescopic cylinder.

7. The microfluidic detection device for pathogenic microorganisms according to claim 1, characterized in that: A crescent groove is provided at one end of the cutting pick, and a plurality of mutually parallel cutting edges are provided on the inner wall of the crescent groove; a hollow tube is coaxially welded at the opening of the adsorption channel, and the length of the hollow tube is smaller than the radius of the crescent groove.

8. The microfluidic detection device for pathogenic microorganisms according to claim 1, characterized in that: A micro pressure gauge is installed on the top of the air storage bag, a sampling tube adapted for the sample liquid entry channel is connected to the top of the substrate on the microfluidic chip, and a fluorescence detection probe and a sample discharge tube adapted for the sample liquid discharge channel are connected to the top of the substrate on the microfluidic chip.

9. The microfluidic detection device for pathogenic microorganisms according to claim 1, characterized in that: The surface of the bellows is equipped with a gas drainage structure, which includes: A drainage channel, which penetrates and communicates with the surface of the corrugated tube, is a hollow cylindrical tube, and has a diameter smaller than the inner diameter of the corrugated tube; A micro air valve is installed at the connection between the drainage channel and the bellows.

10. A method for detecting pathogenic microorganisms using a microfluidic detection device according to any one of claims 1 to 9, characterized in that: The steps include: Step 1: After making the mask, take the clean silicon wafer substrates of the microfluidic chip lower substrate and the microfluidic chip upper substrate and perform plasma cleaning. Spin-coat photoresist to make the microfluidic channel template. Silanize the silicon wafer with the channel after 3-5 minutes. Pour 1:10 polydimethylsiloxane potting glue and let it stand horizontally for 25-35 minutes. Step 2: Place the silicon wafer substrate with the potting compound prepared in step 1 in a baking oven, adjust the baking temperature to 80°C, and dry it by electric heating for 30 minutes. Then, press the prepared potting compound silicon wafer substrate onto the cycloolefin polymer at a temperature of 120°C. Step 3: Use the engraved optically transparent adhesive to irreversibly bond the two silicon wafer substrates made in step 2, so that the bonding area on the silicon wafer substrate and the bonding groove, the sample injection tube and the sample liquid inlet channel, and the sample liquid discharge channel and the sample discharge tube are adapted and connected one by one. Place them in an 80°C electric drying oven and bake for 10-15 minutes to complete the production of the detection chip; Step 4: Connect the chip made in step 3 to an external control device. Specifically, use a PVC tube with a micro lift pump to connect it between the sample discharge tube and the waste liquid collection bottle; Step 5: Air-tightly connect the sample injection tube to one end of the delivery channel, and connect one end of the delivery channel to the sample liquid injection pump. A syringe pump monitor is installed on the side of the sample liquid injection pump, and the syringe pump monitor is electrically connected to the computer using a data cable; Step 6: Connect the fluorescence detection probe to the signal transmission line, which is connected to the input interface of the computer. The sample liquid to be tested is pumped into the sample liquid entry channel through the sample injection tube by the sample liquid injection pump for detection. When the sample liquid flows through the microfluidic channel corresponding to the fluorescence detection probe, the fluorescence detection probe catalyzes the addition reaction between 3-azido-7-hydroxycoumarin and 3-butyn-1-ol to generate a fluorescent product. The detection result signal is transmitted to the computer terminal through the signal transmission line to complete the detection of the pathogenic bacteria Escherichia coli.

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