Nucleic acid detection microfluidic chip and detection system with ultrasonic micro-vibration cavity

Abstract

The embodiment of the present invention provides a nucleic acid detection microfluidic chip and detection system with an ultrasonic micro-vibration cavity, the chip includes a chip body, the chip body includes a reaction chamber, a washing liquid pre-storage chamber, an eluent pre-storage chamber, a lysis liquid pre-storage chamber, a waste liquid chamber and an extract detection chamber, wherein the reaction chamber is designed as a closed suspension bridge structure and a piezoelectric ceramic excitation plate is installed on the back, a micro-concave structure and a flow guide unit are provided in the reaction chamber, and magnetic beads are placed, which are respectively connected to the waste liquid chamber and the extract detection chamber through the flow guide unit. For each mixing step in the nucleic acid extraction process, the sound field generated by the ultrasonic device is used for resonance mixing treatment, and because the micro-concave structure is provided, the sound field forms a strong acoustic micro-flow at the micro-concave structure by the bubble effect, thereby making the mixing more complete; and the liquid in the reaction chamber is flowed to the waste liquid chamber or the extract detection chamber by a double push method, so as to effectively realize the nucleic acid extraction and detection process.

Description

Technical Field

[0001] The present invention relates to the field of, but is not limited to, data processing technology, and in particular to a nucleic acid detection microfluidic chip and a detection system with an ultrasonic micro-vibration cavity. Background Art

[0002] Traditional nucleic acid detection methods, such as Southern blot hybridization and agarose gel electrophoresis, are time-consuming and labor-intensive, have low sensitivity, are difficult to implement for high-throughput testing, and even require the use of radioactive reagents. Traditional laboratory-based pathogen detection methods require specialized personnel in a well-equipped laboratory to transfer and add reagents. These procedures are cumbersome and can expose samples and reagents to air, making them highly susceptible to contamination. Furthermore, the difficulty of lysing samples, such as cells, viruses, serum, and plasma, varies, requiring different reagent compositions. This makes it difficult to detect diverse sample types and results in poor universal adaptability. Furthermore, fixed chips lack centrifugal force, making it difficult to achieve uniform mixing of the beads when extracting nucleic acids using magnetic beads, which can lead to nucleic acid loss and low extraction efficiency. Summary of the Invention

[0003] The following is a summary of the subject matter described in detail herein. This summary is not intended to limit the scope of the claims.

[0004] The main purpose of the embodiments of the present invention is to provide a nucleic acid detection microfluidic chip and detection system with an ultrasonic micro-vibration cavity, which can effectively improve the efficiency of nucleic acid extraction.

[0005] In a first aspect, an embodiment of the present invention provides a nucleic acid detection microfluidic chip with an ultrasonic micro-vibration cavity, which is applied to a detection system. The detection system includes an ultrasonic device, a switch control unit, an exhaust control unit, and an air pump unit. The nucleic acid detection microfluidic chip includes a chip body, which includes:

[0006] A reaction chamber, the reaction chamber is arranged near the ultrasonic device, a micro-concave structure is arranged in the reaction chamber, the reaction chamber is provided with a first exhaust unit and a flow guide unit, and magnetic beads are placed in the reaction chamber;

[0007] a lysis solution pre-storage chamber, one end of which is connected to the reaction chamber via a third switch unit, and the other end of which is connected to a third one-way gas valve;

[0008] a washing liquid pre-storage chamber, one end of which is connected to the reaction chamber via a first switch unit, and the other end of which is connected to a first one-way air valve;

[0009] an eluent pre-storage chamber, one end of which is connected to the reaction chamber via the second switch unit, and the other end of which is connected to the second one-way gas valve;

[0010] a waste liquid chamber, one end of which is in communication with the first end of the guide unit, and the other end of which is in communication with the fourth one-way air valve, and the waste liquid chamber is also in communication with the second exhaust unit;

[0011] an extract detection chamber, one end of which is in communication with the second end of the guide unit and the other end of which is in communication with the fifth one-way air valve, and the extract detection chamber is also in communication with the third exhaust unit;

[0012] The first one-way gas valve, the second one-way gas valve, the third one-way gas valve, the fourth one-way gas valve and the fifth one-way gas valve are used to be connected to the air pump unit, the first switch unit, the second switch unit and the third switch unit are used to be connected to the switch control unit, and the first exhaust unit and the second exhaust unit are controlled by the exhaust control unit;

[0013] By controlling the gas input of the third one-way gas valve, the fourth one-way gas valve, and the fifth one-way gas valve and controlling the opening and closing of the first exhaust unit and the second exhaust unit, the liquid in the reaction chamber is controlled to flow to the waste liquid chamber or the extraction liquid detection chamber.

[0014] In one embodiment, when gas is input to the third one-way gas valve and the fourth one-way gas valve, the third switch unit is opened, the fifth one-way gas valve is closed, the first exhaust unit and the second exhaust unit are closed, and the third exhaust unit is opened, the liquid in the reaction chamber flows into the extraction liquid detection chamber;

[0015] or,

[0016] When gas is input to the third one-way gas valve and the fifth one-way gas valve, the third switch unit is opened, the fourth one-way gas valve is closed, the first exhaust unit and the third exhaust unit are closed, and the second exhaust unit is opened, the liquid in the reaction chamber flows to the waste liquid chamber.

[0017] In one embodiment, the reaction chamber is a closed cantilever vibration chamber, the ultrasonic device is provided on the outer side of the bottom surface of the reaction chamber, and the other side surfaces of the reaction chamber are suspended.

[0018] In one embodiment, the bottom of the reaction chamber is a U-shaped structure, and the reaction chamber is provided with a drainage channel, the drainage channel is used to introduce liquid into the bottom of the reaction chamber, and the guide unit is provided at the bottom of the reaction chamber.

[0019] In one embodiment, the top of the reaction chamber is connected to the washing solution pre-storage chamber, the eluent pre-storage chamber, and the lysis solution pre-storage chamber through different pipes, and the first exhaust unit is disposed at the top of the reaction chamber.

[0020] In one embodiment, the detection system includes a magnet, and when the reaction chamber needs to be drained of liquid, the magnetic beads are adsorbed on the side wall of the reaction chamber by the magnet.

[0021] In one embodiment, an overflow chamber is provided on the pipeline connecting the reaction chamber and the extract detection chamber, the top of the overflow chamber is connected to a guide unit provided at the bottom of the reaction chamber through a pipeline, and an overflow outlet connected to the extract detection chamber is provided at a first preset height on one side of the overflow chamber, the first volume from the overflow outlet of the overflow chamber to the bottom of the reaction chamber is less than the volume of the eluent added to the reaction chamber, and the volume difference is the amount of nucleic acid extract required for amplification, and the volume difference is the difference between the volume of the eluent and the first volume.

[0022] In one embodiment, a baffle structure is provided at the outlet of the waste liquid chamber, and the baffle structure is used to prevent the liquid entering the waste liquid chamber from directly diffusing to the outlet of the waste liquid chamber. The vertical distance between the top of the waste liquid chamber and the bottom of the reaction chamber is greater than the maximum depth of the liquid stored in the reaction chamber.

[0023] In one embodiment, the first switch unit, the second switch unit, and the third switch unit are all plunger valves, a groove structure is provided in the middle area of ​​the plunger valve, and a first sealing ring and a second sealing ring are provided on the upper and lower sides of the groove structure;

[0024] The first one-way air valve, the second one-way air valve, the third one-way air valve, the fourth one-way air valve and the fifth one-way air valve are all duckbill valves.

[0025] In one embodiment, the extract detection chamber is a conical tube, and the conical tube is used to pre-store freeze-dried beads of PCR amplification reagents.

[0026] In a second aspect, an embodiment of the present invention provides a detection system, comprising the nucleic acid detection microfluidic chip with the ultrasonic micro-vibration cavity described in the first aspect.

[0027] The beneficial effects of the present invention include: each mixing step performs resonance mixing treatment on the reaction chamber through an ultrasonic device, so that the liquid in the reaction chamber vibrates more violently, and some micro-concave structures are processed in the reaction chamber, and the sound field forms a strong acoustic micro-flow at the micro-concave structures, thereby making the vibration effect better and the mixing more complete; and then during the extraction process, the air pump unit of the detection system is used to pressurize different chambers to promote the transfer of reagents for liquid addition, liquid discharge, collection and other processes, so that the gas input can be controlled by the third one-way air valve, the fourth one-way air valve, and the fifth one-way air valve, and the opening and closing of the first exhaust unit and the second exhaust unit can be controlled by the exhaust control unit to control the liquid in the reaction chamber to flow to the waste liquid chamber or the extraction liquid detection chamber, so as to realize the nucleic acid extraction and detection micro-process.

[0028] Other features and advantages of the present invention will be described in the following description, and in part will become apparent from the description, or will be understood by practicing the present invention. The purposes and other advantages of the present invention can be realized and obtained by the structures particularly pointed out in the description, claims and drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 This is a schematic diagram of the internal structure of a nucleic acid detection microfluidic chip with an ultrasonic micro-vibration cavity provided by one embodiment of the present invention;

[0030] Figure 2 This is a front view of a nucleic acid detection microfluidic chip with an ultrasonic micro-vibration cavity provided by one embodiment of the present invention;

[0031] Figure 3 This is a rear view of a nucleic acid detection microfluidic chip with an ultrasonic micro-vibration cavity provided by one embodiment of the present invention;

[0032] Figure 4 This is a partially enlarged schematic diagram of a reaction chamber of a nucleic acid detection microfluidic chip with an ultrasonic micro-vibration chamber provided by one embodiment of the present invention;

[0033] Figure 5 Schematic diagram of an ultrasonic-magnetic hybrid mode of a nucleic acid detection microfluidic chip with an ultrasonic micro-vibration cavity provided by one embodiment of the present invention;

[0034] Figure 6 This is a schematic structural diagram of an ultrasonic device for a nucleic acid detection microfluidic chip with an ultrasonic micro-vibration cavity provided by one embodiment of the present invention;

[0035] Figure 7 This is a schematic diagram of the forces acting on microspheres of a nucleic acid detection microfluidic chip with an ultrasonic micro-vibration cavity provided by one embodiment of the present invention;

[0036] Figure 8This is a schematic diagram of a double-push nucleic acid collection method of a nucleic acid detection microfluidic chip with an ultrasonic micro-vibration cavity provided by one embodiment of the present invention;

[0037] Figure 9 This is a schematic diagram of a double-push waste liquid discharge method of a nucleic acid detection microfluidic chip with an ultrasonic micro-vibration cavity provided by one embodiment of the present invention;

[0038] Figure 10 Schematic diagram of a microwell in a reaction chamber of a nucleic acid detection microfluidic chip with an ultrasonic micro-vibration chamber provided by one embodiment of the present invention;

[0039] Figure 11 This is a schematic diagram of the size of the reaction chamber of a nucleic acid detection microfluidic chip with an ultrasonic micro-vibration cavity provided by one embodiment of the present invention;

[0040] Figure 12 Schematic diagram of Reynolds stress around microbubbles in a nucleic acid detection microfluidic chip with an ultrasonic microvibration cavity provided by one embodiment of the present invention;

[0041] Figure 13 Schematic diagram of the flow field around microbubbles of a nucleic acid detection microfluidic chip with an ultrasonic micro-vibration cavity provided by one embodiment of the present invention;

[0042] Figure 14 Schematic diagram of the dispersion effect of microbubbles on magnetic beads in a nucleic acid detection microfluidic chip with an ultrasonic micro-vibration cavity provided by one embodiment of the present invention;

[0043] Figure 15 This is a schematic diagram of magnetic beads moving around microbubbles in a nucleic acid detection microfluidic chip with an ultrasonic micro-vibration cavity provided by one embodiment of the present invention;

[0044] Figure 16 It is a schematic diagram of the flow field changes around microbubbles of different shapes in a nucleic acid detection microfluidic chip with an ultrasonic micro-vibration cavity provided by one embodiment of the present invention.

[0045] Reference numerals:

[0046] Chip body 100, reaction chamber 110, washing solution pre-storage chamber 120, eluent pre-storage chamber 130, lysis solution pre-storage chamber 140, waste liquid chamber 150 and extraction solution detection chamber 160;

[0047] dimple structure 111, first exhaust unit 112, flow guide unit 113;

[0048] A first switch unit 121 and a first one-way gas valve 122;

[0049] A second switch unit 131 and a second one-way gas valve 132;

[0050] A third switch unit 141 and a third one-way gas valve 142;

[0051] Baffle structure 151, fourth one-way air valve 152, second exhaust unit 153;

[0052] Overflow chamber 161, fifth one-way air valve 162, third exhaust unit 163;

[0053] Ultrasonic device 310 , magnetic beads 510 , and microbubbles 1010 . DETAILED DESCRIPTION

[0054] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0055] It should be noted that although the device schematics illustrate functional module divisions and the flowcharts illustrate logical sequences, in certain circumstances, the steps shown or described may be performed in a sequence that differs from the module divisions in the device or the sequence in the flowcharts. The terms "first," "second," and the like in the specification, claims, or accompanying drawings are used to distinguish similar items and are not necessarily used to describe a specific sequence or precedence.

[0056] Traditional nucleic acid detection methods, such as Southern blot hybridization and agarose gel electrophoresis, are time-consuming and labor-intensive, have low sensitivity, are difficult to implement for high-throughput testing, and even require the use of radioactive reagents. Traditional laboratory-based pathogen detection methods require specialized personnel in a well-equipped laboratory to transfer and add reagents. These procedures are cumbersome and can expose samples and reagents to air, making them highly susceptible to contamination. Furthermore, the difficulty of lysing samples, such as cells, viruses, serum, and plasma, varies, requiring different reagent compositions. This makes it difficult to detect diverse sample types and results in poor universal adaptability. Furthermore, fixed chips lack centrifugal force, making it difficult to achieve uniform mixing of the beads when extracting nucleic acids using magnetic beads, which can lead to nucleic acid loss and low extraction efficiency.

[0057] To solve the above-mentioned problems, an embodiment of the present invention provides a nucleic acid detection microfluidic chip with an ultrasonic micro-vibration cavity, which is applied to a detection system. The detection system includes an ultrasonic device, a switch control unit, an exhaust control unit and an air pump unit. The nucleic acid detection microfluidic chip includes a chip body, and the chip body includes a reaction chamber, a washing liquid pre-storage chamber, an eluent pre-storage chamber, a lysis liquid pre-storage chamber, a waste liquid chamber and an extraction liquid detection chamber. The reaction chamber is arranged close to the ultrasonic device, and a micro-concave structure is arranged in the reaction chamber, the reaction chamber is provided with a first exhaust unit and a guide unit, and magnetic beads are placed in the reaction chamber; one end of the lysis solution pre-storage chamber is connected to the reaction chamber through the third switch unit, and the other end is connected to the third one-way air valve; one end of the washing liquid pre-storage chamber is connected to the reaction chamber through the first switch unit, and the other end is connected to the first one-way air valve; one end of the eluent pre-storage chamber is connected to the reaction chamber through the second switch unit, and the other end is connected to the second one-way air valve; one end of the waste liquid chamber is connected to the first end of the guide unit, and the other end is connected to the fourth one-way air valve, and the waste liquid chamber is also connected to the second exhaust unit; one end of the extract detection chamber is connected to the second end of the guide unit The other end is connected to the fifth one-way air valve, and the extract detection chamber is also connected to the third exhaust unit; the first one-way air valve, the second one-way air valve, the third one-way air valve, the fourth one-way air valve and the fifth one-way air valve are used to be connected to the air pump unit, the first switch unit, the second switch unit and the third switch unit are used to be connected to the switch control unit, and the first exhaust unit and the second exhaust unit are controlled by the exhaust control unit; by controlling the gas input of the third one-way air valve, the fourth one-way air valve and the fifth one-way air valve and controlling the opening and closing of the first exhaust unit and the second exhaust unit, the liquid in the reaction chamber is controlled to flow to the waste liquid chamber or the extract detection chamber.

[0058] In the technical solution of this embodiment, each mixing step uses an ultrasonic device to perform resonance mixing treatment on the reaction chamber, so that the liquid in the reaction chamber vibrates more violently, and some micro-concave structures are processed in the reaction chamber, and the sound field forms a strong acoustic micro-flow at the micro-concave structures, thereby making the vibration effect better and the mixing more complete; then, during the extraction process, the air pump of the detection system is used to pressurize different chambers to promote the transfer of reagents for liquid addition, drainage, collection and other processes, so that the gas input can be controlled by the one-way air valves corresponding to different chambers and the opening and closing of different exhaust units can be controlled to control the liquid in the reaction chamber to flow to the waste liquid chamber or the extraction liquid detection chamber, so as to realize the nucleic acid extraction and detection micro-process.

[0059] The embodiments of the present invention are further described below with reference to the accompanying drawings.

[0060] like Figure 1-5 As shown, Figure 1This is an internal schematic diagram of a nucleic acid detection microfluidic chip with an ultrasonic micro-vibration cavity provided by an embodiment of the present application. The nucleic acid detection microfluidic chip with an ultrasonic micro-vibration cavity is applied to a detection system, which includes an ultrasonic device 310, a switch control unit, an exhaust control unit and an air pump unit. The nucleic acid detection microfluidic chip includes a chip body 100, which includes a reaction chamber 110, a washing liquid pre-storage chamber 120, an eluent pre-storage chamber 130, a lysate pre-storage chamber 140, a waste liquid chamber 150 and an extract detection chamber 160, wherein the reaction chamber 110 is arranged near the ultrasonic device 310, and the reaction chamber 110 is arranged near the ultrasonic device 310. A micro-concave structure 111 is provided inside the reaction chamber 110, a first exhaust unit 112 and a flow guide unit 113 are provided, and magnetic beads 510 are placed in the reaction chamber 110; one end of the lysis solution pre-storage chamber 140 is connected to the reaction chamber 110 through the third switch unit 141, and the other end is connected to the third one-way air valve 142, and the third one-way air valve 142 is used to connect to the air pump unit in the detection system; one end of the washing solution pre-storage chamber 120 is connected to the reaction chamber 110 through the first switch unit 121, and the other end is connected to the first one-way air valve 1 22 is connected, the first one-way air valve 122 is used to connect to the air pump unit in the detection system; one end of the eluent pre-storage chamber 130 is connected to the reaction chamber 110 through the second switch unit 131, and the other end is connected to the second one-way air valve 132, and the second one-way air valve 132 is used to connect to the air pump unit in the detection system; one end of the waste liquid chamber 150 is connected to the first end of the diversion unit 113, and the other end is connected to the fourth one-way air valve 152, and the waste liquid chamber 150 is also connected to the second exhaust unit 153, and the fourth one-way air valve 152 is used to connect to the air pump unit in the detection system. The extract detection chamber 160 is connected to the air pump unit in the detection system; one end of the extract detection chamber 160 is connected to the second end of the guide unit 113, and the other end is connected to the fifth one-way air valve 162. The extract detection chamber 160 is also connected to the third exhaust unit 163. The fifth one-way air valve 162 is used to connect to the air pump unit in the detection system; the first switch unit 121, the second switch unit 131, and the third switch unit 141 are used to connect to the switch control unit, and the first exhaust unit 112 and the second exhaust unit 153 are controlled by the exhaust control unit.

[0061] During the nucleic acid extraction process, the gas input of the third one-way gas valve 142, the fourth one-way gas valve 152, and the fifth one-way gas valve 162 is controlled, and the opening and closing of the first exhaust unit 112 and the second exhaust unit 153 are controlled to control the flow of liquid in the reaction chamber 110 to the waste liquid chamber 150 or the extraction liquid detection chamber 160.

[0062] For example: Refer to Figure 8When gas is input into the third one-way gas valve 142 and the fourth one-way gas valve 152, the third switch unit 141 is opened, the fifth one-way gas valve 162 is closed, the first exhaust unit 112 and the second exhaust unit 153 are closed, and the third exhaust unit 163 is opened, the liquid in the reaction chamber 110 flows to the extraction liquid detection chamber 160 through the double push action of the third one-way gas valve 142 and the fourth one-way gas valve 152.

[0063] Another example: refer to Figure 9 When gas is input to the third one-way gas valve 142 and the fifth one-way gas valve 162, the third switch unit 141 is opened, the fourth one-way gas valve 152 is closed, the first exhaust unit 112 and the third exhaust unit 163 are closed, and the second exhaust unit 153 is opened, the liquid in the reaction chamber 110 flows to the waste liquid chamber 150 through the double push action of the third one-way gas valve 142 and the fifth one-way gas valve 162.

[0064] The above two examples utilize the dual push of the air pump unit to achieve the discharge and collection of the liquid in the reaction chamber 110 .

[0065] It should be noted that, in the detection system, the air pumps connected to the first one-way air valve 122, the second one-way air valve 132, the third one-way air valve 142, the fourth one-way air valve 152, or the fifth one-way air valve 162 can be two different air pumps, or different air pumps corresponding to different one-way air valves. If there are two different air pumps (a first air pump and a second air pump), the first air pump can be connected to the third one-way valve, and the second air pump can be controlled to connect to the first one-way air valve 122, the second one-way air valve 132, the fourth one-way air valve 152, or the fifth one-way air valve 162 according to different steps. This embodiment does not specifically limit this.

[0066] In the technical solution of this embodiment, when it is necessary to extract nucleic acid from the sample through the nucleic acid detection microfluidic chip, the nucleic acid detection microfluidic chip is placed in the detection system. At this time, the one-way air valve of each chamber is connected to the air pump of the detection system, the switch unit in each chamber is in a closed state, the exhaust unit in each chamber is in a closed state, the washing liquid pre-storage chamber 120 is pre-stored with washing liquid, the eluent pre-storage chamber 130 is pre-stored with eluent, and the lysis liquid pre-storage chamber 140 is pre-stored with lysis liquid. The sample to be extracted is added to the lysis liquid pre-storage chamber 140, and after standing for a period of time, a lysis mixture is obtained. The first exhaust unit 112 is first opened to and the third switch unit 141, and pumping gas into the third one-way air valve 142 through the air pump to increase the air pressure in the lysate pre-storage chamber 140, so that the lysate mixed solution in the lysate pre-storage chamber 140 flows into the reaction chamber 110; starting the ultrasonic device 310 outside the reaction chamber 110, the ultrasonic device 310 transmits ultrasonic waves into the reaction chamber 110, forming an acoustic field in the reaction chamber 110, and the fluid in the reaction chamber 110 generates acoustic microflow under the action of the acoustic field, driving the magnetic beads 510 to move violently, so that the magnetic beads 510 are dispersed and mixed, so that the magnetic beads 510 fully adsorb nucleic acids, and the micro-concave structure set in the reaction chamber 110 111, the micro-concave structure 111 can enhance the acoustic micro-flow effect and further accelerate the mixing of the magnetic beads 510; after a period of time, the ultrasonic device 310 is stopped, and the detection system adsorbs the magnetic beads 510 through the magnet 520, and adsorbs the magnetic beads 510 on the side wall of the reaction chamber 110; the third switch unit 141 and the second exhaust unit 153 are opened, and gas is pumped into the third one-way air valve 142 and the fifth one-way air valve 162 through the air pump to flow the waste liquid in the reaction chamber 110 to the waste liquid chamber 150; then the second exhaust unit 153 and the third switch unit 141 are closed, and the first switch unit 121 and the first exhaust unit 112 are opened. An air pump is used to pump gas into the first one-way air valve 122 to flow the washing liquid in the washing liquid pre-storage chamber 120 into the reaction chamber 110. The ultrasonic device 310 is started for mixing. After a period of time, the ultrasonic device 310 is stopped. The detection system attracts the magnetic beads 510 via the magnet 520, closes the first switch unit 121 and the first exhaust unit 112, opens the third switch unit 141 and the second exhaust unit 153, and pumps gas into the third one-way air valve 142 and the fifth one-way air valve 162 via the air pump to flow the waste liquid in the reaction chamber 110 into the waste liquid chamber 150. The washing process can be repeated multiple times (generally twice).Then, the second exhaust unit 153 and the third switch unit 141 are closed, the second switch unit 131 and the second exhaust unit 153 are opened, and gas is pumped into the second one-way valve 132 through the air pump to flow the eluent in the eluent pre-storage chamber 130 to the reaction chamber 110, and the ultrasonic device 310 is started for mixing. After a period of time, the ultrasonic device 310 is stopped, and the detection system adsorbs the magnetic beads 510 through the magnet 520, closes the second switch unit 131 and the second exhaust unit 153, and opens the third switch unit 1 41. Third exhaust unit 163 uses an air pump to pump gas into third one-way valve 142 and fourth one-way valve 152 to move the eluted nucleic acid extract in reaction chamber 110 to extract detection chamber 160. The temperature control module then activates to adjust the temperature in extract detection chamber 160 to perform a denaturation-annealing-extension cycle on the 45 DNA samples and perform nucleic acid amplification. During this cycle, the optical module illuminates and detects the fluorescence intensity in extract detection chamber 160 to generate a curve and determine the Ct value.

[0067] Specifically, the nucleic acid extraction process of the nucleic acid detection microfluidic chip for the sample is as follows: first, the reagents required for the reaction, magnetic beads 510, and freeze-dried beads are pre-stored on the nucleic acid detection microfluidic chip, and then 50 μL of sample is drawn from the sample addition port 143 using a pipette and added to the lysis solution pre-storage chamber 140, the sample is lysed for 5 minutes, the plunger valve (third switch unit 141) of the lysis solution pre-storage chamber is opened, and the pump air pushes 150 μL of the lysis mixture into the reaction chamber 110, allowing the magnetic beads 510 in the reaction chamber 110 to adsorb nucleic acids, the ultrasonic device 310 is turned on and mixed for 25 seconds, the magnetic beads 510 are adsorbed with a magnet 520, and the waste liquid is discharged into the waste liquid chamber 150; the plunger valve (first switch unit 121) of the washing solution pre-storage chamber 120 is opened, and the air pump unit pushes about 200 μL of washing solution into The reaction chamber 110 is opened, the ultrasonic device 310 is turned on and mixed for 25 seconds, the magnetic beads 510 are adsorbed by the magnet 520, the waste liquid is discharged into the waste liquid chamber 140, and the washing is repeated twice; the plunger valve of the eluent pre-storage chamber 130 is opened, 50 μL of eluent is pushed into the reaction chamber 110 by pumping air, the ultrasonic device 310 is turned on and mixed for 25 seconds, the magnetic beads 510 are adsorbed by the magnet 520, and the eluted nucleic acid extract is discharged into the freeze-dried bead conical tube (extract detection chamber 160) pre-stored with PCR amplification reagents; the temperature control module is turned on and the temperature of the conical tube (extract detection chamber 160) is adjusted to perform 45 cycles of DNA denaturation-annealing-extension for nucleic acid amplification. During the cycle, the optical module illuminates the fluorescence intensity in the detection tube to generate a curve and obtain the Ct value.

[0068] It should be noted that the detection principle of real-time fluorescence quantitative PCR is: real-time fluorescence quantitative PCR is to add fluorescent groups to the PCR reaction system, and use the changes in the fluorescence signal to monitor the changes in the amount of amplified products in each cycle of the PCR amplification reaction in real time, and quantitatively analyze the starting template through the relationship between the Ct value and the standard curve to obtain the detection results.

[0069] According to the above extraction and detection process, in order to better control the aggregation and dispersion of the magnetic beads 510, the ultrasonic-magnetic hybrid mode is used to extract the nucleic acid, such as Figure 5 As shown, a magnet 520 is used to gather magnetic beads 510 that have absorbed nucleic acids. Once aggregated, the magnetic beads 510 cannot disperse on their own without external force. To evenly distribute the magnetic beads 510 in the reaction chamber 110, external field excitation is required. Ultrasonic waves have the characteristic of vibration mixing, which can disperse the magnetic beads 510 and promote the nucleic acid extraction reaction.

[0070] The structure of ultrasonic mixing device is as follows Figure 6 As shown. In order to improve the efficiency of ultrasonic energy transmission, the bottom wall thickness of the reaction chamber 110 is designed to be 0.5mm to reduce energy loss. In order to prevent gaps between the piezoelectric transducer and the chamber wall, the piezoelectric transducer is densely pasted on the side wall of the reaction chamber 110 with AB glue, and then a waveform generator is connected to excite ultrasonic waves. The sound waves pass through the thin wall and enter the reaction chamber. Under the action of ultrasonic waves, the liquid in the cavity generates resonance-induced acoustic micro-flow. The magnetic beads 510 will move with the vortex of the micro-acoustic flow, gradually dispersed and evenly distributed in the reaction chamber 110. In the reaction chamber 110 excited by ultrasonic vibration, the magnetic beads 510 are simultaneously affected by flow field force, gravity, buoyancy, interaction force, acoustic radiation force and Brownian force, as shown in FIG. Figure 7 shown.

[0071] The effect of ultrasound in the mixing process is explained through a specific embodiment. For example, the reagent in the chamber is a homogeneous isotropic fluid, and the NS equation and momentum equation of the fluid are:

[0072]

[0073]

[0074] Where ρ is the fluid density, t is the time, u is the fluid velocity, p is the pressure, μ is the fluid dynamic coefficient, μ b is the bulk viscosity coefficient.

[0075] For modeling the boundary-driven flow field in the acoustic streaming device, the numerical method used is based on perturbation theory, assuming that the second-order time-averaged acoustic streaming velocity is superimposed on the first-order acoustic velocity field. According to this theory, the density, pressure, and velocity of the fluid can be expressed as:

[0076] ρ=ρ0+ρ1+ρ2+..., (3.3)

[0077] p=p0+p1+p2+..., (3.4)

[0078] u=u1+u2+..., (3.5)

[0079] The subscript numbers represent static, first-order and second-order quantities respectively.

[0080] Substituting equations (3.3)(3.4)(3.5) into equations (3.1)(3.2) and considering the equations as first order, we can obtain the expression of the first-order sound velocity field:

[0081]

[0082] Repeat the above process, consider the equation as second order, and take the time average of the equation. The continuity equation and momentum equation for solving the second order time average acoustic streaming velocity can be expressed as:

[0083]

[0084] Among them, Reynolds stress The acoustic flow modes in the chamber can be calculated using formulas (3.8) and (3.9). This method is the Reynolds stress method.

[0085] It should be noted that the micro-concave structure 111 in the reaction chamber 110 is a micro-pore with a diameter of 10 μm. Figure 10 When the reaction chamber 110 is filled with liquid, the liquid cannot enter the micropores due to surface tension, and a micro bubble 1010 film is generated on the surface of the micropores. The diameter of the micro bubble 1010 is also 10 μm.

[0086] The role of micropores in the mixing process is explained through specific examples, for example Figure 11 As shown, the reaction chamber 110 is a rectangular chamber with a size of 3 mm × 0.5 mm. The diameter of the microbubble 1010 is 0.01 mm. The one-dimensional half-wavelength standing wave field in the rectangular domain is established by harmonic excitation of the bottom boundary at a frequency of f≈118 kHz. At this frequency, the thickness of the viscous boundary layer in water is given by the formula Calculation shows that it is about 1.69 μm, where v = μ / ρ0 is the kinematic viscosity coefficient of the fluid, and ω = 2πf is the angular frequency.

[0087] The model includes the Thermoviscous Acoustics Module and the Creeping Flow Module. First, the COMSOL "Thermoviscous Acoustics, Frequency Domain" interface is used to solve the first-order acoustic pressure and velocity fields. The bottom boundary of the rectangular channel is set to harmonic excitation, and the remaining boundaries are set to acoustic reflection boundary conditions. Velocity excitation is selected to reflect the boundary vibrations generated by the piezoelectric transducer in the actual device. The acoustic flow field is then simulated using the "Creeping Flow, Stationary" interface. In this step, the two Reynolds stress components are calculated, and all boundaries are set to no-slip boundary conditions.

[0088] The Reynolds stress generated around the microbubble 1010 is obtained through simulation as follows Figure 12 As shown in FIG, a relatively strong Reynolds stress is generated around the microbubble 1010, and vortexes are formed on both sides of the microbubble 1010 with different rotation directions; the flow field around the microbubble 1010 is as shown in FIG. Figure 13 As shown, the flow field lines are consistent with the Reynolds stress direction. This can be explained as the microbubbles 1010 absorbing ultrasonic energy in the acoustic field, generating vibrations, and forming an internal flow field around the microbubbles 1010. This internal flow field then drives the external liquid to flow, forming a vortex motion flow field.

[0089] Under the action of ultrasound, strong flow field disturbances are generated around the microbubbles 1010. When the magnetic beads 510 gather near the micropores, they are subjected to the flow field viscosity force exerted by the vortex flow field, and are driven by it to move violently and disperse from the aggregated state. Figure 14 This is the dispersion effect of the microporous microbubbles 1010 on the magnetic beads 510. The magnetic beads 510 gathered around the microporous microbubbles 1010 are gradually dispersed and moved away under the action of the acoustic flow field.

[0090] The movement of the magnetic beads 510 around the microbubbles 1010 is observed under a microscope. Figure 15 As shown in FIG, the acoustic vortex can be clearly observed, and the magnetic beads 510 are drawn into the vortex, moving and dispersing. Figure 16 As shown, during the ultrasonic vibration process, the shape of the acoustic flow vortex is constantly changing, and the movement trajectory of the magnetic bead 510 will also change accordingly. This can be explained as the microbubble 1010 is constantly vibrating in the sound field, and the shape of the microbubble 1010 membrane is also constantly changing, thereby affecting the flow field around the microbubble 1010, resulting in a change in the acoustic flow vortex. However, this change in the vortex shape can make the movement of the magnetic bead 510 more disordered, and to a certain extent, can make the magnetic bead 510 more evenly dispersed.

[0091] It should be noted that ultrasonic vibration refers to a mechanical vibration wave with a frequency higher than 20kHz, which is usually generated by an ultrasonic generator. Ultrasonic waves can generate shear force, pressure and ion effects in liquids to achieve mixing. Among them, for shear force mixing: when ultrasonic waves propagate in liquids, they will generate high-frequency vibration shear force, causing tiny particles and molecules in the liquid to shear, rotate and vibrate, accelerating the mixing process. For pressure effect mixing: when ultrasonic waves propagate in liquids, they will generate periodic high-pressure and low-pressure fluctuations. In high-pressure areas, the distance between liquid molecules becomes shorter, and the compression becomes denser, while in low-pressure areas, the distance between liquid molecules becomes longer, resulting in expansion and swelling. The alternating fluctuations of high and low pressure cause microbubbles 1010 in the liquid to instantly contract and expand, generating strong eddies and turbulence, which promote mixing. Regarding ion-effect mixing, when ultrasound propagates through liquid, it produces a high-frequency piezoelectric effect, polarizing and rearranging ions in the liquid, accelerating their diffusion and mixing. Furthermore, when ultrasound reaches a certain intensity, microbubbles 1010 are generated in the liquid. With the continued action of ultrasound, microbubbles 1010 continue to grow and expand, eventually bursting, releasing extremely high energy and forming cavitation. Using ultrasound to generate cavitation microbubbles 1010 in the liquid creates a powerful impact that disturbs the liquid and promotes more thorough mixing.

[0092] It should be noted that ultrasound has a certain effect on cell lysis. After the sample to be tested is added to the nucleic acid detection microfluidic chip, chemical lysis, thermal lysis, and ultrasound-assisted lysis can be carried out simultaneously, quickly and thoroughly lysing the sample cells. Even viruses wrapped in nucleocapsids can be lysed. It can also be applied to respiratory research samples, serum, plasma, urine, whole blood samples, and free DNA / RNA. It has good adaptability to multiple samples. At the same time, the heat generated by ultrasound can also promote the extraction reaction of nucleic acids.

[0093] In some optional embodiments, referring to Figure 1 The bottom of the reaction chamber 110 is a U-shaped structure, and the reaction chamber 110 is provided with a drainage channel for introducing liquid into the bottom of the reaction chamber 110. The drainage unit 113 is provided at the bottom of the reaction chamber 110. The lower portion of the reaction chamber 110 is U-shaped, and the bottom surface of the reaction chamber 110 is inclined. Under the action of gravity and air pump pressure, it can ensure that the liquid in the reaction chamber 110 is drained cleanly, which can reduce the loss of nucleic acid. In addition, the drainage channel designed in the reaction chamber 110 can guide the added liquid into the bottom of the reaction chamber 110, thereby preventing the liquid from leaking from the exhaust port of the reaction chamber 110.

[0094] In some optional embodiments, referring to Figure 1The top of the reaction chamber 110 is connected to the washing liquid pre-storage chamber 120, the eluent pre-storage chamber 130, and the lysis liquid pre-storage chamber 140 via separate pipes. A first exhaust unit 112 is disposed at the top of the reaction chamber. Placing the connecting pipes for the input liquid at the top of the reaction chamber 110 effectively prevents the input liquid from remaining in the pipes, allowing the input liquid to enter the reaction chamber 110 efficiently under the influence of air pressure and gravity.

[0095] In some optional embodiments, referring to Figure 1 The chip's air intake method uses the air pump's air filling needle and a one-way air valve, which can prevent gas leakage and liquid reflux, as well as prevent sample reagents from contaminating the air filling needle; there is a gas collection device at the exhaust port, and the entire air path is sealed and leak-free, safe and clean.

[0096] In some optional embodiments, referring to Figure 1 The reaction chamber 110 is a cantilever structure. An ultrasonic device 310 is provided on the outer side of one side of the reaction chamber 110, and the other sides of the reaction chamber 110 are suspended. When the reaction chamber 110 is a rectangular body, its three-sided suspended design gives the reaction chamber 110 a greater degree of freedom. Under the action of ultrasonic waves, the reaction chamber 110 and the ultrasonic device 310 resonate, and the liquid in the reaction chamber 110 vibrates more violently. In addition, some micro-convex structures and / or micro-concave structures 111 can be processed at the bottom of the chamber, and the sound field forms a strong acoustic micro-flow at the micro-structure, which can make the vibration effect better.

[0097] In some optional embodiments, reaction chamber 110 is a closed-end cantilever vibration chamber. By arranging piezoelectric ceramic excitation plates along the walls of reaction chamber 110 and utilizing the different sizes and input frequencies of the ceramic piezoelectric plates, a hybrid acoustic flow field is formed within reaction chamber 110, where multiple acoustic fields are superimposed. This hybrid acoustic flow field generates a repetitive and intense acoustic convection shock, which disperses and evenly distributes the magnetic beads, allowing them to fully react with the reagents, thereby improving nucleic acid extraction efficiency. This solution can also utilize the ultrasonic effect to enhance the lysis process during the lysis process, meeting the needs of different sample types.

[0098] In some optional embodiments, referring to Figure 1An overflow chamber 161 is provided on the pipeline connecting the reaction chamber 110 and the extract detection chamber 160. The top of the overflow chamber 161 is connected to the guide unit 113 provided at the bottom of the reaction chamber 110 through a pipeline. An anti-overflow outlet connected to the extract detection chamber 160 is provided at a first preset height on one side of the overflow chamber 161. The vertical distance between the top of the overflow chamber 161 and the bottom of the reaction chamber 110 is greater than the maximum depth of the liquid stored in the reaction chamber 110; a baffle structure 151 is provided at the outlet of the waste liquid chamber 150. The baffle structure 151 is used to prevent the liquid entering the waste liquid chamber 150 from directly diffusing to the outlet of the waste liquid chamber 150. The vertical distance between the top of the waste liquid chamber 150 and the bottom of the reaction chamber 110 is greater than the maximum depth of the liquid stored in the reaction chamber 110. When the nucleic acid detection microfluidic chip is in use, it is placed in a vertical fixed position. The air pump unit of the detection system is used to pressurize the reaction chamber 110, promote the transfer of liquid in the reaction chamber 110, and control the direction of liquid flow through the cooperation of the reversing valve and the group valve. The flow channels on both sides of the reaction chamber 110 are designed in an M shape to prevent the liquid from flowing to other chambers under the action of gravity. The double push of the air pump unit can realize the discharge and collection of the liquid in the reaction chamber 110. The waste liquid chamber 150 is used to store waste liquid. The volume of the waste liquid chamber 150 is sufficient to accommodate all waste liquids. There is a baffle at the top air outlet to prevent the waste liquid from directly diffusing from the inlet to the outlet and causing leakage.

[0099] In some optional embodiments, referring to Figure 1 The first switch unit 121, the second switch unit 131, and the third switch unit 141 can all be configured as plunger valves. A groove structure is provided in the middle area of ​​the plunger valve, and a first sealing ring and a second sealing ring are provided on the upper and lower sides of the groove structure. A circle of groove structure is provided in the middle of the plunger valve, which is used for liquid circulation. When the plunger valve is switched to this position, the plunger valve is in the open state. There is a sealing ring on the upper and lower sides of the groove structure. In normal state, the lower sealing ring blocks the flow channel, and the plunger valve is in the closed state. When in use, the electric push rod is moved downward to press the plunger valve downward, so that the groove structure connects to the flow channel, causing the plunger valve to switch to the open state.

[0100] In some optional embodiments, referring to Figure 1 The first one-way air valve 122, the second one-way air valve 132, the third one-way air valve 142, the fourth one-way air valve 152, and the fifth one-way air valve 162 can all be configured as duckbill valves, with the tops of the duckbill valves being sealed. During use, the air pump unit has a needle at its outlet, which pierces the tops of the duckbill valves. The air pump unit pumps gas into the chambers in the chip through the needles to push the liquid to other chambers for reaction. At this time, the needles are in close contact with the duckbill valves, effectively preventing air leakage. The duckbill valves prevent the liquid in the chambers from flowing back and contaminating the needles.

[0101] It should be noted that, referring to Figure 2 The washing liquid pre-storage chamber 120 can be provided with a fourth exhaust unit 211, and the elution liquid pre-storage chamber 130 can be provided with a fifth exhaust unit 212. The fourth exhaust unit 211 and the fifth exhaust unit 212 are controlled by the exhaust switch unit of the detection system. This embodiment does not specifically limit the structure, number and opening size of the exhaust unit of the chip, and can be set according to actual conditions.

[0102] It should be noted that, referring to Figure 2 The washing liquid pre-storage chamber 120 can be provided with a first infusion port 221, the eluent pre-storage chamber 130 can be provided with a second infusion port 222, and the extraction liquid detection chamber 160 can be provided with a third infusion port 223. This embodiment does not specifically limit the structure, number and opening size of the infusion ports of the chip, and can be set according to actual conditions.

[0103] In addition, a detection system includes the nucleic acid detection microfluidic chip with an ultrasonic micro-vibration cavity in the above-mentioned embodiment, the ultrasonic device 310, the switch control unit, the exhaust control unit and the air pump unit. The ultrasonic device 310, the switch control unit, the exhaust control unit and the air pump unit of the detection system are designed to cooperate with the structure of the nucleic acid detection microfluidic chip with an ultrasonic micro-vibration cavity. The technical means in the above-mentioned embodiment can solve the technical problems in the above-mentioned embodiment and can also achieve the technical effects in the above-mentioned embodiment, which will not be repeated here.

[0104] The above is a specific description of the preferred implementation of the present application, but the present application is not limited to the above implementation mode. Technical personnel familiar with the art can also make various equivalent modifications or substitutions under the shared conditions that do not violate the spirit of the present application. These equivalent modifications or substitutions are all included in the scope defined by the claims of the present application.

Claims

1. A nucleic acid detection microfluidic chip with an ultrasonic micro-vibration cavity, characterized in that: Applied to a detection system, the detection system includes an ultrasonic device, a switch control unit, an exhaust control unit and an air pump unit, and the nucleic acid detection microfluidic chip includes a chip body, the chip body includes: A reaction chamber, the reaction chamber is arranged near the ultrasonic device, a micro-concave structure is arranged in the reaction chamber, the reaction chamber is provided with a first exhaust unit and a flow guide unit, and magnetic beads are placed in the reaction chamber; a lysis solution pre-storage chamber, one end of which is connected to the reaction chamber via a third switch unit, and the other end of which is connected to a third one-way gas valve; a washing liquid pre-storage chamber, one end of which is connected to the reaction chamber via a first switch unit, and the other end of which is connected to a first one-way air valve; an eluent pre-storage chamber, one end of which is connected to the reaction chamber via the second switch unit, and the other end of which is connected to the second one-way gas valve; a waste liquid chamber, one end of which is in communication with the first end of the guide unit, and the other end of which is in communication with the fourth one-way air valve, and the waste liquid chamber is also in communication with the second exhaust unit; an extract detection chamber, one end of which is in communication with the second end of the guide unit and the other end of which is in communication with the fifth one-way air valve, and the extract detection chamber is also in communication with the third exhaust unit; The first one-way gas valve, the second one-way gas valve, the third one-way gas valve, the fourth one-way gas valve and the fifth one-way gas valve are used to be connected to the air pump unit, the first switch unit, the second switch unit and the third switch unit are used to be connected to the switch control unit, and the first exhaust unit and the second exhaust unit are controlled by the exhaust control unit; By controlling the gas input of the third one-way gas valve, the fourth one-way gas valve, and the fifth one-way gas valve and controlling the opening and closing of the first exhaust unit and the second exhaust unit, the liquid in the reaction chamber is controlled to flow to the waste liquid chamber or the extraction liquid detection chamber.

2. The nucleic acid detection microfluidic chip with an ultrasonic micro-vibration cavity according to claim 1, characterized in that: When gas is input to the third one-way gas valve and the fourth one-way gas valve, the third switch unit is opened, the fifth one-way gas valve is closed, the first exhaust unit and the second exhaust unit are closed, and the third exhaust unit is opened, the liquid in the reaction chamber flows into the extraction liquid detection chamber; or, When gas is input to the third one-way gas valve and the fifth one-way gas valve, the third switch unit is opened, the fourth one-way gas valve is closed, the first exhaust unit and the third exhaust unit are closed, and the second exhaust unit is opened, the liquid in the reaction chamber flows to the waste liquid chamber.

3. The nucleic acid detection microfluidic chip with an ultrasonic micro-vibration cavity according to claim 1, characterized in that: The reaction chamber is a closed cantilever vibration chamber, the ultrasonic device is arranged on the outer side of the bottom surface of the reaction chamber, and the other side surfaces of the reaction chamber are suspended.

4. The nucleic acid detection microfluidic chip with an ultrasonic micro-vibration cavity according to claim 3, characterized in that: The bottom of the reaction chamber is a U-shaped structure, and the reaction chamber is provided with a drainage channel, the drainage channel is used to introduce liquid into the bottom of the reaction chamber, and the guide unit is provided at the bottom of the reaction chamber.

5. The nucleic acid detection microfluidic chip with an ultrasonic micro-vibration cavity according to claim 4, characterized in that: The top of the reaction chamber is connected to the washing liquid pre-storage chamber, the eluent pre-storage chamber, and the lysis liquid pre-storage chamber through different pipelines, and the first exhaust unit is arranged on the top of the reaction chamber.

6. The nucleic acid detection microfluidic chip with an ultrasonic micro-vibration cavity according to claim 1, characterized in that: The detection system includes a magnet. When the reaction chamber needs to drain liquid, the magnetic beads are adsorbed on the side wall of the reaction chamber by the magnet.

7. The nucleic acid detection microfluidic chip with an ultrasonic micro-vibration cavity according to claim 1, characterized in that: An overflow chamber is provided on the pipeline connecting the reaction chamber and the extract detection chamber, the top of the overflow chamber is connected to the guide unit provided at the bottom of the reaction chamber through a pipeline, and an overflow outlet connected to the extract detection chamber is provided at a first preset height on one side of the overflow chamber, the first volume from the overflow outlet of the overflow chamber to the bottom of the reaction chamber is less than the volume of the eluent added to the reaction chamber, and the volume difference is the amount of nucleic acid extract required for amplification, and the volume difference is the difference between the volume of the eluent and the first volume.

8. The nucleic acid detection microfluidic chip with an ultrasonic micro-vibration cavity according to claim 7, characterized in that: A baffle structure is provided at the outlet of the waste liquid chamber, and the baffle structure is used to prevent the liquid entering the waste liquid chamber from directly diffusing to the outlet of the waste liquid chamber. The vertical distance between the top of the waste liquid chamber and the bottom of the reaction chamber is greater than the maximum depth of the liquid stored in the reaction chamber.

9. The nucleic acid detection microfluidic chip with an ultrasonic micro-vibration cavity according to claim 2, characterized in that: The first switch unit, the second switch unit, and the third switch unit are all plunger valves, a groove structure is provided in the middle area of ​​the plunger valve, and a first sealing ring and a second sealing ring are provided on the upper and lower sides of the groove structure; The first one-way air valve, the second one-way air valve, the third one-way air valve, the fourth one-way air valve and the fifth one-way air valve are all duckbill valves.

10. The nucleic acid detection microfluidic chip with an ultrasonic micro-vibration cavity according to claim 1, characterized in that: The extraction liquid detection chamber is a conical tube, and the conical tube is used for pre-storing freeze-dried beads containing PCR amplification reagents.

11. A detection system, characterized in that: A nucleic acid detection microfluidic chip with an ultrasonic micro-vibration cavity according to any one of claims 1 to 10.

Images