Microfluidic chip and application thereof

Abstract

The application relates to a micro-fluidic chip and application thereof, and belongs to the technical field of micro-fluidics. The micro-fluidic chip comprises a bottom plate layer and a cover plate layer in sealing cooperation with the bottom plate layer, a rotation center at the center of a circle is arranged on the bottom plate layer, and a sample pool, a transition flow channel, a detection unit, a balance flow channel and a waste pool are sequentially connected; the detection unit comprises a transition pool, a first flow channel, a first reaction pool, a second flow channel, a second reaction pool and a third flow channel which are sequentially connected; the transition flow channel and the balance flow channel are arranged around the rotation center; the transition flow channel is closer to the rotation center than the balance flow channel; and the detection unit is arranged in a region between the transition flow channel and the balance flow channel. A sample adding hole and a detection hole are arranged on the cover plate layer; the sample adding hole is located in correspondence with the sample pool; and the detection hole is located in correspondence with the second reaction pool. The micro-fluidic chip only needs to be matched with a simple mechanical structure, and can quickly complete nucleic acid RPA amplification and CRISPR accurate detection of various nucleic acid molecules including pathogenic bacteria under constant temperature by utilizing centrifugal force.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of microfluidic technology, in particular to a microfluidic chip and application thereof. BACKGROUND

[0002] With the development of detection technology, the current detection chip can use microfluidic technology to complete different biochemical reactions to meet the detection requirements. The conventional test chip has a disc shape, a sheet shape and a cartridge shape, etc. Microfluidics refers to the science and technology of using microchannels (size of tens to hundreds of microns) to process or manipulate microfluids (volume of nanoliters to attoliters). It is a new cross-disciplinary subject involving chemistry, fluid physics, microelectronics, new materials, biology and biomedical engineering. Because of miniaturization and integration, microfluidic devices are often referred to as microfluidic chips, also known as lab-on-a-chip and micro-total analytical system.

[0003] In the microfluidic detection technology, the disc-shaped centrifugal chip has the advantages of simple driving structure, avoiding complicated mechanical design, reducing the probability of mechanical error and reducing the cost of instrument manufacturing, because it can be driven by a single motor. Under the driving of centrifugal force, the liquid spreads outwards along the radial direction from the near center, and flows into the chamber gradually away from the center. It has the characteristics of simple driving force and clear liquid motion direction.

[0004] The existing microfluidic detection chip mainly detects biochemical indicators, and it is difficult to complete the complex pathogen nucleic acid amplification and detection reaction process. At present, the microfluidic chip used to complete the molecular detection project generally adopts the PCR (Polymerase Chain Reaction) method, which has high requirements for the temperature change of the equipment.

[0005] Recombinase Polymerase Amplification (RPA) is considered as a nucleic acid detection technology that can replace PCR. The RPA technology mainly uses three enzymes: a recombinase that can bind to single-stranded nucleic acids (oligonucleotide primers), a single-stranded DNA binding protein (SSB), and a strand displacement DNA polymerase. The mixture of the three enzymes is also active at room temperature, and the optimal reaction temperature is around 37°C. Compared with PCR, RPA is isothermal amplification, which does not require temperature changes and does not require the use of complex temperature modules for instruments. Compared with another isothermal amplification method LAMP (Loop-mediated Isothermal Amplification), RPA has the advantages of requiring fewer primers, shorter reaction time, easy storage of reagents, lower reaction temperature, and higher accuracy of results.

[0006] However, the molecular detection microfluidic chip based on isothermal amplification technology has many manual operation steps and low throughput, because it needs to accommodate multiple steps of molecular amplification detection reaction, and the design difficulty is greatly improved. Therefore, the number of detection items at a time is small, and there are problems of high cost and low efficiency.

[0007] The CRISPR detection (Clustered Regularly Interspaced Short Palindromic Repeats) technology has the characteristics of high sensitivity, strong specificity, short detection time, strong anti-interference, and fast development speed. It can improve the overall sensitivity of the amplification detection reaction.

[0008] Researchers have proposed that RPA amplification and CRISPR detection can be concentrated on a disc centrifugal chip, and then controlled by microfluidic technology, which can realize micro-total analysis of nucleic acid detection and has excellent application prospects.

[0009] However, the existing isothermal amplification molecular detection microfluidic chip has the disadvantages of low accuracy, low sensitivity, short and inaccurate control process. And when multiple indicators are detected at the same time, the sample amount of each reaction in the conventional microfluidic chip is not uniform, and there are problems of mutual interference and contamination.

[0010] Currently, there is no microfluidic centrifugal chip that can be put into practical application, which combines the advantages of high-throughput microfluidic technology, centrifugal disc, RPA amplification and CRISPR detection. SUMMARY

[0011] Therefore, it is necessary to provide a microfluidic chip in view of the above problems, which can quickly and accurately complete nucleic acid RPA amplification and CRISPR detection under constant temperature conditions by matching a simple mechanical structure and using centrifugal force. The microfluidic chip can be applied in various fields, such as pathogen molecular diagnosis of respiratory tract infection.

[0012] A microfluidic chip comprises a bottom plate layer and a cover plate layer in sealing cooperation with the bottom plate layer, a rotation center for connecting a rotating shaft is arranged on the bottom plate layer, and a sample pool, a transition flow channel, a detection unit, a balance flow channel and a waste liquid pool are sequentially connected, the detection unit comprises a transition pool, a first flow channel, a first reaction pool, a second flow channel, a second reaction pool and a third flow channel which are sequentially connected, the transition flow channel and the balance flow channel are arranged around the rotation center, the transition flow channel is closer to the rotation center than the balance flow channel, and the detection unit is arranged in a region between the transition flow channel and the balance flow channel.

[0013] A sample adding hole and a detection hole are arranged on the cover plate layer, the sample adding hole is located in correspondence with the sample pool, and the detection hole is located in correspondence with the second reaction pool.

[0014] The detection principle of the microfluidic chip is as follows: the sample to be detected is injected into the sample pool through the sample adding hole, the microfluidic chip is controlled to rotate, the sample flows through the transition flow channel into the transition pool, and a predetermined volume is measured; the microfluidic chip is controlled to rotate again, the sample flows through the first flow channel into the first reaction pool, and fully reacts with the reaction reagent pre-embedded in the first reaction pool; the microfluidic chip is controlled to rotate again, the sample flows through the second flow channel into the second reaction pool, and fully reacts with the reaction reagent pre-embedded in the second reaction pool to generate a detection signal, and the detection mechanism reads the detection signal through the detection hole. During the detection process, the excess waste liquid sequentially passes through the third flow channel and the balance flow channel into the waste liquid pool under the action of centrifugal force.

[0015] In the microfluidic chip, the balance flow channel is connected to each second reaction pool, so that the pressure at the connection position is the same, thereby effectively maintaining the force balance of the overall structure of the microfluidic chip. The detection hole and the balance flow channel are arranged on the upper and lower cover plate layer and bottom plate layer respectively, so that the spatial structure interference between the two is avoided.

[0016] It can be understood that the above microfluidic chip is made of conventional chip materials, such as PMMA (polymethylmethacrylate), PP, PC, and other polymer materials. For the corresponding position of the detection hole and the second reaction pool, the signal collection device can collect the detection reaction signal. According to the actual application, it can be adjusted and set, such as setting the detection hole and the second reaction pool on the vertical section of the radial extending from the rotation center to the outer periphery, that is, the detection hole and the second reaction pool of the same reaction unit correspond to the same radial. The setting position of the rotation center can also be adjusted according to the overall shape of the microfluidic chip, such as the microfluidic chip being circular, the rotation center can be located at the center of the circle, and the microfluidic chip being a fan, the rotation center can be located at the center of the circle matched by the fan, that is, only the rotation axis of the microfluidic chip needs to be set.

[0017] The above microfluidic chip can be used in the combined detection of RPA isothermal amplification and CRISPR detection technology. RPA isothermal amplification is carried out in the first reaction pool, and CRISPR detection is realized in the second reaction pool. The RPA-CRISPR (RPA, Recombinase Polymerase Amplification; CRISPR, Clustered Regularly Interspaced Short Palindromic Repeats) method is used, and the temperature control requirement of the device is low, only about 40 degrees Celsius is required for temperature control, and the control is relatively simple.

[0018] If there is no suitable flow channel design, the liquid in each channel cannot uniformly flow into the respective reaction pool when the air pressure is unbalanced and unstable, resulting in uncontrollable differences in each reaction volume, which will seriously affect the reaction result and cannot guarantee the accuracy and reliability of the result.

[0019] The microfluidic chip has the advantages of high accuracy and high sensitivity. Due to the delicate layout design of the flow channels and reaction pools in the microfluidic chip, the control process is long, and the liquid flow is accurately controlled from liquid inlet to RPA and CRISPR step-by-step reaction. Because of the delicate layout of the flow channels, the separation between different processes and channels is clear, and the liquid flow is accurately controlled. There is no mutual interference such as liquid mixing in the chip, which effectively avoids contamination between reagents.

[0020] In one embodiment, the detection unit is evenly distributed along the transition flow channel, each detection unit is in communication with the transition flow channel through the transition pool, and is in communication with the balance flow channel through the third flow channel.

[0021] By accurate design of each reaction pool and flow channel, the purpose of setting multiple detection units on the same chip is achieved, each detection unit can complete an independent detection, high-throughput detection is achieved, and different detection items can be detected. For example, reagents required for detection of several different pathogens are pre-embedded in the first reaction pool and the second reaction pool of the corresponding detection unit, so that detection of several different pathogens can be realized. Moreover, each detection unit in the chip is an independent unit, which can effectively avoid pollution between different items, and the chip as a whole has the same property, so that the nucleic acid amplification detection reaction can be completed synchronously under the driving of centrifugal force. Moreover, the microfluidic chip not only has the characteristics of high throughput and large number of detection reactions, but also controls the volume of a single reaction, which can be only 10 microliters, and the amount of pre-embedded chemical reagents is small, so that the chip has the advantages of low cost, high efficiency, easy normal temperature preservation and transportation.

[0022] In one of the embodiments, the detection hole and the second reaction pool are arranged on the vertical section of the radial extending from the rotation center to the outer periphery. The above arrangement is conducive to the reading of subsequent detection signals.

[0023] In one of the embodiments, the balance flow channel is arranged in a wavy circular ring, and the wavy balance flow channel protrudes away from the center of the circle at a position corresponding to the second reaction pool. The detection hole overlaps with the protruding end of the balance flow channel away from the center of the circle. Through the wavy design of the balance flow channel, the position protruding away from the center of the circle overlaps with the detection hole in the vertical direction, which can better complete the positioning of each reaction channel. Specifically, the positioning function is that the balance flow channel and the detection hole are obviously different from other parts of the chip in terms of light sensitivity, and the light sensor is used to identify the difference in light sensitivity to achieve accurate positioning control of the chip in the corresponding detection equipment.

[0024] In one of the embodiments, the third flow channel and the balance flow channel are communicated at the position of the wavy balance flow channel protruding towards the center of the circle. The communication position is arranged at a different radius from the detection hole, which can normally realize the communication function without affecting the positioning of the detection hole, and save space position without expanding the radius of the chip for different structures.

[0025] In one of the embodiments, the balance flow channel further comprises a communication flow channel, one end of the communication flow channel is communicated with the waste pool through a valve hole, and the other end is communicated with the balance flow channel. The radial passage width of the valve hole is greater than the radial passage width of the communication flow channel. By changing the radial passage width, the siphon effect that may occur when the liquid in the waste pool is excessive can be blocked, the remaining sample waste liquid is prevented from leaking, and aerosol pollution is effectively avoided. Preferably, the radial passage width of the valve hole is more than 5-15 times the radial passage width of the communication flow channel, which has a better cutting effect.

[0026] In one embodiment, the first flow channel has an S-shaped passage, and the third flow channel has a C-shaped passage. The S-shaped passage is used to block the sample from flowing from the transition pool to the first reaction pool, and the S-shaped arc with large curvature is used to control the microfluidic chip to enter the first reaction pool only at a predetermined rotation speed. Similarly, the C-shaped passage is used to increase the difficulty of the sample flowing through the third flow channel into the balance flow channel, so that the sample preferentially enters the second reaction pool for reaction, thereby maintaining the detection effect.

[0027] In addition, the first flow channel and the balance flow channel both adopt S-shaped and wavy arcs with large curvature, which can maintain the balance of air pressure in the chip and stably control the liquid flow, and ensure that the channels do not interfere with each other.

[0028] In one embodiment, the transition pool, the first reaction pool and the second reaction pool are arranged in the order of being away from the rotation center. The above arrangement is conducive to the sample preferentially taking the required sample amount in the transition pool by using the volume of the transition pool, and then sequentially entering the first reaction pool and the second reaction pool for reaction.

[0029] In one embodiment, the first flow channel outlet and the third flow channel inlet are both arranged on the side close to the center of the first reaction pool, and the second flow channel inlet is arranged on the side away from the center of the first reaction pool. The above arrangement is conducive to the sample preferentially entering the second reaction pool through the second inlet under the action of centrifugal force, and then entering the balance flow channel through the third flow channel when the second reaction pool is filled.

[0030] In one embodiment, the sample pool is arranged between the rotation center and the transition flow channel, and the waste liquid pool is arranged between the transition flow channel and the balance flow channel.

[0031] In one embodiment, the bottom plate layer is further provided with a bottom plate air hole, and the cover plate layer is respectively provided with a cover plate air hole corresponding to the bottom plate air hole. The bottom plate air hole and the cover plate air hole are both air holes, which have the function of maintaining the communication between the chip and the atmosphere and maintaining the balance of air pressure in the chip.

[0032] In one embodiment, the thickness of the bottom plate layer is 2-5 mm, the diameter is 80-150 mm, the pore size of the transition flow channel is 0.8-1.3 mm, the pore sizes of the first flow channel, the second flow channel, the third flow channel and the balance flow channel are independently selected from 0.1-0.5 mm, the holding volume of the transition pool is 10-40 μL, the holding volume of the first reaction pool is 20-50 μL, and the holding volume of the second reaction pool is 10-30 μL. It can be understood that the above specifications can be adjusted according to specific reaction conditions and detection requirements, but according to the above size specifications, the liquid flow can be precisely controlled, the different reaction pools and channels are clearly separated and do not interfere with each other, and the size of the chip is reduced.

[0033] In one embodiment, the first reaction pool and the second reaction pool are respectively pre-embedded with reaction reagents. It can be understood that the above-mentioned reaction reagents are set according to specific reaction requirements. For example, if the first reaction pool is a new crown nucleic acid RPA amplification reaction, the new crown RPA amplification reagent is pre-embedded, if the first reaction pool is a tuberculosis nucleic acid RPA amplification reaction, the tuberculosis RPA amplification reagent is pre-embedded, and so on. If the second reaction pool is a new crown CRISPR reaction, the new crown CRISPR detection reagent is pre-embedded, if the second reaction pool is a tuberculosis CRISPR reaction, the tuberculosis CRISPR detection reagent is pre-embedded, and so on.

[0034] The application further discloses a microfluidic-based constant-temperature amplification detection device.

[0035] The constant-temperature amplification detection device adopts the microfluidic chip for detection, and can only need to be matched with a simple mechanical structure, and can quickly complete nucleic acid RPA amplification and CRISPR accurate detection of various nucleic acid molecules including pathogens under constant-temperature conditions by using centrifugal force.

[0036] In one embodiment, the microfluidic-based constant-temperature amplification detection device further comprises:

[0037] The centrifugal mechanism comprises a driving mechanism for connecting the rotation center and driving the microfluidic chip to rotate;

[0038] The heating mechanism comprises a heat source mechanism for heating the detection unit;

[0039] The detection mechanism comprises a detection mechanism for reading the reaction signal of the first reaction pool and / or the second reaction pool; and

[0040] The control mechanism comprises a control mainboard, and the control mainboard is electrically connected or signal-connected with the centrifugal mechanism, the heating mechanism and the detection mechanism, and is used for controlling the centrifugal mechanism, the heating mechanism and the detection mechanism.

[0041] It can be understood that the above-mentioned detection mechanism can be adjusted according to the specific signal type generated, for example, if the optical signal such as fluorescence is collected as the detection means, the detection mechanism is an optical detection mechanism. The signal of the first reaction pool or the second reaction pool is also adjusted according to the signal link generated in the specific detection process.

[0042] The application further discloses a microfluidic-based detection method using the microfluidic chip, which comprises the following steps: injecting a sample to be detected into the sample pool through the sample injection hole, rotating the microfluidic chip, and making the sample flow through the transition flow channel and into the transition pool; rotating the microfluidic chip again, making the sample flow through the first flow channel and into the first reaction pool, and fully reacting with the reaction reagent pre-embedded in the first reaction pool; rotating the microfluidic chip again, making the sample flow through the second flow channel and into the second reaction pool, fully reacting with the reaction reagent pre-embedded in the second reaction pool, generating a detection signal, and reading the detection signal through the detection hole by the detection mechanism.

[0043] It can be understood that the detection method can be used for isothermal amplification detection of nucleic acid molecules and has good effects, and can also be applied to other types of multi-step reaction detection.

[0044] In one of the embodiments, the excess waste liquid in the first reaction pool sequentially flows into the waste liquid pool through the third flow channel and the balance flow channel under the action of centrifugal force.

[0045] Compared with the prior art, the application has the following beneficial effects:

[0046] The microfluidic chip can be used for combined detection of RPA isothermal amplification and CRISPR detection technology, RPA isothermal amplification is carried out in the first reaction pool, CRISPR detection is realized in the second reaction pool, and the RPA-CRISPR (RPA, Recombinase Polymerase Amplification, CRISPR, Clustered Regularly Interspaced Short Palindromic Repeats) method is used, so that the temperature control requirement of the device is low, only about 40 degrees Celsius, and the control is simple.

[0047] The microfluidic chip has the advantages of high accuracy and high sensitivity, because the flow channels and reaction pools in the microfluidic chip are precisely laid out and designed, the control process is long, and the liquid flow in the step-by-step reaction from liquid inlet to RPA and CRISPR can be accurately controlled. Because the layout of the flow channels is precise, the separation between different processes and channels is clear, the liquid flow is accurately controlled, there is no mutual interference such as liquid mixing in the chip, and contamination between reagents is effectively avoided.

[0048] And through the first flow channel and the balance flow channel clever design, using the curvature of the larger arc, can maintain the chip pressure balance and stable control of liquid flow, and ensure that each channel will not interfere with each other. When the liquid through the transition pool, flow through the first flow channel, at a certain speed into the first reaction pool for full reaction; then at another speed into the second reaction pool to complete the next reaction independently.

[0049] The microfluidic chip combines the advantages of high-throughput microfluidic technology, centrifugal disc, RPA amplification and CRISPR detection. Only need to match a simple mechanical structure, use centrifugal force, under constant temperature conditions, can quickly complete the nucleic acid RPA amplification and CRISPR accurate detection of pathogens. And pre-embedded chemical reagents are easy to save and transport. Also because the reaction is carried out in a sealed chip, it has the advantages of avoiding aerosol, reducing manual operation and improving detection sensitivity. BRIEF DESCRIPTION OF DRAWINGS

[0050] Figure 1 It is a schematic diagram of the bottom layer structure of the microfluidic chip in Example 1;

[0051] Figure 2 It is a schematic diagram of the cover layer structure of the microfluidic chip in Example 1;

[0052] Figure 3 It is a schematic diagram of the sample solution position of the microfluidic chip in Example 3 in detection;

[0053] Figure 4 It is a schematic diagram of the microfluidic chip result in Example 4;

[0054] Figure 5 It is a schematic diagram of the microfluidic chip result in Example 5;

[0055] Figure 6 It is a schematic diagram of the bottom layer structure of the microfluidic chip in Comparative Example 1;

[0056] Figure 7 It is a schematic diagram of the cover layer structure of the microfluidic chip in Comparative Example 1;

[0057] Figure 8 It is a schematic diagram of the sample solution position of the microfluidic chip in Comparative Example 2 in detection;

[0058] Figure 9 It is a schematic diagram of the sample solution position of the microfluidic chip in Comparative Example 3 in detection.

[0059] Wherein: 100, bottom layer; 110, rotation center; 120, sample cell; 130, transition flow channel; 141, transition cell; 142, first flow channel; 143, first reaction cell; 144, second flow channel; 145, second reaction cell; 146, third flow channel; 150, balance flow channel; 151, communication flow channel; 152, valve hole; 160, waste cell; 171, first hole; 172, second hole; 200, cover layer; 210, sample adding hole; 220, detection hole; 231, third hole; 232, fourth hole. DETAILED DESCRIPTION

[0060] For the purpose of promoting an understanding of the principles of the application, reference will now be made to the embodiments illustrated in the drawings. There can, of course, be many embodiments of the application and the specific embodiments described herein are not intended to limit the application but merely to illustrate it. It is further understood that the drawings are not necessarily to scale and that the various features of the structures illustrated in the drawings can be combined in various manners and / or separated into multiple structures.

[0061] It is to be understood that the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. It is to be understood that the term "comprising" is not limiting, but rather, is intended to mean that the compositions and methods include any additional steps and / or ingredients as appropriate. It is further to be understood that the terms "comprise", "comprising", "comprises" and "comprised of" when used in this specification, specify the presence of stated features, integers, steps, components, but do not preclude the presence or addition of one or more other features, integers, steps, components or groups thereof.

[0062] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used in the description of the application herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety.

[0063] Example 1

[0064] A microfluidic chip comprises a bottom layer 100 and a cover layer 200 sealed with the bottom layer 100, the bottom layer 100 is provided with a rotation center 110 at the center of the bottom layer 100 for connecting a rotating shaft, and a sample cell 120, a transition flow channel 130, a detection unit, a balance flow channel 150 and a waste cell 160 connected in sequence.

[0065] In the embodiment, the detection units are 30, each of which comprises a transition pool 141, a first flow channel 142, a first reaction pool 143, a second flow channel 144, a second reaction pool 145 and a third flow channel 146 connected in sequence, the transition flow channel 130 and the balance flow channel 150 are arranged around the rotation center 110, the transition flow channel 130 is closer to the rotation center 110 than the balance flow channel 150, and the detection units are arranged in the region between the transition flow channel 130 and the balance flow channel 150; and a plurality of detection units are uniformly distributed between the transition flow channel 130 and the balance flow channel 150 with the transition flow channel 130 as the inner circular ring and the balance flow channel 150 as the outer circular ring, each detection unit extends generally in the radial direction of the bottom plate layer 100, which is conducive to the sample entering the pre-set flow channel and reaction pool under the action of centrifugal force. The sample pool 120 is arranged between the rotation center 110 and the transition flow channel 130, and the waste liquid pool 160 is located between the transition flow channel 130 and the balance flow channel 150.

[0066] Specifically, the transition pool 141, the first reaction pool 143 and the second reaction pool 145 are arranged away from the rotation center 110 in sequence. The above arrangement is conducive to the sample taking the required sample amount for reaction in the transition pool 141 by using the volume of the transition pool 141 (i.e. the sample amount can be controlled by controlling the volume of the transition pool 141), and then entering the first reaction pool 143 and the second reaction pool 145 in sequence for reaction.

[0067] More specifically, the first flow channel 142 has an S-shaped passage at the end communicating with the transition pool 141, and the third flow channel 146 has a C-shaped passage (also referred to as a U-shaped passage, i.e. the flow channel has a 180° turning). The S-shaped passage is used to block the flow of the sample from the transition pool 141 to the first reaction pool 143, and a large curvature S-shaped arc is adopted to control that the microfluidic chip can only enter the first reaction pool 143 at a specific rotation speed. Similarly, the C-shaped passage is used to increase the difficulty of the sample flowing through the third flow channel 146 into the balance flow channel 150, so that the sample preferentially enters the second reaction pool 145 for reaction, thereby maintaining the detection effect.

[0068] Further, the outlet of the first flow channel 142 and the inlet of the third flow channel 146 are arranged on the near-center side of the first reaction pool 143, and the inlet of the second flow channel 144 is arranged on the far-center side of the first reaction pool 143. The above arrangement is conducive to the sample preferentially entering the second reaction pool 145 through the second inlet under the action of centrifugal force, and then entering the balance flow channel 150 through the third flow channel 146 when the second reaction pool 145 is filled.

[0069] In this embodiment, the balance flow channel 150 is arranged in a wavy circular ring, and the wavy balance flow channel 150 protrudes away from the center of the circle at a position corresponding to the second reaction pool 145, and the detection hole 220 overlaps with the protruding end of the balance flow channel 150 away from the center of the circle. Through the wavy design of the balance flow channel 150, the protruding position away from the center of the circle overlaps with the detection hole 220 in the vertical direction, which can better complete the positioning of each reaction channel. And the third flow channel 146 and the balance flow channel 150 are communicated at the protruding position of the wavy balance flow channel 150 to the center of the circle. The communication is arranged at a different radius from the detection hole 220, which can normally realize the communication function, will not affect the positioning of the detection hole 220, and saves space position, and does not need to expand the radius of the chip for different structures.

[0070] The balance flow channel 150 further comprises a communication flow channel 151 at one end communicated with the waste liquid pool 160, and the other end is communicated with the balance flow channel 150. The radial passage width of the valve hole 152 is greater than the radial passage width of the communication flow channel 151. By changing the radial passage width, the siphon effect that may occur when the liquid in the waste liquid pool 160 is excessive can be blocked, ensuring that the remaining sample waste liquid is not leaked, and effectively avoiding aerosol pollution. Preferably, the radial passage width of the valve hole 152 in this embodiment is 8 times the radial passage width of the communication flow channel 151.

[0071] The first flow channel 142 and the balance flow channel 150 both adopt S-shaped and wavy arcs with large curvature, which can maintain the balance of air pressure in the chip and stably control the flow of liquid, and ensure that each channel will not interfere with each other. When the air pressure is unbalanced and the control is unstable, the liquid in each channel cannot flow uniformly into the respective reaction pool, causing uncontrollable differences in each reaction volume, which will seriously affect the reaction result and cannot guarantee the accuracy and reliability of the result. Therefore, the S-shaped flow channel design of the first flow channel 142 and the wavy design of the balance flow channel 150 in this embodiment have good effects on maintaining the balance of air pressure in the chip and stably controlling the flow of liquid.

[0072] The cover layer 200 is provided with a sample adding hole 210 and a detection hole 220, and the sample adding hole 210 is positioned corresponding to the sample pool 120, and the detection hole 220 is positioned corresponding to the second reaction pool 145. In this embodiment, the detection hole 220 and the second reaction pool 145 are both arranged on the vertical section of the radial extending from the rotation center 110 to the outer periphery.

[0073] And the bottom plate layer 100 is also provided with bottom plate air holes, two in this embodiment, specifically first hole 171 and second hole 172, and the cover plate layer 200 is respectively provided with cover plate air holes corresponding to the bottom plate air holes, specifically third hole 231 and fourth hole 232. The above-mentioned bottom plate air holes and cover plate air holes have the function of maintaining the communication of the chip with the atmosphere and keeping the air pressure balance in the chip.

[0074] In this embodiment, the thickness of the bottom plate layer 100 is 3mm, and the diameter is 120mm, the pore size of the transition flow channel 130 is 1.0mm, the pore size of the first flow channel 142, the second flow channel 144, the third flow channel 146 and the balance flow channel 150 is independently selected from 0.3mm, the containing volume of the transition pool 141 is 20μL, the containing volume of the first reaction pool 143 is 40μL, and the containing volume of the second reaction pool 145 is 20μL. It can be understood that the above-mentioned specifications can be adjusted according to the specific reaction conditions and detection requirements, but according to the above-mentioned size specifications, the advantages of precise control of liquid flow, clear separation between different reaction pools and channels without interference, and reduction of chip size are achieved.

[0075] The first reaction pool 143 and the second reaction pool 145 are respectively pre-embedded with reaction reagents. It can be understood that the above-mentioned reaction reagents are set according to specific reaction requirements, such as the first reaction pool 143 is a new crown nucleic acid RPA amplification reaction, and the new crown RPA amplification reagent is pre-embedded, such as the first reaction pool 143 is a tuberculosis nucleic acid RPA amplification reaction, and the tuberculosis RPA amplification reagent is pre-embedded, etc., such as the second reaction pool 145 is a new crown CRISPR reaction, and the new crown CRISPR detection reagent is pre-embedded, such as the second reaction pool 145 is a tuberculosis CRISPR reaction, and the tuberculosis CRISPR detection reagent is pre-embedded, etc.

[0076] The detection principle of the above-mentioned microfluidic chip is: the sample to be detected is injected into the sample pool 120 through the sample hole 210, the microfluidic chip is controlled to rotate, the sample flows through the transition flow channel 130 into the transition pool 141; the microfluidic chip is controlled to rotate again, the sample enters the first reaction pool 143 through the first flow channel 142 and reacts with the reaction reagent pre-embedded in the first reaction pool 143; the microfluidic chip is controlled to rotate again, the sample enters the second reaction pool 145 through the second flow channel 144 and reacts with the reaction reagent pre-embedded in the second reaction pool 145 to generate a detection signal, which is read by the detection mechanism through the detection hole 220. And in the detection process, the excess waste liquid enters the waste liquid pool 160 through the third flow channel 146 and the balance flow channel 150 under the action of centrifugal force.

[0077] Embodiment 2

[0078] A microfluidic-based isothermal amplification detection device, comprising: a microfluidic chip of embodiment 1, a centrifugal mechanism, a heating mechanism, a detection mechanism and a control mechanism.

[0079] The centrifugal mechanism comprises a driving mechanism for connecting the rotation center and driving the microfluidic chip to rotate, such as a conventional driving motor, e.g. a stepper motor, a servo motor, etc.

[0080] The heating mechanism comprises a heat source mechanism for heating the detection unit, such as a heating wire, a heating film, a heating plate, etc., which can maintain a temperature of about 40 degrees Celsius within 1 hour.

[0081] The detection mechanism comprises an optical detection mechanism for reading the reaction signal of the second reaction pool.

[0082] The control mechanism comprises a control mainboard electrically or signal connected with the centrifugal mechanism, the heating mechanism and the detection mechanism, for controlling the centrifugal mechanism, the heating mechanism and the detection mechanism.

[0083] The above-mentioned isothermal amplification detection device is used for detection, comprising the following steps: injecting the sample to be tested into the sample pool through the sample injection hole, controlling the rotation of the microfluidic chip, and flowing the sample through the transition flow channel into the transition pool; then controlling the rotation of the microfluidic chip, and flowing the sample through the first flow channel into the first reaction pool to fully react with the reaction reagent pre-embedded in the first reaction pool; then controlling the rotation of the microfluidic chip, and flowing the sample through the second flow channel into the second reaction pool to fully react with the reaction reagent pre-embedded in the second reaction pool, to generate a detection signal, which is read by the detection mechanism through the detection hole. Excess waste liquid generated during the detection process flows into the waste liquid pool through the third flow channel and the balance flow channel under the action of centrifugal force.

[0084] Embodiment 3

[0085] Taking sputum liquefaction liquid, alveolar lavage fluid, nasopharyngeal swab samples and other types of samples, using the detection device of embodiment 2, nucleic acid detection of 30 kinds of pathogens is carried out, comprising the following steps:

[0086] The above-mentioned sample is pretreated, 820 microliters of nucleic acid sample is taken through the sample injection hole, and flows into the sample pool, as shown in Figure 3 A, where the dark part is the sample solution. Under the driving of centrifugal force, the sample flows through the transition flow channel into the transition pool. 20 microliters of sample is taken in each transition pool, as shown in Figure 3 B, and then flows into the first reaction pool through the first flow channel with large curvature S-shaped arc control at a suitable rotating speed, as shown in Figure 3As shown in C, the flow rate is controlled by the rotation speed. When the flow rate is too fast, the sample solution cannot stay in the first reaction pool for reaction and will flow into the second reaction pool in advance. When the flow rate is too slow, the sample solution cannot flow into the first reaction pool for reaction. The appropriate rotation speed can be adjusted by observing the liquid flow process.

[0087] The sample is fully mixed with the pre-embedded RPA reagent in the first reaction pool at 40 degrees Celsius, and the reaction lasts for 30 minutes. Subsequently, the control microfluidic chip is rotated to make the sample solution enter the second reaction pool, as shown in Figure 3 D, complete the CRISPR detection reaction with the pre-embedded CRISPR reagent at 40 degrees Celsius, and the reaction emits fluorescence, which can be received, detected and analyzed by the detection mechanism through the detection hole.

[0088] The waste liquid also flows into the waste liquid pool under the action of centrifugal force. Due to the setting of the valve hole, the width of the radial channel is increased, the size of the centrifugal force acting on the liquid at this position is changed, and the siphon effect when the liquid is too much is blocked, which can effectively ensure that the waste liquid pool does not leak and effectively avoid aerosol pollution.

[0089] Example 4

[0090] A microfluidic chip similar to the chip of Example 1, except that the thickness of the bottom layer is 2 mm, the diameter is 80 mm, the pore size of the transition flow channel is 0.8 mm, the pore size of the first flow channel, the second flow channel, the third flow channel and the balance flow channel is 0.1 mm, the containing volume of the transition pool is 10 μL, the containing volume of the first reaction pool is 20 μL, and the containing volume of the second reaction pool is 10 μL.

[0091] The microfluidic chip with the above size rule is tested, which basically meets the experimental requirements, and the sample solution can enter each flow channel and containing pool according to the expected procedure, as shown in Figure 4 .

[0092] Example 5

[0093] A microfluidic chip similar to the chip of Example 1, except that the thickness of the bottom layer is 4 mm, the diameter is 150 mm, the pore size of the transition flow channel is 1.3 mm, the pore size of the first flow channel, the second flow channel, the third flow channel and the balance flow channel is 0.5 mm, the containing volume of the transition pool is 40 μL, the containing volume of the first reaction pool is 50 μL, and the containing volume of the second reaction pool is 30 μL.

[0094] The microfluidic chip with the above size rule is tested, which basically meets the experimental requirements, and the sample solution can enter each flow channel and containing pool according to the expected procedure, as shown in Figure 5 .

[0095] Comparative Example 1

[0096] A microfluidic chip similar to the chip of Example 1, except that the detection hole and the flow channel are on the same side of the chip, and the detection hole and the wave-shaped protrusion of the balance flow channel are interlaced with each other, as shown in Figures 6-7 .

[0097] The positioning detection effect of this microfluidic chip is poor, and the protruding part of the balance flow channel will interfere with the accurate positioning of the detection hole during rotation. In the case of misreading the detection hole, the detection fails, and the result is unusable.

[0098] Comparative Example 2

[0099] A microfluidic chip similar to the chip of Example 1, except that the size of the flow channel is 3 times the size of the flow channel of Example 1.

[0100] The liquid control effect is poor, the first reaction pool and the second reaction pool are filled at the same time, the liquid sample amount of each detection unit is not uniform, which seriously affects the reaction, as shown in Figure 8 .

[0101] Comparative Example 3

[0102] A microfluidic chip similar to the chip of Example 1, according to the following formula for calculating the centrifugal force, the volumes of the transition pool, the first reaction pool and the second reaction pool of each detection unit are all 2 times of the corresponding volumes in Example 1.

[0103]

[0104] θp c = pressure difference; ρ = density; ω = rotational speed; r = distance from the center of the circle.

[0105] The results are shown in Figure 9 , the change in the volume of the transition pool makes it difficult to accurately control the flow of liquid when the size of the flow channel is constant, resulting in different and uneven liquid amounts in different reaction pools. Moreover, the liquid amount of the transition pool is too large, being twice the standard reaction volume, but the amount of pre-embedded reagent remains unchanged, and the concentration is diluted. Under the condition that the performance of the detection mechanism remains unchanged, the final detection fluorescence value is low, which affects the test analysis results.

[0106] The technical features of the above-described embodiments can be combined in any way. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described, but as long as the combinations of the technical features do not contradict, they should be considered as within the scope of the present disclosure.

[0107] The above embodiments only express several implementation manners of the present application, and the description is more specific and detailed, but it should not be understood as a limitation on the scope of the patent. It should be noted that for ordinary skilled persons in the art, without departing from the concept of the present application, several modifications and improvements can be made, which are within the scope of protection of the present application. Therefore, the protection scope of the patent of the present application should be subject to the appended claims.

Claims

1. A microfluidic chip, characterized by, The microfluidic chip comprises a base plate layer and a cover plate layer in sealing cooperation with the base plate layer, the base plate layer is provided with a rotation center for connecting a rotating shaft, and a sample pool, a transition flow channel, a detection unit, a balance flow channel and a waste liquid pool are sequentially communicated, the detection unit comprises a transition pool, a first flow channel, a first reaction pool, a second flow channel, a second reaction pool and a third flow channel which are sequentially communicated, the first flow channel has an S-shaped passage, the transition flow channel and the balance flow channel are both arranged around the rotation center, the transition flow channel is closer to the rotation center than the balance flow channel, the detection unit is arranged in a region between the transition flow channel and the balance flow channel, the transition pool, the first reaction pool and the second reaction pool are arranged in an order of sequentially moving away from the rotation center, the first flow channel outlet and the third flow channel inlet are both arranged on a near-circular center side of the first reaction pool, and the second flow channel inlet is arranged on a far-circular center side of the first reaction pool. The cover plate layer is provided with a sample adding hole and a detection hole, the sample adding hole is located in correspondence with the sample pool, and the detection hole is located in correspondence with the second reaction pool. The detection unit is a plurality of units which are uniformly distributed along the transition flow channel, each detection unit is communicated with the transition flow channel through the transition pool and communicated with the balance flow channel through the third flow channel. The detection hole and the second reaction pool are both arranged on a vertical section of a radial line extending from the rotation center to an outer periphery. The balance flow channel is arranged in a wave-shaped circular ring, and the wave-shaped balance flow channel protrudes in a direction away from the circular center at a position corresponding to the second reaction pool, and the detection hole overlaps with a protruding end of the balance flow channel in the direction away from the circular center. The third flow channel is communicated with the balance flow channel at a position where the wave-shaped balance flow channel protrudes in the direction of the circular center. The balance flow channel further comprises a communication flow channel, one end of the communication flow channel is communicated with the waste liquid pool through a valve hole, and the other end is communicated with the balance flow channel, and a radial passage width of the valve hole is greater than a radial passage width of the communication flow channel.

2. The microfluidic chip of claim 1, wherein, The third flow channel has a C-shaped passage.

3. The microfluidic chip of claim 1, wherein, The sample pool is arranged between the rotation center and the transition flow channel, and the waste liquid pool is arranged between the transition flow channel and the balance flow channel.

4. The microfluidic chip of claim 1, wherein, The base plate layer is further provided with a base plate air hole, and the cover plate layer is provided with a cover plate air hole corresponding to the base plate air hole.

5. The microfluidic chip of claim 1, wherein, The base plate layer has a thickness of 2-4 mm and a diameter of 80-150 mm, the transition flow channel has a pore size of 0.8-1.3 mm, the first flow channel, the second flow channel, the third flow channel and the balance flow channel have pore sizes independently selected from 0.1-0.5 mm, the transition pool has a containing volume of 10-40 μL, the first reaction pool has a containing volume of 20-50 μL, and the second reaction pool has a containing volume of 10-30 μL.

6. The microfluidic chip of claim 1, wherein, Reaction reagents are respectively pre-embedded in the first reaction pool and the second reaction pool.

7. The microfluidic chip of claim 1, wherein, The microfluidic chip of claim 1 is included.

8. A microfluidic-based isothermal amplification detection device, characterized by, Further comprising:

9. The microfluidic-based isothermal amplification detection device according to claim 8, characterized in that, A centrifugal mechanism comprising a driving mechanism for connecting the rotation center and driving the microfluidic chip to rotate; A heating mechanism comprising a heat source mechanism for heating the detection unit. ​ a detection mechanism, comprising a detection mechanism for reading the reaction signal of the first reaction pool and / or the second reaction pool; and a control mechanism, comprising a control mainboard, which is electrically connected or signal connected with the centrifugal mechanism, the heating mechanism and the detection mechanism, for controlling the centrifugal mechanism, the heating mechanism and the detection mechanism.

10. A microfluidic-based detection method, characterized in that, The microfluidic chip of any one of claims 1-7, comprising the following steps: injecting the sample to be tested into the sample pool through the sample injection hole, controlling the rotation of the microfluidic chip, and making the sample flow through the transition flow channel into the transition pool; then controlling the rotation of the microfluidic chip, and making the sample flow through the first flow channel into the first reaction pool to fully react with the reaction reagent pre-embedded in the first reaction pool; then controlling the rotation of the microfluidic chip, and making the sample flow through the second flow channel into the second reaction pool to fully react with the reaction reagent pre-embedded in the second reaction pool, to generate a detection signal, which is read by the detection mechanism through the detection hole.

11. The microfluidic-based isothermal amplification detection method according to claim 10, wherein, The excess waste liquid in the first reaction pool enters the waste liquid pool through the third flow channel and the balance flow channel in turn under the action of the centrifugal force.

Images