Centrifugal micro-fluidic chip for nucleic acid detection

Abstract

The utility model provides a centrifugal micro-fluidic chip for nucleic acid detection, which comprises a detection unit provided with a quantitative cavity, an elution cavity and a detection area, the elution cavity is used for eluting an object to be detected from the solid-phase carrier; the quantifying cavity is used for temporarily storing added liquid and quantifying the added liquid, and the liquid comprises eluent; the quantitative cavity and the elution cavity are connected through a fluid channel; the detection area is used for detecting the content of an object to be detected, and the detection area is connected with the fluid channel through the capillary channel. The chip is simple in structure, low in cost, convenient to assemble and easy to layer and sterilize, can simultaneously realize high-throughput detection of more samples and more detection items, and improves the detection efficiency.

Description

[0001] This application claims the priority of the Chinese prior application, application number: 2024107898334, filing date June 19, 2024; all its contents are regarded as part of the present utility model. Technical Field

[0002] The utility model relates to the field of microfluidic control, and in particular to a centrifugal microfluidic chip for nucleic acid detection. Background Art

[0003] Microfluidic chips are also often called "Lab-on-chip". They can manipulate fluids in micron-scale space and only require trace amounts of samples to complete biological, chemical and other experiments. They have the advantages of integration, high throughput and high efficiency, and are therefore widely used in scientific fields such as environment, chemistry and medicine.

[0004] With the rapid development of technologies such as genetic diagnosis and personalized medicine, the importance of nucleic acid detection technology with high specificity and accuracy is increasing. Existing microfluidic chips have integrated multiple steps such as sample processing, nucleic acid amplification, and nucleic acid detection on a small chip, quickly completing the "sample in-result out" detection process. Since microfluidic chips are free from dependence on precision pipetting devices and professionals, they have great potential in the field of nucleic acid sample detection.

[0005] Centrifugal microfluidic chips drive the flow of liquid by rotating the centrifugal microfluidic chip, which can use centrifugal force to manipulate liquid on a submillimeter scale. Compared with traditional microfluidic chips, the entire device is more compact and simple to handle, so it is gradually being used in the field of nucleic acid sample detection. In the centrifugal microfluidic chip, the microchannels are distributed along the radial direction of the disc. When the chip rotates, the liquid to be tested moves along multiple microchannels to each reaction pool under the action of centrifugal force, realizing the distribution and addition of the liquid to be tested, making the nucleic acid detection process more efficient.

[0006] Nucleic acid testing requires a pretreatment process of lysis and purification, and the gentle and efficient magnetic bead extraction method has attracted attention. The magnetic bead method usually includes four steps: lysis, binding, washing, and elution. The core of the magnetic bead is made of Fe3O4, which has superparamagnetism. The outer layer is coated with silica and has good stability. Its surface is modified with special groups. Under specific ionic conditions, it can specifically adsorb with the nucleic acid molecules released from the sample through electrostatic effects, hydrogen bonds, etc. to form a nucleic acid-magnetic bead complex, while impurities such as proteins remain in the sample liquid, which can achieve rapid extraction and separation of nucleic acids. However, the production process of magnetic beads is complicated and requires professional equipment personnel and technicians. Its high price has greatly increased the cost of detection. The relatively low economic benefits limit the application of magnetic beads in large-scale liquid samples, especially in remote areas. Therefore, it is necessary to find a more economical method.

[0007] The centrifugal microfluidic chips currently on the market usually lyse and purify samples outside the chip before adding them to the microfluidic chip for nucleic acid detection. Nucleic acid extraction is rarely integrated into a single chip because this will make the chip structure complex, increase control difficulty, and increase costs.

[0008] CN116144458A provides a centrifugal microfluidic chip for nucleic acid extraction and detection by magnetic beads, but in the process of nucleic acid elution, it is necessary to set up an additional magnet to adsorb the magnetic beads, which makes the use of the centrifugal microfluidic chip more cumbersome, highly dependent on equipment, and also increases the cost. CN117551540A provides a centrifugal microfluidic chip for nucleic acid extraction and detection by magnetic beads without magnets, but its structure is very complicated, and it is necessary to specially set up structures such as magnetic bead temporary storage chamber, flow channel stop valve, hot melt valve, etc., and one chip can only be used for the detection of one sample. In addition, the above two centrifugal microfluidic chips can only be used for nucleic acid extraction by magnetic beads, and are not suitable for other solid phase carriers to extract, elute and detect nucleic acids.

[0009] In response to these technical problems, there is an urgent need to find a centrifugal microfluidic chip that can quickly and conveniently complete the elution and detection of nucleic acids. It is not only suitable for magnetic beads, but also for other lower-cost solid phase carriers, and can significantly improve the efficiency of nucleic acid detection. Utility Model Content

[0010] In order to solve the above problems, the utility model provides a centrifugal microfluidic chip for nucleic acid detection. For the solid phase carrier used for adsorbing nucleic acid, a centrifugal microfluidic chip that can complete the whole process of nucleic acid elution, amplification and detection is designed, including a quantitative chamber, an elution chamber and a detection area; the elution chamber is used to elute the nucleic acid to be tested from the biochar, and the quantitative chamber is used to temporarily store the added eluent, and quantify the added eluent to accurately complete the elution process; the quantitative chamber and the elution chamber are connected through a fluid channel. By improving the structure of the quantitative chamber, the elution chamber and the fluid channel, and adding steps in the fluid channel, the sample to be tested can smoothly enter the detection area through the capillary under the action of surface tension, and can prevent the biochar from clogging the capillary, thereby ensuring the accuracy of the test results, and can also simultaneously realize high-throughput detection of more samples and more test items, thereby improving the detection efficiency. The chip has a simple structure, effectively reduces the difficulty of preparation and processing, and has a lower production cost.

[0011] On the one hand, the utility model provides a centrifugal microfluidic chip, which includes a detection unit, and the detection unit is provided with a quantitative chamber, an elution chamber and a detection area; the elution chamber is used to elute the analyte from the solid phase carrier; the quantitative chamber is used to temporarily store the added liquid and quantify the added liquid, and the liquid includes an eluent; the quantitative chamber and the elution chamber are connected by a fluid channel; the detection area is used to detect the content of the analyte, and the detection area is connected to the fluid channel through a capillary channel.

[0012] The centrifugal microfluidic chip provided by the utility model is provided with a plurality of detection units, each of which is provided with a quantitative cavity, an elution cavity and a detection area, and can be applicable to the simultaneous detection of a plurality of different target analytes, such as the detection of a plurality of different nucleic acid targets, and is also applicable to the detection of nucleic acid targets eluted from a plurality of different solid phase carriers.

[0013] During use, the centrifugal microfluidic chip (hereinafter referred to as the chip) needs to be kept horizontal with the sample port facing upward. In some methods, the chip can be inserted into a matching detection device, so that the chip is kept horizontally with the sample port facing upward, and the center of the chip is buckled and fixed to the rotating shaft of the detection device for centrifugation, so that the rotating shaft can drive the chip to rotate centrifugally; in some methods, the detection device can provide a heat source for heating the detection area of ​​the chip to help it complete the nucleic acid amplification reaction; in some methods, the detection device can also detect the fluorescent substance produced after the nucleic acid amplification reaction in the chip and read the detection results.

[0014] The chip provided by the utility model is provided with a quantitative cavity and an elution cavity respectively. In the initial stage of sequentially adding a sample containing a solid phase carrier and an eluent, the sample containing a solid phase carrier and the eluent are first preliminarily separated and quantified. Then, as the centrifugation continues, the elution process begins, and the eluent in the quantitative cavity continuously enters the elution cavity to collide and mix with the solid phase carrier. Under the action of centrifugal force, the solid phase carrier will diverge outward as much as possible and get close to the inner wall of the elution cavity farthest from the center of the circle, and the remaining liquid is continuously mixed in the elution cavity or squeezed out of the elution cavity. It can be seen that the setting of the quantitative cavity can not only play a quantitative role, but also make the mixed elution more sufficient, improve the elution efficiency, and thus improve the detection sensitivity.

[0015] The quantitative chamber and the elution chamber are connected via a fluid channel, which refers to a channel connecting the outlet of the quantitative chamber and the inlet of the elution chamber.

[0016] Furthermore, the solid phase carrier includes one or more of biochar, magnetic beads, gel, resin, and cellulose; and the analyte is nucleic acid.

[0017] In some embodiments, the solid support is biochar.

[0018] Biochar has the characteristics of low cost and easy availability of raw materials. When biochar is used as a solid phase carrier for nucleic acid detection, the cost can be significantly reduced. In addition, the density of biochar is smaller than that of magnetic beads, it is looser, non-magnetic, and cannot be separated by a magnetic field, making it more difficult to separate biochar from the sample to be tested after elution than magnetic beads. Therefore, it is necessary to design the structure of various chambers and flow channels in the chip to help the nucleic acid samples on the biochar to be eluted smoothly, and the eluted sample to be tested can smoothly enter the detection area for detection. The biochar is gathered in the area close to the external source of the chip due to the centrifugal effect, and is blocked in the elution chamber by the steps to ensure that it will not block or affect the detection of the sample to be tested.

[0019] Furthermore, the biochar is prepared using rice straw and corn cobs as carbon sources.

[0020] Studies have shown that biochar prepared using rice straw and corn cobs as carbon sources has better nucleic acid adsorption efficiency.

[0021] Furthermore, it also includes a rotation center, which is used to connect with a centrifugal driving device to drive the centrifugal microfluidic chip to centrifuge; the number of the detection units is one or more, which are arranged around the rotation center.

[0022] In some embodiments, the number of the detection units is four, which are evenly arranged around the rotation center, and each detection unit can be used to detect one sample, so that the detection throughput of the chip is higher.

[0023] Furthermore, the capillary channel includes a first section and a second section; the first section is a channel that meanders from the liquid outlet of the fluid channel to the rotation center, and the second section is a channel that meanders from the end of the first section to the detection area.

[0024] When the chip is centrifuged, the flow direction of the fluid sample under the action of centrifugation is emitted from the center of the circle to the outside. The setting of the first section in the capillary channel is such that if the sample wants to enter the first section, its flow direction is opposite to the centrifugal direction. Therefore, during the centrifugation process, the sample cannot enter the capillary channel. The setting of the first section of the capillary channel is equivalent to adding a threshold for the sample to enter the capillary channel, and strengthens the protection to prevent the entry of biochar. First, it ensures that the sample will not enter the capillary channel during the centrifugal mixing and elution process, and the biochar will not be able to block the capillary channel; secondly, when the centrifugation is stopped, the supernatant liquid sample can penetrate into the capillary channel under the action of surface tension, and the biochar is thrown to the bottom of the elution chamber by the centrifugal force, so it cannot contact or block the capillary channel.

[0025] Furthermore, a barrier block is provided below the fluid channel so that the quantitative cavity or the elution cavity is at least partially blocked; a step is also provided on the fluid channel, and the step is provided on one side of the fluid channel close to the liquid outlet leading to the capillary channel.

[0026] The fluid channel connects the quantitative chamber and the elution chamber, but due to the setting of the barrier block, liquid cannot flow below the fluid channel, which is equivalent to only the upper chamber of the quantitative chamber and the elution chamber being fluidically connected, and the lower part is blocked. Such a setting also helps to prevent biochar from clogging the capillary channel, because the biochar will basically sink to the bottom of the elution chamber under the action of centrifugal force.

[0027] The step is located above the barrier block and close to one side of the liquid outlet leading to the capillary channel, so that the liquid surface near the liquid outlet forms a U-shaped liquid shape similar to the residual liquid surface, thereby raising the liquid surface here and helping the sample to penetrate into the capillary channel under the action of surface tension.

[0028] In some embodiments, the width of the fluid channel is 1.0-1.25 mm, and the height of the quantitative cavity and the elution cavity is 4.5-5.5 mm, which can be adjusted according to the required sample volume. The height of the fluid channel is reduced to 1.0-1.5 mm because it is blocked by the blocking block at the bottom.

[0029] In some embodiments, the step has a height of 0.5-0.7 mm and a width of 0.4-0.6 mm.

[0030] Furthermore, the quantitative cavity is provided with a first inlet and a first outlet, and the size of the quantitative cavity gradually decreases near the first outlet.

[0031] In some embodiments, the quantitative cavity is an inverted pentagonal chamber, which is similar to a funnel shape as a whole.

[0032] The shape design of the quantitative cavity helps the sample containing biochar to quickly gather and enter the elution cavity under the action of centrifugal force after entering, and helps the sample containing biochar to remain stably in the elution cavity after entering the elution cavity and is not easy to flow back. It can be seen that the structural design of the quantitative cavity can help prevent the backflow of biochar.

[0033] The quantitative chamber is used to quantitatively store the added liquid, and the volume of the quantitative chamber can be adjusted according to the specific purpose and sample amount.

[0034] In some embodiments, the quantitative cavity has a width of 6.25±0.5 mm, a length of 4.8±0.5 mm, and a volume of 200±30 μL.

[0035] Furthermore, an exhaust channel and a waste liquid discharge channel are also provided on the quantitative chamber; the exhaust channel extends from the quantitative chamber toward the rotation center, and the exhaust port of the exhaust channel is connected to the outside world, which is used to control the flow of gas in the quantitative chamber and / or the elution chamber; the waste liquid discharge channel extends from the quantitative chamber toward the direction away from the rotation center and is connected to the waste liquid pool.

[0036] When the liquid enters the chip from the sample addition area and enters the corresponding chamber under the action of centrifugal force, the gas in the chamber needs to be discharged from the exhaust channel. The quantitative chamber and the elution chamber share an exhaust channel to make the preparation process easier. The direction of the exhaust channel is opposite to the centrifugal direction. Therefore, when the liquid enters the quantitative chamber or the elution chamber under the action of centrifugal force, the liquid will not be discharged from the exhaust channel. Only when the elution chamber and the quantitative chamber are full, the liquid will enter the exhaust channel. The channel coming out of the quantitative chamber is provided with branches. The first branch is opposite to the centrifugal direction and leads to the exhaust port, which is the exhaust channel. The second branch is the same as the centrifugal direction and leads to the waste liquid pool, which is the waste liquid discharge channel. Under the action of centrifugation, the liquid will definitely choose the waste liquid discharge channel in the same direction as the centrifugal direction to enter the waste liquid pool. Therefore, when you see liquid starting to enter the waste liquid pool, it means that the quantitative chamber has been filled. It can be seen that the waste liquid pool also has the effect of helping to quantify the liquid in the quantitative chamber.

[0037] The exhaust port is a circular through structure with a diameter of 2.5 mm, which is used to exhaust the gas in the chip, balance the internal and external air pressure, and prevent bubbles from clogging the microchannel and affecting the flow of liquid.

[0038] In some embodiments, the surface of the exhaust port is pre-covered with a transparent film to seal it. When exhaust is required during use, the transparent film can be punctured for exhaust; if sealing is required later, the transparent film can be added for sealing.

[0039] Furthermore, the elution chamber is provided with a second inlet, and the size of the elution chamber gradually decreases near the second inlet.

[0040] In some embodiments, the elution chamber is a pentagonal chamber with a narrow entrance, which can help prevent the biochar from flowing out of the elution chamber when the sample enters the elution chamber under centrifugal action. The elution chamber is used to elute the nucleic acid to be tested from the biochar and store the eluted liquid. The size and volume of the elution chamber can be set according to the specific needs of the product and can be fine-tuned within an appropriate range.

[0041] In some embodiments, the elution cavity has a width of 5.8±0.5 mm, a length of 4.8±0.5 mm, and a volume of 230±30 μL.

[0042] Furthermore, the detection area includes a detection channel and a detection cavity connected to the detection channel; the detection channel is connected to the capillary channel.

[0043] Furthermore, a second exhaust channel is provided on the detection channel, and the second exhaust channel extends from the detection channel toward the rotation center, and the exhaust port of the second exhaust channel is connected to the outside world for controlling the flow of gas in the detection area; the number of the detection chambers is one or more.

[0044] When there are multiple detection chambers, multiple target nucleic acids can be detected simultaneously. In some embodiments, the number of detection chambers is seven.

[0045] In some embodiments, the detection channel is an arc-shaped channel arranged along a circumference, and the detection cavity is a circular room with a diameter of 1.5-2.5 mm, which matches the spot size of the fluorescence detection module in the detection device.

[0046] In some embodiments, the fluorescence detection process of the chip is performed by slowly rotating the chip during a slow centrifugation process, so that each detection cavity is aligned with the fluorescence detection channel in turn to complete the fluorescence detection.

[0047] In some methods, a line is established from the center of rotation to the center of the detection cavity, and the angle between the lines connecting two adjacent detection cavities is 7 degrees, which is an integer multiple of the single-grid rotation degree of the stepper motor in the centrifugal device (0.7 degrees), and at this angle, fluorescence interference from adjacent detection cavities can be avoided, thereby facilitating the calculation of the position of each detection cavity and recording the reading results, which is beneficial to improving the positioning accuracy during centrifugation and detection.

[0048] In some embodiments, nucleic acid amplification reagents and / or nucleic acid detection reagents need to be pre-installed in the detection chamber.

[0049] In some embodiments, the nucleic acid amplification reagent and / or nucleic acid detection reagent is a solid dry reagent.

[0050] In some embodiments, the amplification reaction includes but is not limited to EPA, RPA, LAMP, NEAR, NASBA, HAD, TMA, SAT, RCR, etc., and the solid dry reagent includes but is not limited to spherical, powdered, flaky or block preparations in the form of freeze-dried, dried, air-dried, etc.

[0051] In some embodiments, the nucleic acid detection includes but is not limited to a variety of detection systems established by ERA, RPA, LAMP, NEAR, NASBA, HAD, TMA, SAT, PCR, hybridization probe technology, CRISPR, Ago, RNase H, etc., and the detection reagent is a solid detection reagent, including but not limited to spherical, powdered, flaky or block preparations in the form of freeze-dried, dried, air-dried, etc.

[0052] Furthermore, it also includes a sample adding area, which is connected to the quantitative cavity through a sample adding channel; the sample adding area, the quantitative cavity and the elution cavity are arranged in sequence along the rotating centrifugal direction of the centrifugal microfluidic chip.

[0053] In some embodiments, the sample loading area is an elliptical chamber with a major axis of 12±0.5mmmm and a minor axis of 7.5±0.5mmmm. There is a sample loading hole with a diameter of 2mm on the top for adding sample liquid and eluent, and an air hole with a diameter of 2mm for exhausting the air in the chamber when adding liquid to balance the air pressure. Compared with the circular structure, the elliptical structure can utilize the area of ​​the chip close to the center of the circle as much as possible, thereby maximizing the sample loading volume under the same disk diameter, and effectively improving the space utilization of the chip.

[0054] In some embodiments, the surface of the sample loading area of ​​the chip is covered with a transparent film for sealing. When loading the sample, the transparent film is pierced by a syringe to load the sample, and after the sample fills the main channel, the surface of the sample loading hole can be covered with a transparent film for sealing.

[0055] Furthermore, the rotation center is a fixing hole, and the fixing hole enables the centrifugal microfluidic chip to be clamped on the rotating shaft of the centrifugal driving device, thereby performing centrifugal rotation.

[0056] In some embodiments, the fixing hole is used for good fit between the chip and the driving member of the centrifugal driving device. The fixing hole is approximately elliptical and can be combined with the driving member of the centrifugal driving device to fix the chip and provide centrifugal power.

[0057] Furthermore, it comprises a plastic upper layer, a silicone inner membrane and a plastic lower layer, and the detection unit is located in the plastic upper layer.

[0058] The chip provided by the utility model is a sandwich structure, including a plastic upper layer, a silicone inner membrane and a plastic lower layer. The sandwich structure is a commonly used processing technology for microfluidic chips, which consists of three layers. The middle layer is usually soft silicone with microchannels, while the top and bottom layers are usually made of hard materials such as plastic, glass, polymer materials, etc. This structure can reduce the cost of consumables and optimize the process flow, achieving a dual improvement in cost-effectiveness and technical performance.

[0059] The plastic upper layer is a polydimethylsiloxane (PDMS) disk processed by injection molding, with a thickness of 1.8-2mm and a diameter of 75mm, and a convex microfluidic structure downward, wherein the width of the capillary channel is 0.3mm, and the width of the remaining microfluidic channels is 0.8-1mm, and the two outer sides of the channel have a 5-10° slope and a support structure with a width of 0.3-0.5mm, which is used to improve the pressure-bearing performance of the microfluidic channel and is conducive to the demoulding of the injection molding. The plastic upper layer includes a fixing hole, a vent hole, a sample loading chamber, a quantitative chamber, an elution chamber, a sample loading channel, a capillary, and a detection chamber.

[0060] The silicone inner membrane is a round silicone diaphragm with a thickness of 0.1mm. It is a soft silicone material that can fill tiny irregular surfaces under pressure, provide better elasticity and sealing, and reduce the leakage rate of liquids. Under normal use and storage conditions, the silicone inner membrane has a very long service life, good aging resistance, heat resistance and chemical resistance, and can be used in a variety of usage scenarios.

[0061] The plastic lower layer is a plastic disc with a fixed hole, with a thickness of 0.1-0.2mm. The injection molding process uses a mixed material (10:10:1) of polymethyl methacrylate (PMMA), polycarbonate (PC), and dioctyl sebacate (DOS) as the base. The processed plastic disc still has good rigidity in an extremely thin condition, and can support and protect the microchannel structure of the plastic upper layer, while preventing the soft silicone inner membrane from being scratched and damaged. The disc has strong drop resistance and light transmittance, and can maintain excellent light transmittance even under accelerated aging conditions, without affecting the subsequent fluorescence detection in the reaction chamber.

[0062] In some methods, the microfluidic channel of the upper plastic layer is a hexahedron, the upper plastic layer provides five faces, and the silicone and the lower plastic layer provide the bottom surface to complete the sealing. It can be seen that the microfluidic channel of the upper plastic layer is open and needs to be sealed from below. The existing methods are to directly assemble the lower plastic layer and the upper plastic layer, which is equivalent to directly gluing or bonding the hard-hard structure. The operation is relatively complicated, and it is also easy for glue to enter the microfluidic channel and affect sterilization. Moreover, because these traditional assemblies are performed after sterilization, it is easy to be contaminated again. After installation, the gas or liquid used for sterilization in the flow channel is either difficult to enter or difficult to come out. In order to achieve rapid assembly, the utility model creatively adopts a hard-soft-hard structure to seal directly through clamping, which has a better sealing effect and will not be contaminated after sterilization.

[0063] Furthermore, it also includes a first buckle, a second buckle, a third buckle and a fourth buckle; the first buckle and the second buckle are assembled into one, and are used to fix and seal the outer periphery of the centrifugal microfluidic chip; the third buckle and the fourth buckle are assembled into one, and are used to fix and seal the inner periphery of the centrifugal microfluidic chip, and the inner periphery is the arc-shaped hole where the rotation center of the centrifugal microfluidic chip is located.

[0064] The first buckle, the second buckle, the third buckle and the fourth buckle are two groups of semicircular acrylic buckles of different sizes. The side is designed with a groove structure with a height of 0.8mm. The total thickness of the chip is 1mm. Acrylic has good toughness and elasticity and is not easy to break or crack. The height difference of 0.2mm is used to make the buckle elastically extrude the three-layer structure of plastic-silicone-plastic to achieve the sealing of the chip.

[0065] The first clip is an acrylic semicircular arc with a groove on the side, the arc width is 2mm, it can fit with the outside of the chip, and the upper and lower ends have female grooves for assembly with the second clip.

[0066] The second buckle is an acrylic semicircular arc with a groove on the side, the size is the same as the first buckle, and the upper and lower ends are provided with male grooves for assembly with the first buckle.

[0067] The third buckle is an acrylic semicircular arc with a groove on the side, the arc width is 2mm, and can fit into the fixing hole of the chip. The upper and lower ends are provided with female grooves for assembly with the fourth buckle.

[0068] The fourth buckle is an acrylic semicircular arc with a groove on the side, the size is consistent with the third buckle, and the upper and lower ends are provided with male grooves for assembly with the third buckle.

[0069] Common assembly methods for microfluidic chips include bonding and gluing. The bonding method has disadvantages such as low alignment accuracy, high scrap rate, and high cost. It also needs to be left to stand for a long time after preparation, otherwise it will affect the strength of the bond. At the same time, bonding technology requires suitable materials, which greatly limits the flexibility of the actual application of the chip. The gluing assembly method has the problem of glue clogging the microchannel and reducing the light transmittance of the chip. At the same time, it is also necessary to consider the biocompatibility of the glue with the sample to be tested.

[0070] This patent adopts a quick assembly buckle method for assembly, which is simple in process, easy to assemble, low in cost, and can significantly improve production efficiency. External assembly can completely avoid contact with samples and biological reagents, eliminating the adverse effects of potential biochemical reactions. At the same time, installing the buckle does not change the shape and transparency of the chip, and no additional polishing is required. It can be widely used in microfluidic chips of various detection principles.

[0071] On the other hand, the utility model provides a method for preparing a micro flow channel on a plastic, the method comprising the following steps:

[0072] (1) Cover the silicone mold with micro-channels on the plastic;

[0073] (2) Injecting a corrosive liquid into the mold, wherein the corrosive liquid comprises dimethylformamide, acetone and tetrahydrofuran.

[0074] There are certain technical difficulties in preparing tiny microfluidic structures on materials such as plastic and glass. Traditional methods can only prepare microfluidic structures on silicone materials through micromachining techniques such as photolithography and etching, and then bond or glue them to plastic, glass, etc. through oxygen plasma bonding. The SU-8 used in photolithography is sensitive to thermal expansion and difficult to separate from the substrate material, so it needs to be processed using an anti-sticking layer process. At the same time, the preparation of SU-8 glue molds involves multiple precise steps, which must be accurately executed in strict accordance with the process. The processing requirements are high, the environmental conditions are many, and the processing techniques are complex. The whole process takes a long time. Moreover, the SU-8 mold will continue to fall off during the preparation of silicone, and its service life is limited. The assembly method of oxygen plasma bonding has high requirements for processing accuracy, and it is necessary to consider the impact of the process on the biochemical reactions in the flow channel, etc. Its reliability and stability need to be improved.

[0075] The utility model can smoothly prepare micro flow channels on plastics by a method of dripping corrosive liquid into a mold. The method has low requirements on processing accuracy and can complete flow channel preparation in a very short time.

[0076] In some embodiments, the etching solution comprises: dimethylformamide (DMF): acetone: tetrahydrofuran = 5:4:1

[0077] In another aspect, the utility model provides a method for preparing a capillary channel on a centrifugal microfluidic chip, the method comprising the following steps:

[0078] (1) Covering a silicone mold with a capillary channel on the plastic upper layer of a centrifugal microfluidic chip so that the capillary channel on the mold is positioned in the area connecting the fluid channel and the detection channel;

[0079] (2) injecting a corrosive liquid into the mold, wherein the corrosive liquid comprises dimethylformamide, acetone and tetrahydrofuran;

[0080] (3) Remove the silicone mold and form a capillary channel on the upper layer of the plastic.

[0081] The production of sandwich-structured microfluidic chips has certain technical requirements. The utility model adopts the above-mentioned method of preparing microchannels on plastic to prepare capillary channels on the upper layer of plastic. The specific method is: cover the silicone mold with capillaries on the unprocessed area of ​​the upper layer of plastic, and position the capillaries on the mold to connect the sample addition channel and the detection area. After injecting 12μL of mixed corrosive liquid into the mold for 15s, use a needle to absorb and recover the corrosive liquid. Remove the silicone mold and form a capillary with a width of 0.3mm and a depth of 0.5mm on the plastic disc.

[0082] The silicone mold is a capillary silicone mold with a width of 0.3 mm made using photolithography technology. The advantage of this processing method is that it can avoid using photolithography and other methods to obtain capillaries with a size less than 1 mm, can control the processing cost and ensure the repeatability of the processing effect.

[0083] In some embodiments, the etching solution comprises dimethylformamide (DMF), acetone, and tetrahydrofuran (5:4:1).

[0084] In another aspect, the utility model provides a method for nucleic acid detection, wherein the method uses the centrifugal microfluidic chip as described above for detection, and the detection method comprises the following steps:

[0085] (1) Clamp the fixed hole of the centrifugal microfluidic chip onto the rotating axis of the detection device;

[0086] (2) adding the sample to be tested containing the solid phase carrier from the sample loading area to the centrifugal microfluidic chip, and centrifuging to allow the sample to be tested containing the solid phase carrier to enter the elution chamber of the chip;

[0087] (3) adding the eluent from the sample loading area to the centrifugal microfluidic chip, centrifuging, and stopping the centrifugation when liquid appears in the waste liquid pool of the centrifugal microfluidic chip, indicating that the eluent has entered the quantitative chamber;

[0088] (4) Centrifuging again, the eluent enters the elution chamber and mixes with the sample to be tested containing the solid phase carrier, completing the elution of the nucleic acid;

[0089] (5) stopping centrifugation, and the liquid containing nucleic acid enters the detection chamber through the capillary tube for nucleic acid amplification;

[0090] (6) Read the test results.

[0091] In some embodiments, in step (5), the nucleic acid amplification is isothermal amplification, and a heating device is required to provide the reaction chamber with a suitable temperature required for isothermal amplification (eg, 30-65° C.).

[0092] Furthermore, the eluent includes Tris-HCl buffer and PBS.

[0093] In some embodiments, the elution solution is 200 μL 10 mM Tris-HCl buffer (pH 7.5) + 30 μL 0.02 M PBS

[0094] The utility model selects the most suitable eluent formula, which can improve the elution effect of nucleic acids in biochar. At the same time, since the eluent is a low-concentration aqueous solution, it can break the chemical balance in the elution chamber after adding it to the chip. Because the first sample is added with a sample containing a solid phase carrier, and the second sample is added with an aqueous eluent, the concentration is changed, making it easier to elute the sample.

[0095] The chip provided by the utility model is based on automatic elution technology, which integrates the extraction of solid phase carriers (such as biochar) into a single chip, eliminating the need for redundant liquid transfer steps, reducing the risk of aerosol contamination and human error, and building a fast and convenient highly integrated detection platform. At the same time, the chip is equipped with four independent areas, which can detect 4*7 target nucleic acid molecules at the same time, which can reduce the use of samples and reagents and save detection costs.

[0096] The centrifugal microfluidic chip provided by the utility model has the following beneficial effects:

[0097] 1. It can complete the whole process of eluting nucleic acid from solid phase carrier and amplifying and detecting it, and is suitable for a variety of different solid phase carriers, such as biochar, nucleic acid, etc.

[0098] 2. By setting up the quantitative chamber and the elution chamber separately, it can not only play a quantitative role, but also make the mixed elution more complete, improve the elution efficiency, and thus improve the detection sensitivity.

[0099] 3. By designing various chamber shapes and structures in the chip, solid phase carriers such as biochar can be eluted smoothly, and the eluted samples can smoothly enter the detection area for detection, while the biochar is blocked in the elution chamber and will not block or affect the detection of the samples.

[0100] 4. By improving the structure of the fluid channel in the chip, a barrier block is set up to effectively block solid phase carriers such as biochar to avoid clogging the capillary channel. The step above the barrier block makes the liquid surface near the liquid outlet form a U-shaped liquid shape similar to the residual liquid surface, thereby raising the liquid level here and helping the sample to penetrate into the capillary channel under the action of surface tension.

[0101] 5. Combine the corrosive liquid and mask to achieve 0.3mm level channel etching on the plastic disc, effectively reducing the processing cost and difficulty, and can complete the flow channel preparation in a very short time.

[0102] 6. The chip can be quickly assembled through the buckle, which has a simple process, convenient assembly, low cost, and significantly improves production efficiency. It completely avoids contact with samples and biological reagents and eliminates the adverse effects of potential biochemical reactions. At the same time, the installation of the buckle does not change the shape and transparency of the chip, and no additional polishing is required. It can be widely used in microfluidic chips with various detection principles.

[0103] 7. Based on automatic elution technology, the solid phase carrier (such as biochar) extraction is integrated into a single chip, eliminating the need for extra liquid transfer steps, reducing the risk of aerosol contamination and human error, and building a fast and convenient highly integrated detection platform; at the same time, the chip is equipped with four independent areas, which can detect 4*7 target nucleic acid molecules at the same time, which can reduce the use of samples and reagents and save detection costs. BRIEF DESCRIPTION OF THE DRAWINGS

[0104] Figure 1 Schematic diagram of the flow channel structure of the centrifugal microfluidic chip in Example 2;

[0105] Figure 2 Schematic diagram of the structure of the quantitative chamber, elution chamber and fluid channel in Example 2;

[0106] Figure 3 An exploded view of the centrifugal microfluidic chip in Example 2;

[0107] Figure 4 A flow chart of preparing a capillary channel on a plastic upper layer by combining an etching solution and a mask in Example 3;

[0108] Figure 5 Schematic diagram of the liquid flow direction in the centrifugal microfluidic chip in Example 4. DETAILED DESCRIPTION

[0109] The preferred embodiments of the present invention are described in further detail below in conjunction with the accompanying drawings. It should be noted that the embodiments described below are intended to facilitate the understanding of the present invention and do not have any limiting effect on the present invention. The raw materials and equipment used in the specific embodiments of the present invention are all known products and are obtained by purchasing commercially available products.

[0110] In the description of the present utility model, it should be understood that the terms "upper", "lower", "front", "back", "left", "right", "vertical", "horizontal", "top", "bottom", "inside", "outside" and the like indicate positions or positional relationships based on the positions or positional relationships shown in the accompanying drawings, and are only for the convenience of describing the present utility model and simplifying the description, rather than indicating or implying that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and therefore cannot be understood as a limitation on the present utility model. In addition, the terms "first", "second", etc. are only used for descriptive purposes, and cannot be understood as indicating or implying relative importance or implicitly indicating the number of technical features indicated. Therefore, features defined as "first", "second", etc. may explicitly or implicitly include one or more of the features. In the description of the present utility model, unless otherwise specified, "multiple" means two or more.

[0111] In the description of the present invention, it should be noted that, unless otherwise clearly specified and limited, the terms "connected" and "connection" should be understood in a broad sense, for example, it can be a fixed connection, a detachable connection, or an integral connection; it can be a mechanical connection or an electrical connection; it can be a direct connection, or it can be indirectly connected through an intermediate medium, or it can be the internal communication of two components. For ordinary technicians in this field, the specific meanings of the above terms in the present invention can be understood by specific circumstances.

[0112] Example 1. Preparation of biochar

[0113] The biochar in this embodiment is prepared with rice straw and corn cobs as carbon sources, and the preparation process is: take rice straw and corn cobs and mix them in a weight ratio of 1:2 to 1:3 (preferably in this embodiment), wash them with tap water, and then dry them in a forced air drying oven at 60°C, then cut the rice straw into pieces into a length of 2 to 3 cm, and then place the corn cobs and rice straw in an anaerobic tubular furnace under a high-purity N2 atmosphere for high-temperature pyrolysis, heat to 700°C at a heating rate of 5°C / min, and carbonize for 2 hours. After the pyrolysis is cooled, take them out, mix them, grind them, and pass them through a 100-mesh sieve.

[0114] Example 2: Centrifugal microfluidic chip provided by the utility model

[0115] The structure of the centrifugal microfluidic chip provided in this embodiment is as follows Figures 1 to 3 As shown, Figure 1Schematic diagram of the flow channel structure of the centrifugal microfluidic chip. Figure 2 It is a schematic diagram of the structure of the quantitative cavity, the elution cavity and the fluid channel; Figure 3 An exploded view of the centrifugal microfluidic chip.

[0116] like Figure 1 The centrifugal microfluidic chip 1 (hereinafter referred to as chip 1) provided in this embodiment includes a detection unit 2, in which a quantitative chamber 3, an elution chamber 4 and a detection area 5 are provided; the elution chamber 4 is used to elute the analyte from the solid phase carrier; the quantitative chamber 3 is used to temporarily store the added liquid and quantify the added liquid. The quantitative chamber 3 and the elution chamber 4 are connected by a fluid channel 6; the detection area 5 is used to detect the content of the analyte, and the detection area 5 is connected to the fluid channel 6 through a capillary channel 7. The chip 1 also includes a rotation center 8, and the rotation center 8 is used to connect to a centrifugal drive device to drive the chip 1 to centrifuge. Preferably, a plurality of groups of detection units 2 can be provided in the chip 1, and each group of detection units 2 is provided with a quantitative chamber 3, an elution chamber 4 and a detection area 5, which can be suitable for the simultaneous detection of multiple different target analytes, such as the detection of multiple different nucleic acid targets, and is also suitable for eluting nucleic acid targets from multiple different solid phase carriers for detection. In this embodiment, the chip 1 is provided with 4 groups of detection units 2, which are evenly arranged around the rotation center 8, and each detection unit 2 can be used for the detection of a sample, so that the detection throughput of the chip 1 is higher. The quantitative cavity 3 and the elution cavity 4 are connected by a fluid channel 6, which refers to the channel connecting the outlet (first outlet 9) of the quantitative cavity 3 and the inlet (second inlet 10) of the elution cavity 4.

[0117] Preferably, the solid phase carrier includes one or more of biochar, magnetic beads, gel, resin, and cellulose; and the test object is nucleic acid. In the present embodiment, the solid phase carrier is the biochar provided in Example 1. Biochar has the characteristics of low cost and easy availability of raw materials. When biochar is used as a solid phase carrier for nucleic acid extraction and detection, the cost can be significantly reduced compared to magnetic beads. However, the density of biochar is smaller and looser than that of magnetic beads, making it more difficult to separate biochar from the test sample after elution than magnetic beads. Therefore, it is necessary to design the structure of various chambers and flow channels in the chip 1 so that the biochar can be eluted smoothly, and the test sample after elution can smoothly enter the detection area 5 for detection, while the biochar is blocked in the elution chamber 4 and will not block or affect the detection of the test sample.

[0118] like Figure 1, the capillary channel 7 includes a first section 11 and a second section 12; the first section 11 is a channel that meanders from the liquid outlet 13 of the fluid channel 6 to the rotation center 8, and the second section 11 is a channel that meanders from the end 14 of the first section 11 to the detection area 5. When the chip 1 is centrifuged, the flow direction of the fluid sample under the centrifugal action is emitted from the center of the circle to the outside. The setting of the first section 11 in the capillary channel 7 makes it so that if the sample wants to enter the first section 11, its flow direction is opposite to the centrifugal direction, so during the centrifugation process, the sample cannot enter the capillary channel 7. The setting of the first section 11 of the capillary channel 7 is equivalent to adding a threshold for the sample to enter the capillary channel 11, and strengthening the protection for blocking the entry of biochar. First, it ensures that the sample will not enter the capillary channel during the centrifugal mixing and elution process, and the biochar will not be able to block the capillary channel 11; secondly, when the centrifugation is stopped, the supernatant liquid sample can penetrate into the capillary channel 7 under the action of surface tension, and the biochar is thrown to the bottom of the elution chamber 4 by the centrifugal force, so it cannot contact or block the capillary channel 7. The length of the capillary channel 7 can be adjusted according to the size specifications of the chip. In this embodiment, the total length of the capillary channel 7 is 24mm, the width is 0.3mm, and the depth is 0.5mm.

[0119] like Figure 2 , a barrier block 15 is provided below the fluid channel 6, so that the bottom of the quantitative chamber 3 or the elution chamber 4 is at least partially blocked; a step 16 is also provided on the fluid channel 6, and the step 16 is provided on the side of the fluid channel 6 close to the liquid outlet 13 leading to the capillary channel 7. The fluid channel 6 connects the quantitative chamber 3 and the elution chamber 4, but due to the setting of the barrier block 15, the liquid cannot flow below the fluid channel 6, which is equivalent to that only the upper chamber of the quantitative chamber 3 and the elution chamber 4 can be fluidically connected, and the lower part is blocked. Such a setting also helps to prevent biochar from entering or blocking the capillary channel, because the biochar will basically sink below the elution chamber 4 under the action of centrifugal force. The step 16 is located above the barrier block 15 and close to the side of the liquid outlet 13 leading to the capillary channel 7, so that the liquid surface near the liquid outlet 13 forms a U-shaped liquid shape similar to the residual liquid surface, thereby raising the liquid surface here, helping the sample to penetrate into the capillary channel 7 under the action of surface tension. The size of the fluid channel 6 can be adjusted according to actual product needs. In this embodiment, the width of the fluid channel 6 is 1-1.25 mm, the height of the quantitative cavity 3 and the elution cavity 4 is 4.5-5.5 mm, and the height of the fluid channel 6 is blocked by the blocking block 15 at the bottom, and the height of the blocking block 15 is 3-4.5 mm, so that the height of the fluid channel 6 is reduced to 1.0-1.5 mm. The height of the step 16 is 0.5-0.7 mm, and the width is 0.4-0.6 mm.

[0120] The quantitative chamber 3 is provided with a first inlet 17 and a first outlet 9, and the size of the quantitative chamber 3 gradually decreases near the first outlet 9. The quantitative chamber 3 is an inverted pentagonal chamber, and the overall shape is similar to a funnel. The shape design of the quantitative chamber 3 helps the sample containing biochar to quickly gather and enter the elution chamber 4 under the action of centrifugal force after entering, and helps the sample containing biochar to be stably retained in the elution chamber 4 after entering the elution chamber 4, and is not easy to reflux, that is, it can help block the reflux of biochar. The quantitative chamber 3 is used for quantitative storage of the added liquid, and the volume of the quantitative chamber 3 can be adjusted according to the specific purpose and sample amount. In this embodiment, the width of the quantitative chamber 3 is 6.25±0.5mm, the length is 4.8±0.5mm, and the volume is 200±30μL.

[0121] Preferably, an exhaust channel 18 and a waste liquid discharge channel 19 are also provided on the quantitative chamber 3; the exhaust channel 18 extends from the quantitative chamber 3 toward the rotation center 8, and the exhaust port 20 of the exhaust channel 18 is connected to the outside world, which is used to control the flow of gas in the quantitative chamber 3 and / or the elution chamber 4; the waste liquid discharge channel 19 extends from the quantitative chamber 3 in the direction away from the rotation center 8, and is connected to the waste liquid pool 21.

[0122] When the liquid enters the chip 1 from the sample addition area 22 and enters the corresponding chamber under the action of centrifugal force, the gas in the chamber needs to be discharged from the exhaust channel 18. The quantitative chamber 3 and the elution chamber 4 share an exhaust channel 18, which makes the preparation process simpler. The direction of the exhaust channel 18 is opposite to the centrifugal direction. Therefore, when the liquid enters the quantitative chamber 3 or the elution chamber 4 under the action of centrifugal force, the liquid will not be discharged from the exhaust channel 18. Only when the elution chamber 4 and the quantitative chamber 3 are full, the liquid will enter the exhaust channel 18. The exhaust channel 18 and the waste liquid discharge channel 19 share an outlet 23 and a common channel 24 at one end on the quantitative chamber 3. The common channel 24 is provided with branches. The first branch is opposite to the centrifugal direction and leads to the exhaust port 20, which is the exhaust channel 18. The second branch is the same as the centrifugal direction and leads to the waste liquid pool 21, which is the waste liquid channel 19. Therefore, the liquid will choose to enter the waste liquid pool 21 under the action of centrifugation. During actual use, the chip 1 is placed in a detection device. It is difficult to see whether the sample has entered the quantitative cavity 3 through the device, but through the window of the device, it can be seen whether liquid has entered the waste liquid pool 21. When liquid begins to enter the waste liquid pool 21, it means that the quantitative cavity 3 has been filled. It can be seen that the waste liquid pool 21 also has the effect of helping to quantify the liquid in the quantitative cavity 3.

[0123] Preferably, the exhaust port 20 is a circular through-structure with a diameter of 2.5 mm, which is used to exhaust the gas in the chip 1, balance the internal and external air pressure, and prevent bubbles from blocking the microchannel and affecting the flow of liquid. The surface of the exhaust port 20 is pre-covered with a transparent film to play a sealing role. When exhaust is required during use, the transparent film can be punctured for exhaust; if it needs to be closed later, it can also be covered with a transparent film for sealing.

[0124] The elution chamber 4 is provided with a second inlet 10, and the size of the elution chamber 4 gradually decreases near the second inlet 10. The elution chamber 4 is a pentagonal chamber, which is reduced at the second inlet 10. When the sample enters the elution chamber 4 under the action of centrifugation, it can help prevent the biochar from flowing out of the elution chamber 4. The elution chamber 4 is used to elute the nucleic acid to be tested from the biochar and store the eluted liquid. The size and volume of the elution chamber 4 can be set according to the specific needs of the product and can be fine-tuned within an appropriate range. In this embodiment, the elution chamber 4 has a width of 5.8±0.5mm, a length of 4.8±0.5mm, and a volume of 230±30μL.

[0125] Preferably, the detection area 5 includes a detection channel 25, and a detection cavity 26 connected to the detection channel 25; the detection channel 25 is connected to the capillary channel 7. A second exhaust channel 27 is provided on the detection channel 25, and the second exhaust channel 27 extends from the detection channel 25 toward the rotation center 8. The second exhaust port 28 of the second exhaust channel 27 is connected to the outside world for controlling the flow of gas in the detection area 5; the number of the detection cavities 26 is one or more. When the number of the detection cavities 26 is multiple, multiple target nucleic acids can be detected at the same time, and can be set according to product needs. In this embodiment, the number of detection cavities 26 in each detection area 5 is seven, and seven target nucleic acids can be detected on the same sample.

[0126] like Figure 1 , the detection channel 25 is an arc channel arranged along the circumference, and the detection cavity 26 is a circular room with a diameter of 1.5-2.5mm, which can match the spot size of the fluorescence detection module in the detection equipment, slightly larger than the spot diameter and the diameter should not be larger than the spot diameter by more than 0.8mm, and the depth is 0.2-0.5mm. The depth change can control the volume of the reaction, can adapt to a variety of reaction systems, and expand the application scenarios and detection objects. The length of the detection channel 25 can be adjusted according to the product size. In this embodiment, the length of the detection channel 25 is 25mm.

[0127] The fluorescence detection process of the chip 1 is that the chip 1 rotates slowly during the slow centrifugation process, so that each detection cavity 26 is aligned with the fluorescence detection channel in turn to complete the fluorescence detection. In order to enable each detection result to correspond one-to-one with each detection cavity 26, it is necessary to clarify the angle of the position of each detection cavity 26 before detection. In this embodiment, a line is established from the center of rotation to the center of the circle of the detection cavity 26, and the angle between the lines of two adjacent detection cavities 26 is 7 degrees, which is an integer multiple of the single-grid rotation degree of the stepper motor in the centrifugal device (0.7 degrees), and at this angle, the fluorescence interference of adjacent detection cavities 26 can be avoided, thereby facilitating the calculation of the position of each detection cavity 26 and recording the reading results, which is beneficial to improve the positioning accuracy during centrifugation and detection.

[0128] Nucleic acid amplification reagents and / or nucleic acid detection reagents need to be pre-placed in the detection chamber 26. Among them, the amplification reaction includes but is not limited to EPA, RPA, LAMP, NEAR, NASBA, HAD, TMA, SAT, RCR, etc., and the solid dry reagents include but are not limited to spherical, powdered, sheet-like or block-like preparations in the form of freeze-dried, dried, air-dried, etc. Nucleic acid detection includes but is not limited to a variety of detection systems established by ERA, RPA, LAMP, NEAR, NASBA, HAD, TMA, SAT, PCR, hybridization probe technology, CRISPR, Ago, RNase H, etc., and the detection reagents are solid detection reagents, including but not limited to spherical, powdered, sheet-like or block-like preparations in the form of freeze-dried, dried, air-dried, etc.

[0129] like Figure 1 , the detection unit 2 of the chip 1 also includes a sample addition area 22, which is connected to the quantitative cavity 3 through a sample addition channel 30; the sample addition area 22, the quantitative cavity 3 and the elution cavity 4 are arranged in sequence along the rotation centrifugal direction of the chip 1. Preferably, the sample addition area 22 is an elliptical chamber with a major axis of 12±0.5mmmm and a minor axis of 7.5±0.5mmmm. There is a sample addition hole 29 with a diameter of 2mm on the top for adding sample liquid and eluent, and a 2mm diameter pore for exhausting the air in the chamber when adding liquid to balance the air pressure. Compared with the circular structure, the elliptical structure can utilize the area of ​​the chip 1 close to the center of the circle as much as possible, so as to maximize the sample addition volume occupied by the same disc diameter, and effectively improve the space utilization of the chip 1. Preferably, the surface of the sample addition area 22 of the chip 1 is covered with a transparent film to play a sealing role. When adding samples, the transparent film is punctured by a syringe for adding samples. After the sample fills the main channel, the surface of the sample addition hole 29 can also be covered with a transparent film for sealing.

[0130] The rotation center 8 is a fixing hole 32, and the fixing hole 32 can make the chip 1 stuck on the rotating shaft of the centrifugal drive device, so as to perform centrifugal rotation. Preferably, the fixing hole 32 is approximately elliptical, with a major axis of 13 mm and a minor axis of 8 mm, and can be combined with the driving member of the centrifugal drive device to fix the chip and provide centrifugal power.

[0131] like Figure 3 The chip 1 is a sandwich structure, including a plastic upper layer 33, a silicone inner membrane 34 and a plastic lower layer 35, and the detection unit 2 is located on the plastic upper layer 33. The plastic upper layer 33 is a polydimethylsiloxane (PDMS) disc processed by injection molding, with a thickness of 1.8-2mm and a diameter of 75mm. It has a downwardly protruding microfluidic structure, wherein the capillary channel 7 of the plastic upper layer 33 is 0.3mm wide, and the widths of the remaining microfluidic channels are all 0.8-1mm. The two outer sides of the channel have a slope of 5-10 degrees and a support structure with a width of 0.3-0.5mm, which is used to improve the pressure-bearing performance of the microfluidic channel and is also conducive to the demolding of the injection molding. The plastic upper layer 33 is provided with a first fixing hole 132, an exhaust hole 20, a sample addition area 22, a quantitative cavity 3, an elution cavity 4, a fluid channel 6, a capillary channel 7, and a detection cavity 26. The silicone inner membrane 34 is a circular silicone diaphragm with a thickness of 0.1 mm. It is a soft silicone material that can fill tiny irregular surfaces under pressure, provide better elasticity and sealing, and reduce the leakage rate of the liquid. A second fixing hole 232 is provided on the silicone inner membrane 34. Under normal use and storage conditions, the silicone inner membrane 34 has an extremely long service life, has good aging resistance, heat resistance and chemical resistance, and can be applied to a variety of usage scenarios. The plastic lower layer 35 is a plastic disc with a fixing hole, with a thickness of 0.1-0.2 mm, and a third fixing hole 332. The injection molding process uses a mixed material (10:10:1) of polymethyl methacrylate (PMMA), polycarbonate (PC), and dioctyl sebacate (DOS) as the base. The processed plastic disc still has good rigidity in an extremely thin case, which can support and protect the microchannel structure of the plastic upper layer 33, while preventing the soft silicone inner membrane 34 from being scratched and damaged. The disc has strong drop resistance and light transmittance, and can maintain excellent light transmittance even under accelerated aging conditions without affecting subsequent fluorescence detection in the reaction chamber.

[0132] like Figure 3 The chip 1 also includes a quick assembly buckle 36, which is divided into a first buckle 37, a second buckle 38, a third buckle 39 and a fourth buckle 40; the first buckle 37 and the second buckle 38 are assembled into one body, which is used to fix the outer periphery 41 of the sealed chip 1; the third buckle 39 and the fourth buckle 40 are assembled into one body, which is used to fix the inner periphery 42 of the sealed chip 1, and the inner periphery 42 is the arc hole where the fixing hole 32 of the chip 1 (including the first fixing hole 132, the second fixing hole 232 and the third fixing hole 332 combined together) is located.

[0133] The first buckle 37, the second buckle 38, the third buckle 39 and the fourth buckle 40 are two groups of semicircular acrylic buckles of different sizes, and the side is designed with a groove 43 structure with a height of 0.8mm. The total thickness of the chip 1 is 1mm. Acrylic has good toughness and elasticity and is not easy to break or crack. The height difference of 0.2mm is used to make the buckle elastically squeeze the three-layer structure of plastic-silicone-plastic to achieve the sealing of the chip 1. Figure 3 The first buckle 37 is an acrylic semicircular arc with a groove 43 on the side, with an arc width of 2mm, which can fit with the outer periphery 41 of the chip 1, and has female grooves 44 at both ends for assembly with the second buckle 38. The second buckle 38 is an acrylic semicircular arc with a groove 43 on the side, the size of which is the same as that of the first buckle 37, and has male grooves 45 at both ends for assembly with the first buckle 37. The third buckle 39 is an acrylic semicircular arc with a groove 43 on the side, with an arc width of 2mm, which can fit with the fixing hole 32 of the chip 1, and has female grooves 44 at both ends for assembly with the fourth buckle 40. The fourth buckle 40 is an acrylic semicircular arc with a groove 43 on the side, the size of which is the same as that of the third buckle 39, and has male grooves 45 at both ends for assembly with the third buckle 39.

[0134] Example 3. Preparation of centrifugal microfluidic chip

[0135] The preparation method of the centrifugal microfluidic chip 1 provided in this embodiment specifically comprises the following steps:

[0136] 1. The plastic upper layer 33 is prepared by an injection molding one-piece molding method.

[0137] 2. Prepare the capillary channel 7 on the plastic upper layer 33 by combining the etching solution and the mask. The preparation process is shown in Figure 4 :

[0138] (1) Cover the unprocessed area on the plastic upper layer 33 of the chip 1 with the silicone mold 45 with the hollow capillary 46 so that the hollow capillary 46 on the silicone mold 45 is positioned in the area connecting the fluid channel 6 and the detection channel 25 .

[0139] The silicone mold 45 is made by photolithography technology and has a hollow capillary 46 with a width of 0.3 mm.

[0140] (2) injecting 12 μL of the etching solution 47 into the hollow capillary 46 of the silicone mold 45, and after 15 seconds, sucking and recovering the etching solution 47 with a needle;

[0141] Corrosive solution 47: A solution containing 5% dimethylformamide, 4% acetone and 1% tetrahydrofuran by mass concentration is prepared with pure water.

[0142] (3) The silicone mold is removed, and a capillary channel 7 with a width of 0.3 mm and a depth of 0.5 mm is formed on the plastic upper layer 33 .

[0143] 3. Assemble the chip in the order of “plastic upper layer 33 - silicone inner film 34 - plastic lower layer 35”. After alignment, use the first clip 37 and the second clip 38 to press the outside of the chip, and use the third clip 39 and the fourth clip 40 to press the inside of the chip to complete the chip packaging.

[0144] Example 4: Method for using a centrifugal microfluidic chip

[0145] The centrifugal microfluidic chip is used for sample detection. The detection method is as follows (the flow direction of the sample in the chip 1 is as follows: Figure 5 As shown, the black arrow represents the direction of liquid flow):

[0146] (1) Take the chip 1 prepared in Example 3, poke open the exhaust holes 20 and the sample loading holes 31 of the plastic upper layer 33, and clamp the fixing hole 32 of the chip 1 on the centrifugal rotating shaft of the detection device.

[0147] (2) Prepare a sample to be tested containing a solid phase carrier (such as biochar or magnetic beads), inject 200 μL of the sample to be tested from the sample injection hole 31 using a syringe, and start the centrifugation program (1200 r / min, 50 s) to allow the sample to be tested to enter the elution chamber 4 of the chip.

[0148] (3) Use a syringe to inject 230 μL of eluent from the sample injection hole 31 and start the centrifugation program (1200 r / min, 30 s). When liquid appears in the waste liquid pool 21, stop the centrifugation, indicating that the eluent has filled the quantitative chamber 3.

[0149] (4) Start the centrifugation program (1200 r / min, 2 min), the eluent enters the elution chamber 4 and mixes with the sample to be tested, and elutes the cleavage products bound to the solid phase carrier, including proteins, nucleic acids, and bacterial fragments. Among them, the nucleic acid molecules have the lightest mass and float in the supernatant, completing the elution of the nucleic acid.

[0150] (5) Stop centrifugation, the sample containing nucleic acid enters the detection area 5 through the capillary channel 7, and the solid phase carrier is blocked and remains in the elution chamber 4; start the centrifugation program again (1200r / min, 1min), the sample is evenly distributed from the detection channel 25 into each detection chamber 26, and nucleic acid isothermal amplification is performed (isothermal amplification requires the detection equipment to provide each detection chamber 26 with a suitable temperature required for isothermal amplification (such as 30-65°C)); the equipment starts the detection centrifugation program "high speed 1200r / min 45s; low speed 200r / min15s alternating", the detection equipment performs fluorescence detection on the sample amplification products in each detection chamber 26 in turn, obtains a real-time fluorescence detection curve, and completes the entire detection process.

[0151] Example 5: Screening of Carbon Sources for Biochar Preparation

[0152] In this example, biochar was prepared according to the method provided in Example 1, wherein the carbon sources used to prepare the biochar were selected from five groups of carbon sources as shown in Table 1, thereby preparing five groups of biochar, and the five groups of biochar were used to extract nucleic acid from Escherichia coli bacterial liquid, respectively. The specific method was as follows: 300 μL of the liquid sample obtained by sampling was added to a centrifuge tube, 1 mL of biochar (10 mg / mL) was added thereto, and after thermal cracking at 100°C for 5 min in a metal bath, the mixture was evenly mixed on a vortex oscillator, the centrifuge tube was allowed to stand for 7-10 min, and after the cracking product was completely adsorbed by the biochar, the supernatant was removed, and the washing was repeated 3 times, 1 mL of PBST buffer was added to the precipitate, 1 mL of sterile PBST buffer (100 mL of 0.01 M PBS, 50 μL of Tween 20, pH 7.4) was gently shaken to obtain a sample solution containing biochar. The five groups of sample solutions containing biochar were then eluted with 200 μL 10 mM Tris-HCl buffer (pH 7.5) + 30 μL 0.02 M PBS. The nucleic acid extraction yield of each group of biochar was investigated and compared with the magnetic beads. The results are shown in Table 1.

[0153] Table 1. Nucleic acid extraction effects of different biochars

[0154] Solid phase carrier Carbon source Nucleic acid extraction yield (%) Biochar 1 2% Rice Straw 53.2 Biochar 2 6% Corncob 41.0 Biochar 3 2% rice straw + 6% corn straw 86.4 Biochar 4 2% sawdust + 6% corn cob 92.7 Biochar 5 2% rice straw + 6% corn cob 99.2 Magnetic beads / 98.9

[0155] It can be seen from Table 1 that the nucleic acid extraction yields of biochars prepared from different carbon sources vary greatly, so the most preferred biochar is the fifth group, which is biochar prepared using rice straw and corn cobs as carbon sources.

[0156] Example 6: Verification of the detection effect of the centrifugal microfluidic chip

[0157] This example uses the centrifugal microfluidic chip provided in Example 2 for nucleic acid detection, and uses ERA-CRISPR detection technology (enzymatic recombinant amplification combined with CRISPR detection technology) to perform respiratory pathogen nucleic acid detection (influenza A virus, influenza B virus, respiratory syncytial virus, adenovirus, human rhinovirus, Mycoplasma pneumoniae, human metapneumovirus, parainfluenza virus) on blood specimens suspected of respiratory tract infection, and uses qPCR detection method (commercial reagents) for comparative verification. 300 μL of the liquid sample obtained by sampling was added to the centrifuge tube, 1 mL of nano-scale biochar (10 mg / mL, prepared in Example 1) was added thereto, and after thermal cracking in a metal bath at 100°C for 5 min, the mixture was mixed evenly on a vortex oscillator, and the centrifuge tube was allowed to stand for 7-10 min. After the pyrolysis product was completely adsorbed by the biochar, the supernatant was removed, and the washing was repeated 3 times. 1 mL of PBST buffer was added to the precipitate, and 1 mL of sterile PBST buffer (100 mL 0.01 M PBS, 50 μL Tween 20, pH 7.4) was gently shaken to obtain a sample solution containing biochar. The method provided in Example 6 was used for nucleic acid detection, and the detection chamber was pre-loaded with ERA freeze-dried microspheres containing specific amplification primers. The results are shown in Table 1.

[0158] Table 1. Centrifugal microfluidic chip detection performance verification list

[0159]

[0160] As can be seen from Table 1, under the same other conditions, the detection results of the centrifugal microfluidic chip device of Example 2 are consistent with the results of the qPCR detection method, but it is more convenient and quick to use, and has a lower overall use cost.

[0161] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0162] Although the utility model is disclosed as above, the utility model is not limited thereto. For example, the scope of application of the utility model in the field of microfluidics can be expanded. Any person skilled in the art can make various changes and modifications without departing from the spirit and scope of the utility model. Therefore, the scope of protection of the utility model shall be based on the scope defined by the claims.

[0163] The contents of the articles, patents, patent applications, and all other documents and electronically available information described or recorded herein are incorporated by reference in their entirety to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference. Applicants reserve the right to incorporate into this application any and all materials and information from any such article, patent, patent application, or other document.

Claims

1. A centrifugal microfluidic chip for nucleic acid detection, characterized in that: It comprises a detection unit, wherein the detection unit is provided with a quantitative cavity, an elution cavity and a detection area; the elution cavity is used to elute the object to be detected from the solid phase carrier; The quantitative chamber is used to temporarily store the added liquid and quantify the added liquid, wherein the liquid includes an eluent; The quantitative chamber and the elution chamber are connected via a fluid channel; The detection zone is used to detect the content of the substance to be detected, and the detection zone is connected to the fluid channel through a capillary channel.

2. The nucleic acid detection centrifugal microfluidic chip according to claim 1, characterized in that: The solid phase carrier includes one or more of biochar, magnetic beads, gel, resin, and cellulose; and the analyte includes nucleic acid.

3. The nucleic acid detection centrifugal microfluidic chip according to claim 2, characterized in that: The solid phase carrier is biochar.

4. The nucleic acid detection centrifugal microfluidic chip according to claim 1, characterized in that: It also includes a rotation center, which is used to connect with a centrifugal driving device to drive the centrifugal microfluidic chip to centrifuge; the number of the detection units is one or more, which are arranged around the rotation center.

5. The nucleic acid detection centrifugal microfluidic chip according to claim 4, characterized in that: The capillary channel includes a first section and a second section; the first section is a channel that meanders from the liquid outlet of the fluid channel to the rotation center, and the second section is a channel that meanders from the end of the first section to the detection cavity.

6. The nucleic acid detection centrifugal microfluidic chip according to claim 5, characterized in that: A blocking block is provided below the fluid channel to at least partially block the quantitative cavity or the elution cavity; a step is also provided on the fluid channel, and the step is provided on one side of the fluid channel close to the liquid outlet leading to the capillary channel.

7. The nucleic acid detection centrifugal microfluidic chip according to claim 5, characterized in that: The quantitative cavity is provided with a first inlet and a first outlet, and the size of the quantitative cavity gradually decreases near the first outlet; The elution chamber is provided with a second inlet, and the size of the elution chamber gradually decreases near the second inlet.

8. The nucleic acid detection centrifugal microfluidic chip according to claim 7, characterized in that: The quantitative chamber is also provided with an exhaust channel and a waste liquid discharge channel; the exhaust channel extends from the quantitative chamber toward the rotation center, and the exhaust port of the exhaust channel is connected to the outside world for controlling the flow of gas in the quantitative chamber and / or the elution chamber; the waste liquid discharge channel extends from the quantitative chamber away from the rotation center and is connected to the waste liquid pool.

9. The nucleic acid detection centrifugal microfluidic chip according to claim 1, characterized in that: The detection area includes a detection channel and a detection cavity connected to the detection channel; the detection channel is connected to the capillary channel.

10. The nucleic acid detection centrifugal microfluidic chip according to claim 9, characterized in that: The detection channel is provided with a second exhaust channel, which extends from the detection channel toward the rotation center. The exhaust port of the second exhaust channel is connected to the outside world for controlling the flow of gas in the detection area. The number of the detection chambers is one or more.

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