Centrifugal micro-fluidic chip for method for rapidly enriching nucleic acid by microbial biochar

Abstract

The invention provides a centrifugal micro-fluidic chip for a method for rapidly enriching nucleic acid by microbial biochar, and aims at a solid-phase carrier for adsorbing nucleic acid, and the centrifugal micro-fluidic chip capable of completing the whole process of nucleic acid elution, amplification and detection is designed. A quantitative cavity, an elution cavity and a detection area are arranged in the chip, the elution cavity is used for eluting nucleic acid to be detected from biochar, and the quantitative cavity is used for temporarily storing added eluent and quantifying the added eluent to accurately complete the elution process; the structure of the quantitative cavity, the structure of the elution cavity and the structure of the fluid channel are improved, and steps are additionally arranged in the fluid channel, so that a to-be-detected sample smoothly enters a detection area for detection through the capillary tube under the action of surface tension, and the capillary tube can be prevented from being blocked by solid-phase carriers used in pretreatment such as charcoal and magnetic beads; the accuracy of a detection result is ensured; 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] The present application claims priority to the Chinese prior application, application number: 2024107898334, filing date: June 19, 2024; the contents of which are hereby incorporated by reference as part of the present application. TECHNICAL FIELD

[0002] The present application relates to the field of microfluidic control, in particular, to a centrifugal microfluidic chip for rapid enrichment of nucleic acid from microbial biochar. BACKGROUND

[0003] Microfluidic chips, also commonly known as "lab-on-chip", can manipulate fluid in a micron-scale space, complete biological, chemical, and other experiments with only a small amount of sample, and have the advantages of integration, high throughput, and high efficiency, and are widely used in scientific fields such as environment, chemistry, and medicine.

[0004] With the rapid development of genetic diagnosis, personalized medicine, and other technologies, nucleic acid detection technologies with high specificity and accuracy are increasingly important. Existing microfluidic chips have integrated sample processing, nucleic acid amplification, and nucleic acid detection into a small chip, and can quickly complete the "sample in-result out" detection process. Because microfluidic chips do not rely on precise pipetting devices and professional personnel, 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 manipulate liquid on a sub-millimeter scale using centrifugal force. Compared with traditional microfluidic chips, the entire device is more compact and simple to handle, and therefore is gradually being applied in the field of nucleic acid sample detection. In a centrifugal microfluidic chip, microchannels are distributed along the radial direction of the disc. When the chip is rotated, the liquid to be tested moves along the microchannels to each reaction pool under the action of centrifugal force, achieving sample distribution and addition, and making the nucleic acid detection process more efficient.

[0006] Nucleic acid detection needs to be pretreated by lysis and purification. Mild and efficient magnetic bead extraction method is concerned. The magnetic bead method usually includes four steps of 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, which has good stability. The surface is modified by special groups. Under specific ion conditions, it can specifically adsorb free nucleic acid molecules in the sample through electrostatic interaction, hydrogen bond interaction and other ways to form nucleic acid-magnetic bead complex, while proteins and other impurities remain in the sample liquid, which can realize the rapid extraction and separation of nucleic acid. However, the production process of magnetic beads is complex, which needs professional equipment personnel and technical personnel, and its price is high, which greatly increases the cost of detection, and the relatively low economic benefit limits the application of magnetic beads in large-scale liquid samples, especially in remote areas. Therefore, a more economical and practical method needs to be found.

[0007] The centrifugal microfluidic chip on the market usually lyses and purifies the sample outside the chip before adding it into the microfluidic chip for nucleic acid detection. Few nucleic acid extraction is integrated into a single chip, because this will make the chip structure complex, increase the control difficulty and increase the cost.

[0008] CN116144458A provides a centrifugal microfluidic chip for detecting nucleic acid extracted by magnetic beads, but in the nucleic acid elution process, an additional magnet is needed to adsorb the magnetic beads, which makes the use process of the centrifugal microfluidic chip more complicated, has high dependence on equipment, and also increases the cost. CN117551540A provides a centrifugal microfluidic chip for detecting nucleic acid extracted by magnetic beads without magnet, but its structure is very complex, which needs to specially set magnetic bead temporary storage chamber, flow channel stop valve, hot melt valve and other structures, and one chip can only be used for detection of one sample. In addition, the above two kinds of centrifugal microfluidic chips can only be used for nucleic acid extraction by magnetic beads, and cannot be used for other solid phase carriers to extract, elute and detect nucleic acid.

[0009] In view of these technical problems, it is urgent to find a centrifugal microfluidic chip which can quickly and conveniently complete the elution and detection of nucleic acid, not only suitable for magnetic beads, but also suitable for other solid phase carriers with lower cost, and can significantly improve the efficiency of nucleic acid detection. SUMMARY

[0010] To solve the above problems, the application provides a centrifugal microfluidic chip for a method for rapid enrichment of nucleic acid of microbial biochar, a centrifugal microfluidic chip capable of completing the whole process of nucleic acid elution, amplification and detection is designed for a solid phase carrier for adsorbing nucleic acid, and the centrifugal microfluidic chip comprises a quantitative cavity, an elution cavity and a detection zone; the elution cavity is used for eluting the nucleic acid to be detected from the biochar, the quantitative cavity is used for temporarily storing the eluent and quantifying the eluent, and the elution process is accurately completed; the quantitative cavity and the elution cavity are connected through a fluid channel, the structure of the quantitative cavity, the elution cavity and the fluid channel is improved, and a step is additionally arranged in the fluid channel, so that the sample to be detected is smoothly introduced into the detection zone for detection under the action of surface tension, the biochar is prevented from blocking the capillary, the accuracy of the detection result is ensured, high-throughput detection of more samples and more detection items can be simultaneously realized, and the detection efficiency is improved. The chip has simple structure, effectively reduced manufacturing and processing difficulty and lower production cost.

[0011] In one aspect, the application provides a centrifugal microfluidic chip, which comprises a detection unit, the detection unit is provided with a quantitative cavity, an elution cavity and a detection zone; the elution cavity is used for eluting the analyte from a solid phase carrier; the quantitative cavity is used for temporarily storing a liquid and quantifying the liquid, and the liquid comprises an eluent; the quantitative cavity and the elution cavity are connected through a fluid channel; and the detection zone is used for detecting the content of the analyte, and the detection zone is connected with the fluid channel through a capillary channel.

[0012] The centrifugal microfluidic chip provided by the application is provided with a plurality of detection units, each detection unit is provided with a quantitative cavity, an elution cavity and a detection zone, can be suitable for simultaneous detection of a plurality of different target analytes, such as detection of a plurality of different nucleic acid targets, and can also be suitable for eluting the nucleic acid targets from a plurality of different solid phase carriers for detection.

[0013] In the use process of the centrifugal microfluidic chip (referred to as a chip), the chip needs to be kept in a horizontal state and placed with the sample adding port upward. In some modes, the chip can be inserted into a matching detection device, so that the chip is kept in a horizontal state with the sample adding port upward, the center position of the chip is fixed together with the rotating shaft for centrifugation of the detection device, so that the rotating shaft can drive the chip to rotate for centrifugation; in some modes, the detection device can provide a heat source for the detection zone of the chip for heating, helping to complete the nucleic acid amplification reaction; in some modes, the detection device can also detect the fluorescent substance generated after the nucleic acid amplification reaction in the chip, and read the detection result.

[0014] The chip provided by the application is characterized in that: a quantitative cavity and an elution cavity are arranged respectively, in the initial stage of sequentially adding a sample containing a solid carrier and an eluent, the sample containing the solid carrier and the eluent are preliminarily separated and quantified, then, with the continuation of centrifugation, the elution process starts, the eluent in the quantitative cavity continuously enters the elution cavity and collides and mixes with the solid carrier, the solid carrier disperses outward as far as possible under the action of centrifugal force and adheres to the inner wall of the elution cavity farthest from the center, and the rest of the liquid is continuously mixed in the elution cavity or is squeezed out of the elution cavity. It can be seen that the arrangement of the quantitative cavity not only plays a quantitative role, but also makes the mixing and elution more sufficient, improves the elution efficiency, and thus improves the detection sensitivity.

[0015] The quantitative cavity and the elution cavity are connected through a fluid channel, which is a channel connecting the outlet of the quantitative cavity and the inlet of the elution cavity.

[0016] Further, the solid carrier includes one or more of biochar, magnetic beads, gel, resin and cellulose; and the to-be-detected substance is nucleic acid.

[0017] In some modes, the solid carrier is biochar.

[0018] Biochar has the characteristics of low cost and easy-to-obtain raw materials, and when biochar is used as a solid carrier for nucleic acid detection, the cost can be significantly reduced. Moreover, the density of biochar is smaller than that of magnetic beads, and biochar is more loose and non-magnetic, so it cannot be separated by a magnetic field, which makes it more difficult to separate biochar from the to-be-detected sample after elution. Therefore, it is necessary to design the structure of various chambers and flow channels in the chip to help the nucleic acid sample on the biochar to successfully complete elution, and the to-be-detected sample after elution to smoothly enter the detection area for detection, while the biochar is gathered near the outer source of the chip due to the centrifugal effect and is blocked in the elution cavity by the step, so as to ensure that the to-be-detected sample will not be blocked or affected in detection.

[0019] Further, the biochar is prepared by taking rice straw and corn cob as carbon sources.

[0020] Researches have proved that the biochar prepared by taking rice straw and corn cob as carbon sources has better nucleic acid adsorption efficiency.

[0021] Further, a rotation center is further included, the rotation center is used to be connected with a centrifugal driving device, so as to drive the centrifugal microfluidic chip to centrifuge; and the number of the detection units is one or more, and the detection units are arranged around the rotation center.

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

[0023] Further, the capillary channel comprises a first section and a second section; the first section is a channel winding from the liquid outlet of the fluid channel to the rotation center, and the second section is a channel winding 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 centrifugal force is outward from the center. The first section of the capillary channel is arranged such that the flow direction of the sample entering the first section is opposite to the centrifugal direction, so that during the centrifugal process, the sample cannot enter the capillary channel. The arrangement of the first section of the capillary channel is equivalent to adding a threshold for the sample to enter the capillary channel, and it strengthens the protection against the charcoal. First, it ensures that during the centrifugal mixing and elution process, the sample cannot enter the capillary channel, and the charcoal is also unable to block the capillary channel. Second, during the stop of the centrifugal process, the supernatant sample can penetrate into the capillary channel under the action of surface tension, while the charcoal is thrown to the bottom of the elution chamber by the centrifugal force, so it cannot contact or block the capillary channel.

[0025] Further, the lower part of the fluid channel is provided with a blocking block, so that at least part of the lower part of the quantitative cavity or the elution cavity is blocked; and a step is further arranged on the fluid channel, which is arranged on one side of the fluid channel close to the liquid outlet of the capillary channel.

[0026] The fluid channel connects the quantitative cavity and the elution cavity, but due to the arrangement of the blocking block, the lower part of the fluid channel cannot realize liquid flow, which is equivalent to that only the upper chamber of the quantitative cavity and the elution cavity can be in fluid communication, and the lower part is blocked. Such an arrangement also helps to block the charcoal from blocking the capillary channel, because the charcoal will basically sink to the lower part of the elution chamber under the action of the centrifugal force.

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

[0028] In some ways, the width of the fluid channel is 1.0-1.25 mm, 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 amount, and the height of the fluid channel is reduced to 1.0-1.5 mm due to the blocking of the blocking block.

[0029] In some ways, the height of the step is 0.5-0.7 mm, and the width is 0.4-0.6 mm.

[0030] Further, the quantitative cavity is provided with a first inlet and a first outlet, and the size of the quantitative cavity gradually decreases at a position close to the first outlet.

[0031] In some embodiments, the quantification cavity is a pentagonal chamber, which is similar to a funnel in shape.

[0032] The design of the quantification 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 stably stay in the elution cavity after entering the elution cavity, and is not easy to backflow. Therefore, the structure design of the quantification cavity can help to block the backflow of biochar.

[0033] The quantification cavity is used for quantitatively storing the added liquid, and the volume of the quantification cavity can be adjusted according to specific purposes and sample amounts.

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

[0035] Further, the quantification cavity is further provided with an exhaust passage and a waste liquid passage; the exhaust passage extends from the quantification cavity to the direction of the rotation center, the exhaust port of the exhaust passage is connected with the outside, and is used for controlling the flow of gas in the quantification cavity and / or the elution cavity; and the waste liquid passage extends from the quantification cavity to the direction away from the rotation center, and is connected with the waste liquid pool.

[0036] When the liquid enters the chip from the sample adding area and enters the corresponding chamber under the action of centrifugal force, the gas in the chamber needs to be discharged from the exhaust passage. The quantification cavity and the elution cavity share one exhaust passage, so that the preparation process is more convenient. The direction of the exhaust passage is opposite to the centrifugal direction, so when the liquid enters the quantification cavity or the elution cavity under the action of centrifugal force, the liquid will not be discharged from the exhaust passage, but only when the elution cavity and the quantification cavity are filled, the liquid will enter the exhaust passage. The passage from the quantification cavity is provided with branches, the first branch is opposite to the centrifugal direction and leads to the exhaust port, which is the exhaust passage, and the second branch is the same as the centrifugal direction and leads to the waste liquid pool, which is the waste liquid passage. Under the action of centrifugal force, the liquid will definitely choose the waste liquid passage with the same direction as the centrifugal direction to enter the waste liquid pool. Therefore, when the waste liquid pool begins to have liquid entering, it can be seen that the quantification cavity has been filled. It can be seen that the waste liquid pool also has the effect of helping the liquid in the quantification cavity to be quantified.

[0037] The exhaust port is a circular through structure with a diameter of 2.5mm, which is used for discharging the gas in the chip, balancing the internal and external air pressure, and avoiding bubbles from blocking the microfluid channel to affect the flow of liquid.

[0038] In some embodiments, the surface of the exhaust port is pre-covered with a transparent film to play a sealing role. During use, the transparent film can be punctured to discharge air; if subsequent sealing is required, the transparent film can be additionally covered to seal.

[0039] Further, the elution cavity is provided with a second inlet, and the size of the elution cavity gradually decreases at a position close to the second inlet.

[0040] In some embodiments, the elution cavity is a pentagonal chamber, and the size of the elution cavity decreases at the inlet, which can help to prevent the biochar from flowing out of the elution cavity under the centrifugal force when the sample enters the elution cavity. The elution cavity is used for eluting the nucleic acid to be detected from the biochar, and storing the eluted liquid. The size and volume of the elution cavity can be set according to the specific requirements of the product, and can be fine-tuned within a suitable range.

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

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

[0043] Further, the detection channel is provided with a second exhaust channel extending from the detection channel to the direction of the rotation center, and the exhaust port of the second exhaust channel is connected to the outside, which is used to control the flow of gas in the detection area; and the number of detection cavities is one or more.

[0044] When the number of detection cavities is multiple, the detection of multiple target nucleic acids can be realized at the same time. In some embodiments, the number of detection cavities is seven.

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

[0046] In some embodiments, the fluorescence detection process of the chip is carried out in a slow centrifugation process, in which the chip slowly rotates, so that each detection cavity is aligned with the fluorescence detection channel in turn to complete the fluorescence detection.

[0047] In some embodiments, a line is established from the rotation center to the center of the detection cavity, and the included angle between the lines of two adjacent detection cavities is 7 degrees, which is an integer multiple of the single-step rotation angle (0.7 degrees) of the stepping motor in the centrifugal device, and at this angle, the fluorescence interference between adjacent detection cavities can be avoided, so as to facilitate the calculation of the position of each detection cavity and the recording of the reading results, and to improve the positioning accuracy in the centrifugation and detection processes.

[0048] In some embodiments, the nucleic acid amplification reagent and / or nucleic acid detection reagent needs to be preloaded in the detection cavity.

[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, a spherical, powdery, flaky or massive preparation in the form of lyophilization, drying, air-drying, 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, a spherical, powdery, flaky or massive preparation in the form of lyophilization, drying, air-drying, etc.

[0052] Further, a sample adding area is connected to the quantification cavity through a sample adding channel; the sample adding area, the quantification cavity and the elution cavity are arranged in sequence along the rotation direction of the centrifugal microfluidic chip.

[0053] In some embodiments, the sample adding area is an oval chamber with a long axis of 12±0.5mm and a short axis of 7.5±0.5mm, and a sample adding hole with a diameter of 2mm is arranged on the top of the chamber for adding sample liquid and eluent, and an air hole with a diameter of 2mm is arranged for discharging air in the chamber when liquid is added, balancing air pressure, and the oval structure can utilize as much as possible the area close to the center of the chip, so as to maximize the sample adding volume under the same disc diameter, and effectively improve the space utilization of the chip.

[0054] In some embodiments, the surface of the sample adding area of the chip is covered with a transparent film for sealing. When adding sample, a syringe is used to puncture the transparent film for sample adding, and after the sample fills the main flow channel, the surface of the sample adding hole can be further sealed by covering the transparent film.

[0055] Further, the rotation center is a fixed hole, which can make the centrifugal microfluidic chip clamped on the rotation shaft of the centrifugal driving device, so as to rotate.

[0056] In some embodiments, the fixed hole is used for good fitting of the chip and the driving part of the centrifugal driving device, and the fixed hole is approximately oval, which can combine and fix the chip with the driving part of the centrifugal driving device to provide centrifugal power.

[0057] Further, the detection unit is located on the plastic upper layer.

[0058] The chip provided by the application is a sandwich structure, comprising a plastic upper layer, a silica gel inner film and a plastic lower layer. The sandwich structure is a commonly used processing technology of microfluidic chips, which is composed of three layers. The middle layer is usually soft silica gel with microfluid channels, and the top layer and the bottom layer are usually hard materials such as plastic, glass and polymer materials. This structure can reduce the cost of consumables and optimize the process flow, realizing the dual improvement of cost-effectiveness and technical performance.

[0059] The plastic upper layer is a polydimethylsiloxane (PDMS) disc processed by injection molding, with a thickness of 1.8-2mm and a diameter of 75mm. It has a convex microfluid channel structure downward, in which the capillary part channel width is 0.3mm, and the rest of the microfluid channel width is 0.8-1mm. The two outer sides of the channel have a 5-10° slope and a 0.3-0.5mm wide support structure, which can improve the pressure-bearing performance of the microfluid channel and facilitate demolding. The plastic upper layer includes a fixed hole, a vent hole, a sample addition chamber, a quantitative chamber, an elution chamber, a sample addition channel, a capillary, and a detection chamber.

[0060] The silica gel inner film is a circular silica gel film with a thickness of 0.1mm, which belongs to soft silica gel material. It can fill the small irregular surface under pressure, provide better elasticity and sealing performance, and reduce the leakage rate of liquid. Under normal use and storage conditions, the service life of the silica gel inner film is very long, and it has good aging resistance, heat resistance and chemical resistance, and can be applied to various use 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 mixture of polymethyl methacrylate (PMMA), polycarbonate (PC) and dioctyl sebacate (DOS) (10:10:1) as the base material. The plastic disc processed in this way still has good rigidity even in very thin conditions, which can support and protect the microfluid channel structure of the plastic upper layer, and prevent the soft silica gel inner film from being scratched and damaged. The disc has strong anti-falling and light-transmitting properties, and can maintain excellent light-transmitting rate even under accelerated aging conditions, without affecting the subsequent fluorescence detection in the reaction chamber.

[0062] In some ways, the microfluidic channel of the plastic upper layer is hexahedral, the plastic upper layer provides five sides, and the silica gel and plastic lower layer provides the bottom side to complete the sealing. It can be seen that the microfluidic channel of the plastic upper layer is open and needs to be sealed below. The existing way is to directly assemble the plastic lower layer and the plastic upper layer, which is equivalent to hard-hard structure direct glue sticking or bonding assembly, which is relatively complex to operate, is easy to cause glue to enter the microfluidic channel, and affects sterilization, and because sterilization is completed and then these traditional assemblies are easy to be contaminated again, the gas or liquid for sterilization in the flow channel is difficult to enter or difficult to come out. In order to realize rapid assembly, the hard-soft-hard structure is creatively adopted by clamping to directly seal, the sealing effect is better, and the sterilization is not contaminated again.

[0063] Further, 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 body for fixing and sealing the outer periphery of the centrifugal microfluidic chip; the third buckle and the fourth buckle are assembled into one body for fixing and sealing the inner periphery of the centrifugal microfluidic chip, and the inner periphery is an arc 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 with different sizes, the side design height is 0.8mm groove structure, the total thickness of the chip is 1mm, the acrylic has good toughness and elasticity and is not easy to break or crack, the 0.2mm height difference is used to make the buckle elastic extrusion of the three-layer structure of plastic-silica gel-plastic, and the sealing of the chip is realized.

[0065] The first buckle is a semicircular acrylic arc with a groove on one side, the arc width is 2mm, can be fitted with the outer side of the chip, and the upper and lower ends are provided with female slots for assembling with the second buckle.

[0066] The second buckle is a semicircular acrylic arc with a groove on one side, the size is consistent with the first buckle, and the upper and lower ends are provided with male slots for assembling with the first buckle.

[0067] The third buckle is a semicircular acrylic arc with a groove on one side, the arc width is 2mm, can be fitted with the fixing hole of the chip, and the upper and lower ends are provided with female slots for assembling with the fourth buckle.

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

[0069] The commonly used assembly methods of microfluidic chips include bonding and adhesive bonding. The bonding method has the disadvantages of low alignment accuracy, high waste rate, high cost, etc. and needs to be placed for a long time after preparation, otherwise the bonding strength will be affected. At the same time, the bonding technology needs to be adapted to the material, which greatly limits the flexibility of the actual application of the chip. The adhesive bonding method has the problems of blocking the microfluidic channel by the adhesive, reducing the light transmittance of the chip, and also needs to consider the biological compatibility of the adhesive to the sample to be measured, etc.

[0070] The patent adopts a fast assembly buckle assembly method, which is simple in process, convenient to assemble, low in cost and can obviously improve the production efficiency. The external assembly can completely avoid contacting the sample and biological reagent, and eliminate the adverse effects of potential biochemical reactions. At the same time, the installation buckle does not change the shape and transparency of the chip, and does not need additional polishing treatment, and can be widely used in microfluidic chips of various detection principles.

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

[0072] (1) covering a silica gel mold with a micro channel on plastic;

[0073] (2) injecting an etching liquid into the mold, the etching liquid comprising dimethylformamide, acetone and tetrahydrofuran.

[0074] It has certain technical difficulty to prepare a micro channel structure on plastic, glass and other materials. The traditional method can only prepare a micro channel structure on silica gel material through micro processing technologies such as photolithography and etching, and then bond or adhesive bond with plastic, glass and other materials through oxygen plasma bonding method. SU-8 used in photolithography is sensitive to thermal expansion, and it is difficult to separate from the substrate material, so an anti-sticking layer process needs to be used for corresponding treatment; at the same time, the preparation of SU-8 glue mold involves multiple precise steps, which must be accurately executed according to the process, and the processing requirements are high, the environmental conditions are many, and the processing skills are complex, and the whole process needs a long time; and the SU-8 mold will continuously fall off when preparing silica gel, and the service life is limited. The assembly method of oxygen plasma bonding has high processing precision requirements, and needs to consider the influence of the process on the biochemical reaction in the channel, and the reliability and stability need to be improved.

[0075] The application can successfully prepare a micro channel on plastic by the method of adding an etching liquid in the mold, which has low processing precision requirements and can complete the channel preparation in a very short time.

[0076] In some ways, the etching liquid comprises dimethylformamide (DMF), acetone and tetrahydrofuran in a ratio of 5:4:1.

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

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

[0079] (2) injecting an etching solution into the mold, the etching solution comprising dimethylformamide, acetone and tetrahydrofuran;

[0080] (3) removing the silica mold to form a capillary channel on the plastic upper layer.

[0081] The microfluidic chip with sandwich structure has certain technical requirements in manufacturing. The method for preparing a micro channel on plastic is used to prepare a capillary channel on the plastic upper layer, and the specific method is as follows: covering a silica mold with a capillary channel on the unprocessed area of the plastic upper layer, and positioning the capillary channel on the mold at the area connecting the sample adding channel and the detection area. After 12 μL of mixed etching solution is injected into the mold for 15 s, the etching solution is sucked and recovered by using a needle. The silica mold is removed to form a capillary channel with a width of 0.3 mm and a depth of 0.5 mm on the plastic disc. The silica mold is a silica mold with a capillary channel with a width of 0.3 mm, which is made by using a photolithography technology. The advantage of this processing method is that it can avoid using photolithography and other methods to obtain a capillary channel with a size of less than 1 mm, can control the processing cost and ensure the repeatability of the processing effect.

[0082] In some ways, the etching solution comprises dimethylformamide (DMF), acetone and tetrahydrofuran (5:4:1).

[0083] In another aspect, the present application provides a nucleic acid detection method, which uses the centrifugal microfluidic chip as described above for detection, and the detection method comprises the following steps:

[0084] (1) clamping the fixed hole of the centrifugal microfluidic chip on the rotating shaft of the detection device;

[0085] (2) adding the sample to be tested containing a solid carrier into the centrifugal microfluidic chip from the sample adding area, centrifuging, so that the sample to be tested containing the solid carrier enters the elution cavity of the chip;

[0086] (3) adding the eluent into the centrifugal microfluidic chip from the sample adding area, centrifuging, and stopping the centrifugation when the liquid appears in the waste liquid pool of the centrifugal microfluidic chip, which indicates that the eluent has entered the quantitative cavity;

[0087] (4) centrifuging again, so that the eluent enters the elution cavity and mixes with the sample to be tested containing the solid carrier, and the elution of the nucleic acid is completed;

[0088] (5) stopping centrifugation, and the liquid containing nucleic acid enters the detection chamber through capillary, and nucleic acid amplification is carried out;

[0089] (6) reading the detection result.

[0090] In some modes, in step (5), the nucleic acid amplification is isothermal amplification, and a heating device is needed to provide a suitable temperature (such as 30-65 DEG C) required for isothermal amplification to the reaction chamber.

[0091] Further, the eluent includes Tris-HCl buffer and PBS.

[0092] In some modes, the eluent is 200 muL 10mM Tris-HCl buffer (pH 7.5) + 30 muL 0.02M PBS

[0093] The present application screens the most suitable eluent formula, which can improve the elution effect of nucleic acid in biochar, and since the eluent is a low-concentration aqueous solution, the chemical equilibrium in the elution chamber can be broken after adding the chip. Because the first sample addition is a sample containing a solid carrier, and the second sample addition is an aqueous eluent, the concentration is changed, and the sample is more easily eluted.

[0094] The chip provided by the present application is based on automatic elution technology, and the solid carrier (such as biochar) is extracted and integrated in a single chip, without additional liquid transfer steps, reducing the risk of aerosol pollution and human error, and constructing a rapid, convenient and highly integrated detection platform. At the same time, the chip is provided with four independent regions, which can simultaneously detect 4*7 target nucleic acid molecules, thereby reducing the use amount of samples and reagents and saving detection cost.

[0095] The centrifugal microfluidic chip provided by the present application has the following beneficial effects:

[0096] 1. The nucleic acid can be eluted from the solid carrier and the whole process of amplification and detection can be completed, which is suitable for various solid carriers such as biochar, nucleic acid, etc.

[0097] 2. By respectively setting the quantification chamber and the elution chamber, not only the quantification effect can be achieved, but also the mixing and elution can be more sufficient, the elution efficiency is improved, and the detection sensitivity is improved.

[0098] 3. By designing the shape and structure of various chambers in the chip, the solid carrier such as biochar can be smoothly eluted, and the eluted sample to be detected can be smoothly detected in the detection area, while the biochar is blocked in the elution chamber and does not block or affect the detection of the sample to be detected.

[0099] 4. By improving the structure of the fluid channel in the chip, a blocking block is set, so that the solid phase carrier such as biochar is effectively blocked, avoiding the blockage of the capillary channel, and the step above the blocking block makes the liquid surface near the 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.

[0100] 5. The etching of 0.3mm level flow channel on the plastic disc is realized by combining the etching liquid and the mask, which effectively reduces the processing cost and difficulty, and the flow channel preparation can be completed in a very short time.

[0101] 6. The rapid assembly of the chip is realized by the buckle, which is simple in process, convenient in assembly and low in cost, and can significantly improve the production efficiency, completely avoid the contact with the sample and the biological reagent, eliminate the adverse effects of potential biochemical reactions, and at the same time, the installation of the buckle does not change the shape and transparency of the chip, without the need for additional polishing treatment, which can be widely used in microfluidic chips of various detection principles.

[0102] 7. Based on the automatic elution technology, the solid phase carrier (such as biochar) is extracted and integrated in a single chip, without the need for additional liquid transfer steps, reducing the risk of aerosol pollution and human error, and constructing a rapid, convenient and highly integrated detection platform; at the same time, the chip is provided with four independent areas, which can simultaneously detect 4*7 target nucleic acid molecules, thereby reducing the use amount of sample and reagent and saving the detection cost. BRIEF DESCRIPTION OF DRAWINGS

[0103] Figure 1 It is a schematic diagram of the flow channel structure of the centrifugal microfluidic chip in embodiment 2.

[0104] Figure 2 It is a schematic diagram of the structure of the quantitative cavity, elution cavity and fluid channel in embodiment 2.

[0105] Figure 3 It is an exploded view of the centrifugal microfluidic chip in embodiment 2.

[0106] Figure 4 It is a flow chart of preparing the capillary channel on the plastic upper layer by combining the etching liquid and the mask in embodiment 3.

[0107] Figure 5 It is a schematic diagram of the liquid flow direction in the centrifugal microfluidic chip in embodiment 4. DETAILED DESCRIPTION

[0108] The preferred embodiments of the present application will be further described in detail below with reference to the accompanying drawings, and it should be pointed out that the following embodiments are intended to facilitate the understanding of the present application and do not have any limiting effect on the present application. The raw materials and equipment used in the specific embodiments of the present application are known products, which can be obtained by purchasing commercially available products.

[0109] In the description of the present application, it needs to be understood that the terms "upper", "lower", "front", "back", "left", "right", "vertical", "horizontal", "top", "bottom", "inner", "outer" and the like indicate the orientation or positional relationship based on the orientation or positional relationship shown in the drawings, and are only for the convenience of describing the present application and simplifying the description, and do not indicate or imply that the devices or elements referred to must have a particular orientation, be constructed and operated in a particular orientation, and therefore cannot be understood as a limitation on the present application. In addition, the terms "first", "second" and the like are only for descriptive purposes and cannot be understood as indicating or implying relative importance or implicitly indicating the number of the technical features indicated. Therefore, the features defined with "first", "second" and the like can be explicitly or implicitly included one or more. In the description of the present application, unless otherwise specified, the meaning of "a plurality of" is two or more.

[0110] In the description of the present application, it needs to be understood that unless otherwise explicitly specified and limited, the terms "connected", "connected" should be understood broadly, for example, it can be fixedly connected, or it can be detachably connected, or integrally connected; it can be mechanically connected, or it can be electrically connected; it can be directly connected, or it can be indirectly connected through an intermediate medium, or it can be connected inside two elements. For those skilled in the art, the specific meaning of the above terms in the present application can be understood through specific circumstances.

[0111] Example 1, preparation of biochar

[0112] The biochar in this embodiment is prepared by taking rice straw and corn cob as carbon source, and the preparation process is as follows: rice straw and corn cob are mixed in a weight ratio of 1:2 to 1:3 (preferably 1:2 in this embodiment), washed with tap water, dried in a blast drying oven at 60℃, then the rice straw is cut to a length of 2-3cm, and the corn cob and rice straw are placed in an anaerobic tube furnace under high-purity N2 atmosphere for high-temperature pyrolysis, heated to 700℃ at a heating rate of 5℃ / min, and carbonized for 2h. After pyrolysis and cooling, the mixture is taken out, ground and sieved through a 100 mesh sieve.

[0113] Example 2, centrifugal microfluidic chip provided by the present application

[0114] The structure of the centrifugal microfluidic chip provided by the present application is shown in Figures 1-3 Figure 1 is a schematic diagram of the flow channel structure of the centrifugal microfluidic chip, Figure 2 is a schematic diagram of the structure of the quantitative cavity, elution cavity and fluid channel; Figure 3 is an exploded view of the centrifugal microfluidic chip.

[0115] As shown in Figure 1 ​The centrifugal microfluidic chip 1 (hereinafter referred to as chip 1) provided in the embodiment comprises a detection unit 2, and the detection unit 2 is provided with a quantitative cavity 3, an elution cavity 4 and a detection area 5; the elution cavity 4 is used for eluting the target to be detected from a solid carrier; the quantitative cavity 3 is used for temporarily storing the added liquid and quantifying the added liquid. The quantitative cavity 3 and the elution cavity 4 are connected through a fluid channel 6; the detection area 5 is used for detecting the content of the target to be detected, and the detection area 5 is connected with the fluid channel 6 through a capillary channel 7. The chip 1 further comprises a rotation center 8, which is used for connecting with a centrifugal driving device to drive the chip 1 to centrifuge. Preferably, a plurality of groups of detection units 2 can be provided in the chip 1, each group of detection units 2 is provided with a quantitative cavity 3, an elution cavity 4 and a detection area 5, which can be suitable for simultaneous detection of a plurality of different target to be detected, such as detection of a plurality of different nucleic acid targets, and also suitable for eluting nucleic acid targets from a plurality of different solid carriers for detection. In the embodiment, four groups of detection units 2 are provided in the chip 1 and are uniformly arranged around the rotation center 8, each detection unit 2 can be used for detecting one sample, so that the detection throughput of the chip 1 is higher. The quantitative cavity 3 and the elution cavity 4 are connected through a fluid channel 6, which is a channel connected between the outlet (first outlet 9) of the quantitative cavity 3 and the inlet (second inlet 10) of the elution cavity 4.

[0116] Preferably, the solid carrier comprises one or more of biochar, magnetic beads, gel, resin and cellulose; and the target to be detected is nucleic acid. In the embodiment, the solid carrier is the biochar provided in the embodiment 1. The biochar has the characteristics of low cost and easy availability of raw materials, and when the biochar is used as a solid carrier for nucleic acid extraction and detection, the cost can be significantly reduced compared with magnetic beads. However, the density of the biochar is smaller than that of the magnetic beads, and the biochar is more loose, so that it is more difficult to separate the biochar from the target sample after elution than the 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 successfully complete the elution, and the target sample after elution can smoothly enter the detection area 5 for detection, while the biochar is blocked in the elution cavity 4 and does not block or affect the detection of the target sample.

[0117] As Figure 1, the capillary channel 7 comprises a first section 11 and a second section 12; the first section 11 is a channel that is curved from the liquid outlet 13 of the fluid channel 6 to the rotation center 8, and the second section 11 is a channel that is curved 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 force is outward from the center. The first section 11 of the capillary channel 7 is arranged such that, if the sample enters the first section 11, the flow direction of the sample is opposite to the centrifugal direction, so that the sample cannot enter the capillary channel 7 during centrifugation. The arrangement 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 it strengthens the protection against the charcoal entering. First, it ensures that the sample cannot enter the capillary channel during the centrifugal mixing and elution process, so the charcoal is also less likely to block the capillary channel 11. Second, during the stop of the centrifugation process, the supernatant sample can penetrate into the capillary channel 7 under the action of surface tension, while the charcoal 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 24 mm, the width is 0.3 mm, and the depth is 0.5 mm.

[0118] As Figure 2 , a blocking block 15 is arranged below the fluid channel 6, so that at least part of the lower part of the quantitative chamber 3 or the elution chamber 4 is blocked. A step 16 is also arranged on the fluid channel 6, which is arranged on one 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 arrangement of the blocking block 15, the lower part of the fluid channel 6 cannot realize liquid flow, which is equivalent to the quantitative chamber 3 and the elution chamber 4 only having the upper chamber capable of fluid communication, and the lower part is blocked. Such an arrangement also helps to block the charcoal from entering or blocking the capillary channel, because the charcoal will basically sink to the lower part of the elution chamber 4 under the action of the centrifugal force. The step 16 is located above the blocking block 15 and close to one 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 a residual liquid surface, thereby raising the liquid level at this position and 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 chamber 3 and the elution chamber 4 is 4.5-5.5 mm, and the height of the fluid channel 6 is reduced to 1.0-1.5 mm due to the blocking of the blocking block 15. The height of the step 16 is 0.5-0.7 mm, and the width is 0.4-0.6 mm.

[0119] The quantitative cavity 3 is provided with a first inlet 17 and a first outlet 9, and the size of the quantitative cavity 3 gradually decreases at a position close to the first outlet 9. The quantitative cavity 3 is a chamber with a shape of an inverted pentagon, and the overall shape is similar to that of a funnel. The shape of the quantitative cavity 3 helps the sample containing the biochar to quickly gather and enter the elution cavity 4 under the action of centrifugal force after entering, and helps the sample containing the biochar to stably stay in the elution cavity 4 after entering the elution cavity 4 and not easily backflow, that is, helps to block the backflow of the biochar. The quantitative cavity 3 is used for quantitatively storing the added liquid, and the volume of the quantitative cavity 3 can be adjusted according to specific purposes and sample amounts. In this embodiment, the width of the quantitative cavity 3 is 6.25±0.5 mm, the length is 4.8±0.5 mm, and the volume is 200±30 μL.

[0120] Preferably, the quantitative cavity 3 is further provided with an exhaust passage 18 and a waste liquid passage 19; the exhaust passage 18 extends from the quantitative cavity 3 to the direction of the rotation center 8, the exhaust port 20 of the exhaust passage 18 is connected with the outside, and is used for controlling the flow of gas in the quantitative cavity 3 and / or the elution cavity 4; the waste liquid passage 19 extends from the quantitative cavity 3 to the direction away from the rotation center 8, and is connected with the waste liquid pool 21.

[0121] When the liquid enters the chip 1 from the sample adding area 22 and enters the corresponding cavity under the action of centrifugal force, the gas in the cavity needs to be discharged from the exhaust passage 18. The quantitative cavity 3 and the elution cavity 4 share one exhaust passage 18, so that the preparation process is more simple. The direction of the exhaust passage 18 is opposite to the centrifugal direction, so when the liquid enters the quantitative cavity 3 or the elution cavity 4 under the action of centrifugal force, the liquid will not be discharged from the exhaust passage 18, but only when the elution cavity 4 and the quantitative cavity 3 are both filled, the liquid will enter the exhaust passage 18. The exhaust passage 18 and the waste liquid passage 19 share one outlet 23 and one end shared passage 24 on the quantitative cavity 3, the shared passage 24 is provided with branches, a first branch opposite to the centrifugal direction and leading to the exhaust port 20 for the exhaust passage 18, and a second branch same as the centrifugal direction and leading to the waste liquid pool 21 for the waste liquid passage 19. Therefore, the liquid will choose to enter the waste liquid pool 21 under the action of centrifugal force. In actual use, the chip 1 is placed in a detection device, and it is difficult to see whether the sample in the chip 1 has entered the quantitative cavity 3 through the device, but it can be seen through the window of the device whether the liquid enters the waste liquid pool 21, and when it is seen that the liquid begins to enter the waste liquid pool 21, it can be indicated that the quantitative cavity 3 has been filled. It can be seen that the waste liquid pool 21 also has the effect of helping the liquid in the quantitative cavity 3 to be quantified.

[0122] 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 avoid bubbles from blocking the micro flow channel and affecting the liquid flow. The surface of the exhaust port 20 is pre-covered with a transparent film to play a sealing role. During use, when exhaust is needed, the transparent film can be punctured to exhaust; if subsequent sealing is needed, the transparent film can also be re-lidded to seal.

[0123] The elution cavity 4 is provided with a second inlet 10, and the size of the elution cavity 4 gradually decreases at a position close to the second inlet 10. The elution cavity 4 is a pentagonal chamber, and the second inlet 10 is reduced in size, which can help to block the biochar from flowing out of the elution cavity 4 under the action of centrifugation. The elution cavity 4 is used to elute the nucleic acid to be tested from the biochar and store the eluted liquid at the same time. The size and volume of the elution cavity 4 can be set according to the specific needs of the product, and can be fine-tuned within a suitable range. In this embodiment, the width of the elution cavity 4 is 5.8±0.5 mm, the length is 4.8±0.5 mm, and the volume is 230±30 μL.

[0124] 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. The detection channel 25 is provided with a second exhaust channel 27 extending from the detection channel 25 to the direction of the rotation center 8, and a second exhaust port 28 of the second exhaust channel 27 is connected to the outside, which is used to control the flow of gas in the detection area 5; the number of detection cavities 26 is one or more. When the number of detection cavities 26 is multiple, multiple nucleic acid detections of different purposes can be realized at the same time, which can be set according to the needs of the product. In this embodiment, the number of detection cavities 26 in each detection area 5 is seven, which can detect seven kinds of nucleic acids of different purposes for the same sample.

[0125] As Figure 1 , the detection channel 25 is an arc-shaped channel arranged along the circumference, and the detection cavity 26 is a circular room with a diameter of 1.5-2.5 mm, which can match the spot size of the fluorescence detection module in the detection equipment, and the diameter is slightly larger than the spot diameter and not more than 0.8 mm larger than the spot diameter, and the depth is 0.2-0.5 mm, which can control the volume of the reaction, adapt to various reaction systems, and expand the application scenarios and detection objects. The length of the detection channel 25 can be adjusted according to the size of the product. In this embodiment, the length of the detection channel 25 is 25 mm.

[0126] The fluorescence detection process of the chip 1 is completed in a slow centrifugal process, in which the chip 1 is slowly rotated so that each detection cavity 26 is sequentially aligned with the fluorescence detection channel. In order to enable each detection result to correspond to each detection cavity 26, the angle of the position of each detection cavity 26 needs to be determined before detection. In the embodiment, a line is established from the rotation center to the center 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-step motor rotation degree (0.7 degrees) in the centrifugal equipment, and the angle can avoid the fluorescence interference of adjacent detection cavities 26, thereby facilitating the calculation of the position of each detection cavity 26 and the recording of the reading result, and being beneficial to improving the positioning accuracy in the centrifugal and detection processes.

[0127] The nucleic acid amplification reagent and / or nucleic acid detection reagent need to be preloaded in the detection cavity 26. 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, powdery, flaky or block-shaped preparations in the forms of freeze-drying, oven-drying, air-drying, etc. The nucleic acid detection includes but is not limited to various 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, powdery, flaky or block-shaped preparations in the forms of freeze-drying, oven-drying, air-drying, etc.

[0128] As Figure 1 The detection unit 2 of the chip 1 further includes a sample adding area 22 connected with the quantification cavity 3 through a sample adding channel 30; the sample adding area 22, the quantification cavity 3 and the elution cavity 4 are arranged in sequence along the rotation and centrifugal direction of the chip 1. Preferably, the sample adding area 22 is an oval chamber with a long axis of 12±0.5mm and a short axis of 7.5±0.5mm, and has a sample adding hole 29 with a diameter of 2mm for adding sample liquid and eluent, and an air hole with a diameter of 2mm for discharging air in the chamber during liquid adding to balance the air pressure. The oval structure can utilize the area of the part close to the center of the chip 1 as much as possible to maximize the sample adding volume under the same disc diameter, thereby effectively improving the space utilization of the chip 1. Preferably, the surface of the sample adding area 22 of the chip 1 is covered with a transparent film for sealing. During sample adding, the transparent film is punctured by a syringe, and after the sample fills the main flow channel, the surface of the sample adding hole 29 can be further sealed by the transparent film.

[0129] The rotating center 8 is a fixed hole 32, which can make the chip 1 clamped on the rotating shaft of the centrifugal driving device, so as to carry out centrifugal rotation. Preferably, the fixed hole 32 is approximately elliptical, the long axis is 13 mm, and the short axis is 8 mm, which can combine and fix the chip with the driving part of the centrifugal driving device, and provide centrifugal power.

[0130] As Figure 3 , the chip 1 is a sandwich structure, including a plastic upper layer 33, a silica gel inner film 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 1.8-2 mm and a diameter of 75 mm, and has a convex micro-channel structure downward, wherein the width of the capillary channel 7 of the plastic upper layer 33 is 0.3 mm, and the widths of the rest of the micro-channels are all 0.8-1 mm, the two outer sides of the channel are provided with a slope of 5-10 degrees and a support structure with a width of 0.3-0.5 mm, which is used to improve the pressure-bearing performance of the micro-channel, and is also conducive to the demolding of injection molding. The plastic upper layer 33 is provided with a first fixed 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 silica gel inner film 34 is a circular silica gel film with a thickness of 0.1 mm, which belongs to soft silica gel material and can fill the small irregular surface under the action of pressure, providing better elasticity and sealing performance, and reducing the leakage rate of liquid. The silica gel inner film 34 is provided with a second fixed hole 232. Under normal use and storage conditions, the service life of the silica gel inner film 34 is extremely long, and it has good aging resistance, heat resistance and chemical resistance, and can be applied to various use scenarios. The plastic lower layer 35 is a plastic disc with a fixed hole, with a thickness of 0.1-0.2 mm and provided with a third fixed hole 332, and the plastic disc is processed by injection molding with a mixed material (10:10:1) of polymethyl methacrylate (PMMA), polycarbonate (PC) and dioctyl sebacate (DOS) as the base, which still has good rigidity in the case of extremely thin, and can support and protect the micro-channel structure of the plastic upper layer 33, while preventing the soft silica gel inner film 34 from being scratched and damaged. The disc has strong anti-falling and light-transmitting properties, and can maintain excellent light transmittance even under accelerated aging conditions, without affecting the subsequent fluorescence detection in the reaction chamber.

[0131] As Figure 3 , the chip 1 further 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 and 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 and used to fix the inner periphery 42 of the sealed chip 1, and the inner periphery 42 is an arc-shaped hole where the fixed hole 32 (including the first fixed hole 132, the second fixed hole 232 and the third fixed hole 332 combined together) of the chip 1 is located.

[0132] The first, second, third and fourth buckles 37, 38, 39 and 40 are two groups of half-round acrylic buckles with different sizes, and the side design height is a 0.8mm groove 43 structure. The total thickness of the chip 1 is 1mm, the acrylic has good toughness and elasticity, and is not easy to break or crack. The height difference of 0.2mm makes the buckle elastic extrusion of the three-layer structure of plastic-silica gel-plastic, and realizes the sealing of the chip 1. As shown in Figure 3 , the first buckle 37 is a half-round acrylic arc with a groove 43 on one side, with an arc width of 2mm, which can be fitted with the outer periphery 41 of the chip 1. The upper and lower ends are provided with female grooves 44 for assembling with the second buckle 38. The second buckle 38 is a half-round acrylic arc with a groove 43 on one side, which is the same size as the first buckle 37. The upper and lower ends are provided with male grooves 45 for assembling with the first buckle 37. The third buckle 39 is a half-round acrylic arc with a groove 43 on one side, with an arc width of 2mm, which can be fitted with the fixing hole 32 of the chip 1. The upper and lower ends are provided with female grooves 44 for assembling with the fourth buckle 40. The fourth buckle 40 is a half-round acrylic arc with a groove 43 on one side, which is the same size as the third buckle 39. The upper and lower ends are provided with male grooves 45 for assembling with the third buckle 39.

[0133] Example 3, preparation of a centrifugal microfluidic chip

[0134] The preparation method of the centrifugal microfluidic chip 1 provided in this embodiment specifically includes the following steps:

[0135] 1. The plastic upper layer 33 is prepared by an injection molding integrated molding method.

[0136] 2. The capillary channel 7 is prepared on the plastic upper layer 33 by combining the etching liquid and the mask, and the preparation process is shown in Figure 4 :

[0137] (1) The silica gel mold 45 with the hollow capillary 46 is covered on the unprocessed area of the plastic upper layer 33 of the chip 1, so that the hollow capillary 46 on the silica gel mold 45 is positioned at the area connecting the fluid channel 6 and the detection channel 25.

[0138] The silica gel mold 45 is made of photolithography technology and has a 0.3mm wide hollow capillary 46 on it.

[0139] (2) 12μL of etching liquid 47 is injected into the hollow capillary 46 of the silica gel mold 45, and after 15s, the etching liquid 47 is sucked and recovered with a needle;

[0140] The etching liquid 47 is a solution prepared by using pure water to prepare a solution containing 5% dimethylformamide, 4% acetone and 1% tetrahydrofuran.

[0141] (3) Remove the silicone mold to form a capillary channel 7 with a width of 0.3 mm and a depth of 0.5 mm on the plastic upper layer 33.

[0142] 3. Assemble the chip in the order of "plastic upper layer 33-silicone inner film 34-plastic lower layer 35", and press the chip from the outside using the first and second buckles 37 and 38 and from the inside using the third and fourth buckles 39 and 40 after alignment to complete the packaging of the chip.

[0143] Example 4, method for using a centrifugal microfluidic chip

[0144] The sample is detected using the centrifugal microfluidic chip, and the detection method is as follows (the flow direction of the sample in the chip 1 is shown in Figure 5

[0145] (1) Take the chip 1 prepared in Example 3, and prick the exhaust hole 20 and the sample injection hole 31 of the plastic upper layer 33. The fixing hole 32 of the chip 1 is clamped on the centrifugal rotating shaft of the detection device.

[0146] (2) Prepare the sample to be detected containing a solid-phase carrier (such as biological charcoal or magnetic beads), inject 200 μL of the sample to be detected from the sample injection hole 31 using a syringe, and start the centrifugal program (1200 r / min, 50 s) to make the sample to be detected enter the elution cavity 4 of the chip.

[0147] (3) Inject 230 μL of eluent from the sample injection hole 31 using a syringe, and start the centrifugal program (1200 r / min, 30 s). When the liquid appears in the waste pool 21, stop the centrifugation, which indicates that the eluent fills the quantitative cavity 3.

[0148] (4) Start the centrifugal program (1200 r / min, 2 min), and the eluent enters the elution cavity 4 to mix with the sample to be detected, elute the lysate bound to the solid-phase carrier, including proteins, nucleic acids, and bacterial fragments, among which the nucleic acid molecules with the lightest mass float in the supernatant, and the elution of the nucleic acid is completed.

[0149] (5) Stop the centrifugation, and the sample containing the nucleic acid enters the detection area 5 through the capillary channel 7, while the solid-phase carrier is blocked in the elution cavity 4; start the centrifugal program again (1200 r / min, 1 min), and the sample is evenly distributed into each detection cavity 26 from the detection channel 25, and the isothermal amplification of the nucleic acid is carried out (for isothermal amplification, the detection device needs to provide a suitable temperature (such as 30-65°C) required for isothermal amplification for each detection cavity 26); the detection device starts the centrifugal program "high speed 1200 r / min 45 s; low speed 200 r / min 15 s alternately", and detects the amplification product of the sample in each detection cavity 26 in turn to obtain the real-time fluorescence detection curve, and the whole detection process is completed. ​

[0150] Example 5, Screening of carbon source for preparing biochar

[0151] In this example, biochar was prepared according to the method provided in Example 1, wherein the carbon source for preparing biochar was selected from five groups of carbon sources as shown in Table 1, respectively, to prepare five groups of biochar, and then the nucleic acid extraction of E. coli liquid was carried out using the five groups of biochar, respectively. The specific method was as follows: 300 μL of liquid sample obtained by sampling was added to a centrifuge tube, 1 mL of biochar (10 mg / mL) was added, and the sample was mixed uniformly on a vortex shaker after thermal cracking at 100°C for 5 min. The centrifuge tube was left to stand for 7-10 min, and after the lysate was completely absorbed by the biochar, the supernatant was removed, and the washing was repeated for 3 times. 1 mL of PBST buffer was added to the precipitate, and 1 mL of sterile PBST buffer (100 mL of 0.01M PBS, 50 μL of Tween 20, pH 7.4) was gently shaken to obtain a sample solution containing biochar. Then the elution of the five groups of sample solutions containing biochar was carried out, respectively, and the eluent was 200 μL of 10 mM Tris-HCl buffer (pH 7.5) + 30 μL of 0.02M PBS. The nucleic acid extraction yield of each group of biochar was investigated and compared with that of magnetic beads, and the results are shown in Table 1.

[0152] Table 1, Nucleic acid extraction effect of different biochar

[0153] Solid support Carbon source Yield of nucleic acid extraction (%) Biochar 1 2% rice straw 53.2 Biochar 2 6% corn cob 41.0 Biochar 3 2% rice straw + 6% corn cob 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

[0154] According to Table 1, the nucleic acid extraction yield of biochar prepared from different carbon sources is quite different, and therefore the fifth group of biochar is the most preferred, which is prepared from rice straw and corn cob as carbon sources.

[0155] Example 6, Screening of eluent

[0156] In this example, biochar was prepared according to the method provided in Example 1, and the sample solution containing biochar was prepared according to the method provided in Example 4 using the biochar. The elution was carried out using the eluent as shown in Table 2, respectively, to investigate the effect of different eluent formulations on the nucleic acid extraction yield of biochar, and the detection results are shown in Table 2.

[0157] Table 2, Effect of different eluents on nucleic acid extraction of biochar

[0158] Eluent Yield of nucleic acid extraction (%) 200 μL 10 mM Tris-HCl buffer 96.4 0.02 M PBS 95.3 200 μL 10 mM TE buffer 97.9 200 μL 10 mM Tris-HCl buffer (pH 7.5) + 30 μL 0.02 M PBS 99.1

[0159] According to Table 2, for the biochar prepared in Example 1, the most preferred eluent is 200 μL of 10 mM Tris-HCl buffer (pH 7.5) + 30 μL of 0.02M PBS, which can effectively improve the elution efficiency and thus improve the nucleic acid extraction yield of the sample.

[0160] Example 6, screening of etching solution

[0161] This example uses the method provided in Example 3 to prepare capillary channels on the upper layer of plastic, where the etching solution is respectively used in different formulations shown in Table 3. The effects of different etching solutions in preparing capillary channels are compared, and the method is as follows: add etching solution to silica gel molds with hollow capillary channels with widths of 0.3, 0.8, and 1.3 μm, respectively, and investigate whether different etching solutions can successfully prepare capillary channels with corresponding widths (without problems such as uneven width of capillary channels and excessive corrosion by etching solution), i.e., the precision of capillary channel preparation using the etching solution. The results are shown in Table 3.

[0162] Table 3, comparison of effects of different etching solutions

[0163] Etching solution Precision of capillary channel preparation 5% dimethylformamide + 4% acetone + 1% tetrahydrofuran 0.3 μm 10% acetone solution 0.8 μm Concentrated nitric acid mixed with concentrated hydrochloric acid at a ratio of 1:3 1.3 μm

[0164] As can be seen from Table 3, the precision of preparing capillary channels on the upper layer of plastic using different etching solutions is quite different, and the most preferred etching solution formulation is: 5% dimethylformamide + 4% acetone + 1% tetrahydrofuran, which has the highest precision when used to prepare capillary channels.

[0165] Example 7, verification of detection effect of centrifugal microfluidic chip

[0166] This example uses the centrifugal microfluidic chip provided in Example 2 to perform nucleic acid detection, uses ERA-CRISPR detection technology (enzyme-mediated recombination amplification combined with CRISPR detection technology) to detect respiratory pathogen nucleic acids (influenza A virus, influenza B virus, respiratory syncytial virus, adenovirus, human rhinovirus, Mycoplasma pneumoniae, human metapneumovirus, parainfluenza virus) in blood samples suspected of respiratory tract infection, and uses qPCR detection method (commercial reagent) for comparison and verification. Add 300 μL of liquid sample obtained by sampling to the centrifugal tube, add 1 mL of nano-sized biochar (10 mg / mL, prepared in Example 1) to it, heat lyse at 100°C for 5 min in a metal bath, then mix uniformly on a vortex shaker, let the centrifugal tube stand for 7-10 min, remove the supernatant after the lysis product is completely adsorbed by the biochar, repeat the washing for 3 times, add 1 mL of PBST buffer 1 mL of sterile PBST buffer (100 mL of 0.01M PBS, 50 μL of Tween 20, pH 7.4) to the precipitate and gently shake to obtain a sample solution containing biochar. Use the method provided in Example 6 to perform nucleic acid detection, and pre-load the detection chamber with ERA freeze-dried microspheres containing specific amplification primers. The results are shown in Table 1.

[0167] Table 1, list of performance verification of centrifugal microfluidic chip detection

[0168]

[0169]

[0170] From Table 1, under the same 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 fast to use, and the comprehensive use cost is lower.

[0171] The technical features of the above-described embodiments can be combined arbitrarily, and to make the description concise, all possible combinations of the technical features in the above-described embodiments are not described, however, as long as the combinations of the technical features do not exist contradictions, they should be considered as the scope of the present disclosure.

[0172] Although the present disclosure is as described above, the present disclosure is not limited thereto. It can be extended according to its application scope in the microfluidic field. Any person skilled in the art can make various changes and modifications without departing from the spirit and scope of the present disclosure, and therefore the protection scope of the present disclosure should be limited by the scope defined by the claims.

[0173] The content of the articles, patents, patent applications, and all other documents and electronically available information described or identified in this document are hereby incorporated by reference in their entirety as if each individual publication were specifically and individually indicated to be incorporated by reference. Applicants reserve the right to claim priority to any and all currently pending patent applications that have been filed by Applicants and / or their assignees that are related to the present application.

Claims

1. A centrifugal microfluidic chip, characterized in that, The system includes a detection unit, which is equipped with a quantitative chamber, an elution chamber, and a detection area. The elution chamber is used to elute the analyte from the solid support; The metering chamber is used to temporarily store the added liquid and to quantify the added liquid, the liquid including the eluent; The metering chamber and the elution chamber are connected by a fluid channel; The detection zone is used to detect the content of the analyte, and the detection zone is connected to the fluid channel through a capillary channel.

2. The centrifugal microfluidic chip as described in claim 1, characterized in that, The solid support includes one or more of biochar, magnetic beads, gel, resin, and cellulose; the analyte includes nucleic acid.

3. The centrifugal microfluidic chip as described in claim 2, characterized in that, The solid support is biochar.

4. The centrifugal microfluidic chip as described in claim 1, characterized in that, It also includes a rotation center for connecting to a centrifugal drive device to drive the centrifugal microfluidic chip to centrifuge; the number of detection units is one or more, arranged around the rotation center.

5. The centrifugal microfluidic chip as described in claim 4, characterized in that, The capillary channel includes a first section and a second section; the first section is a channel that bends from the outlet of the fluid channel toward the center of rotation, and the second section is a channel that bends from the end of the first section toward the detection chamber.

6. The centrifugal microfluidic chip as described in claim 5, characterized in that, A blocking block is provided below the fluid channel to at least partially block the area below the metering chamber or elution chamber; a step is also provided on the fluid channel, the step being located on the side of the fluid channel near the outlet leading to the capillary channel.

7. The centrifugal microfluidic chip as described in claim 5, characterized in that, The metering cavity has a first inlet and a first outlet, and the size of the metering 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 centrifugal microfluidic chip as described in claim 7, characterized in that, The metering chamber is also provided with an exhaust channel and a waste liquid discharge channel; the exhaust channel extends from the metering chamber toward the rotation center, and the exhaust port of the exhaust channel is connected to the outside, which is used to control the flow of gas in the metering chamber and / or the elution chamber; the waste liquid discharge channel extends from the metering chamber away from the rotation center and is connected to the waste liquid pool.

9. The centrifugal microfluidic chip as described in 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 a capillary channel.

10. The centrifugal microfluidic chip as described in 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 and is used to control the flow of gas in the detection area. The number of detection chambers is one or more.

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