Centrifugal micro-fluidic chip as well as driving method and application thereof

Abstract

The invention discloses a centrifugal micro-fluidic chip as well as a driving method and application thereof, and belongs to the technical field of biomedical treatment. The chip comprises a chip upper layer and a chip lower layer. The chip lower layer comprises a disc body which has a predetermined thickness and is provided with a lower layer centrifugal shaft hole in the center. A sample bin, a washing liquid bin and an eluent bin are arranged on the circumference of the lower-layer centrifugal shaft hole in an array manner. The front end of the extraction channel is connected with a sample bin, the rear end of the extraction channel is connected with a Coriolis valve, the radius of the extraction channel is gradually increased from front to back, and the inner wall of the extraction channel is modified by double-network hydrogel for intercepting and adsorbing nucleic acid. Input ends of the first channel and the second channel are respectively connected with the washing liquid bin and the eluent bin, and output ends of the first channel and the second channel converge at the front end of the extraction channel. The chip is further provided with a waste liquid bin, a collection bin and a plurality of amplification detection areas which are annularly and uniformly distributed on the edge, the amplification detection areas are connected with the collection bin through distribution channels and are all used for quantitative detection, and the chip provides a convenient and efficient microfluidic technology platform for related applications such as on-site instant detection of nucleic acid and the like.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and in particular relates to a centrifugal microfluidic chip and a driving method and application thereof. Background Art

[0002] Rapid on-site nucleic acid detection is fast, does not require cumbersome sample pre-treatment steps, and is not limited to PCR Laboratory, so it has attracted wide attention in scientific research and industrialization.

[0003] In the prior art, Cepei GeneXpert The cartridge design uses ultrasound to lyse cells and release nucleic acids. Liquid drive is achieved through the precise suction movement of the piston and the clever cooperation of the rotary valve. The reagents are pre-stored in the chip cartridge using freeze-drying technology. GeneXpert It plays a huge role in the detection of infectious disease pathogens, such as Mycobacterium tuberculosis and the new coronavirus. GeneXpert The modular design allows for stacking of modules to achieve multi-channel testing. However, the cartridge design has a high manufacturing cost, as the liquid drive is complex, the internal parts must be tightly sealed and there must be no leakage.

[0004] BioMérieux FilmArray Multiple PCR The system also acts as a molecule POCT The classic representative of the product, which automatically completes the entire nucleic acid analysis process in a fully sealed chip, can be up to 1 h It simultaneously detects 24 bloodstream infection-related pathogen targets and 3 antibiotic resistance genes. FilmArray It is a pneumatic extrusion drive method, which uses flexible bags to design various reaction pools, inflates and squeezes the flexible bags to drive liquid movement, and sets extrusion valves to intercept the liquid. However, the flexible bag design is prone to the risk of material rupture, causing laboratory contamination.

[0005] Abbott's IDNOW The isothermal amplification method is used, and a positive result can be obtained in as fast as 5 minutes. However, the anti-interference ability is not good, and false positive results or inhibition of amplification reactions are prone to occur.

[0006] Currently, centrifugal microfluidics is widely used in the field of biochemical testing, mostly in basic medical institutions. However, due to the complexity of chip design, the products of centrifugal microfluidics used in molecular diagnosis are relatively immature. POCT It is mainly used for the detection of infectious pathogens, and is less used in oncology. For example, the detection of drug-resistant genes. Rapid real-time detection of drug-resistant genes is conducive to timely changing treatment plans and the construction of outpatient chemotherapy. Summary of the invention

[0007] In order to solve the technical problems existing in the above-mentioned background technology, the present invention provides a centrifugal microfluidic chip and a driving method and application thereof.

[0008] The present invention adopts the following technical scheme: a centrifugal microfluidic chip, comprising: a chip upper layer and a chip lower layer; the chip lower layer comprises: The disc body has a predetermined thickness; a lower centrifugal shaft hole is opened at the center of the disc body; The sample chamber, the washing liquid chamber and the eluent chamber are arranged in a circular array around the centrifugal shaft hole of the lower layer; An extraction channel is provided on the disc body according to a predetermined path; the front end of the extraction channel is connected to the sample chamber, and the rear end is connected to the Coriolis valve; the radius of the extraction channel gradually increases from the front end to the rear end and circles around the lower centrifugal shaft hole, and the inner wall thereof is modified with a double network hydrogel for interception and adsorption of nucleic acids; The first channel and the second channel have input ends connected to the washing liquid tank and the elution liquid tank respectively, and output ends merged at the front end of the extraction channel; A waste liquid tank and a collection tank are symmetrically arranged on both sides of the Coriolis valve and are connected to the Coriolis valve; A plurality of amplification detection areas are evenly arranged in a ring shape at the edge of the disc body; the plurality of amplification detection areas are connected to the collection chamber through a distribution channel, and each amplification detection area is quantitatively detected.

[0009] In a further embodiment, the shape structure of the upper layer of the chip corresponds to the shape structure of the lower layer of the chip, and the upper layer of the chip is provided with: an upper centrifugal shaft hole, a liquid addition requirement hole, a plurality of groups of amplification requirement holes and a positioning hole; Wherein, the liquid adding demand holes include a sample adding hole, a washing liquid adding hole and an eluent adding hole corresponding to the sample chamber, the washing liquid chamber and the eluent chamber respectively; Each set of expansion requirement holes includes: a refueling hole and an exhaust hole.

[0010] In a further embodiment, the lower layer of the chip further comprises: a buffer chamber connected to the eluent chamber via a second channel; The second passage between the eluent bin and the buffer bin is provided with a first air valve, a first siphon valve and a second air valve in sequence; A second siphon valve and a third air valve are sequentially arranged on the second channel between the cache bin and the extraction channel.

[0011] In a further embodiment, the amplification detection zone comprises: A quantitative bin, the input end of which is connected to the distribution channel; An amplification reagent ball bin is connected to the quantitative bin through a fourth channel; freeze-dried amplification reagent balls are placed in the amplification reagent ball bin; an amplification detection chamber connected to the amplification reagent ball chamber via a fifth channel; A plurality of diamond-shaped support columns are arranged in the amplification detection chamber in a predetermined distribution pattern.

[0012] In a further embodiment, the double network hydrogel comprises: a hydrogel for nucleic acid extraction and a base material for fixing the hydrogel in a channel; wherein the base material is dense (3-glycidyl propoxy) trimethoxy silane; The hydrogel is a double network hydrogel formed by chitosan and polyacrylamide The chitosans have different molecular weights; The polyacrylamide is acrylamide, N , N It is prepared by mixing methylenebisacrylamide, ammonium persulfate and tetramethylethylenediamine.

[0013] In a further embodiment, a fourth air valve, a third siphon valve and a fifth air valve are sequentially arranged on the first channel between the washing liquid tank and the extraction channel.

[0014] In a further embodiment, a fourth siphon valve is provided between the collecting bin and the distribution channel.

[0015] In a further embodiment, the sample chamber, the washing liquid chamber and the eluting liquid chamber are all provided with isolation walls at positions away from the outlet; and each set of isolation walls is configured with a corresponding sixth air valve through the third channel.

[0016] In a further embodiment, the front end of the distribution channel is connected to the collection chamber, and is sequentially connected to the amplification detection area at predetermined intervals starting from the front end to the end; The inner diameter and the outer diameter of the distribution channel gradually increase from the front end to the rear end, and the channel width gradually increases.

[0017] In a further embodiment, it also includes: a residual liquid tank connected to the end of the distribution channel.

[0018] In a further embodiment, the Coriolis valve is symmetrically butterfly-shaped, having a head end and two sets of butterfly-shaped tail ends; the head end is used to connect to the extraction channel, and the two sets of butterfly-shaped tail ends are respectively connected to the waste liquid bin and the collection bin.

[0019] In a further embodiment, a rectangular groove body is provided between the Coriolis valve and the waste liquid bin and the collection bin, and the depth of the rectangular groove body is at least 3 times the depth of the connecting channel.

[0020] In a further embodiment, the fifth channel is configured as a curved portion flowing toward the center of the circle at the connection of the amplification detection chamber.

[0021] In a further embodiment, a refueling port and an exhaust port are respectively provided at designated positions of the amplification detection chamber, and the refueling port and the exhaust port are respectively adapted to the refueling hole and the exhaust hole on the upper layer of the chip.

[0022] In a further embodiment, the upper layer of the chip is processed by a pressure-sensitive adhesive film die-cutting process.

[0023] In a further embodiment, the chip lower layer is made of a polymer material, and the polymer material at least includes: polycarbonate PC , or polymethyl methacrylate PMMA , or cycloolefin copolymer COC One or more of the following.

[0024] The driving method based on the centrifugal microfluidic chip as described above comprises the following steps: Add samples, washing liquid and eluent into the sample chamber, washing liquid chamber and eluent chamber through the sample adding hole, washing liquid adding hole and eluent adding hole respectively; add fluorinated oil into the amplification detection chamber through the filling hole, and the fluorinated oil and the vent hole are sealed with a pressure-sensitive adhesive film; At a predetermined speed and booking duration The centrifugal microfluidic chip is rotated toward the direction of the waste liquid bin, and the sample enters the waste liquid bin through the extraction channel; the first air valve and the fourth air valve corresponding to the eluent bin and the washing liquid bin are opened, the first siphon valve and the third siphon valve are closed, and the eluent and the washing liquid stop moving; After standing, all samples flow into the waste liquid tank; nucleic acid molecules are intercepted and adsorbed by the hydrogel in the extraction channel, the first siphon valve and the third siphon valve are opened, and the eluent and washing liquid are driven by capillaries to flow to the second air valve and the fifth air valve respectively; At a predetermined speed and booking duration Rotate the centrifugal microfluidic chip toward the waste liquid bin, and open the second air valve and the fifth air valve, so that the eluent flows into the buffer bin, and the washing liquid flows into the waste liquid bin through the extraction channel; Let it stand until all the washing liquid flows into the waste liquid tank, and open the second siphon valve to allow the eluent to flow to the third air valve under capillary drive; At a predetermined speed and booking duration Rotate the centrifugal microfluidic chip toward the collection chamber and open the third air valve, so that the eluent flows into the collection chamber through the extraction channel; After the mixture is left to stand, the fourth siphon valve connected to the collection chamber is opened, and the eluent is driven to the front end of the distribution channel by capillary force; At a predetermined speed and booking duration The centrifugal microfluidic chip is rotated toward the direction of the collection chamber, and the eluent enters the amplification detection area through the distribution channel, and the excess eluent enters the residual liquid chamber; At a predetermined speed and booking duration The centrifugal microfluidic chip is rotated in the direction of the collection chamber, and the eluent enters the amplification reagent ball chamber through the quantitative chamber and dissolves the amplification reagent freeze-dried balls in the amplification reagent ball chamber, and is left to stand for a predetermined time. ; At a predetermined speed and booking duration With continuous rotation, the amplification reagent enters the amplification detection chamber and stably exists in the amplification detection chamber in the form of "water-in-oil" droplets; The centrifugal microfluidic chip is heated to a predetermined temperature, and the fluorescence image of the droplet array is detected using an endpoint method.

[0025] In a further embodiment, the following steps are also included: The fluorescent signal of the droplets containing the target gene is defined as bright, and the number is recorded as , the fluorescence signal of the droplet without the target gene is dark, and the number is recorded as ; The copy number of the mutant was calculated using the following formula: : ; In the formula, is the total volume of amplification reagents, is the volume of the droplet.

[0026] Based on the application of the centrifugal microfluidic chip as described above, it is applied to drug-resistant gene detection, low-abundance gene expression detection, gene mutation detection, copy number variation analysis and liquid biopsy.

[0027] Beneficial effects of the present invention: The centrifugal microfluidic chip disclosed in the present invention is simple to operate and can complete the washing, elution, quantitative distribution, droplet formation, amplification and digital detection of nucleic acids in a fully integrated manner.

[0028] The double network hydrogel is used to ensure the mechanical properties of the microfluidic chip at a high centrifugal speed, with both high strength and good stretchability. A large number of amino and hydroxyl groups provide good extraction efficiency for nucleic acids.

[0029] The centrifugal microfluidic chip of the present invention has strong scalability and can be subsequently matched with an instrument for high-throughput processing chips, so it can be suitable for high-throughput detection; the upper limit of chip design is multi-target detection, and researchers can detect any combination of multiple targets.

[0030] The microfluidic chip of the present invention is a fully enclosed design, without PCRThe laboratory can complete the nucleic acid test, and the supporting single-test instrument is simple, which is convenient for use in areas with underdeveloped medical facilities. It is very suitable for the detection of tumor resistance gene targets in outpatients, and has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 1 is a diagram showing the overall structure of the centrifugal microfluidic chip of Example 1.

[0032] Figure 2 This is a physical picture of the centrifugal microfluidic chip of Example 1.

[0033] Figure 3 This is a structural diagram of the chip upper layer of Example 1.

[0034] Figure 4 This is a structural diagram of the lower layer of the chip of Example 1.

[0035] Figure 5 The local discharge of the lower layer of the chip of Example 1 Figure 1 .

[0036] Figure 6 The local discharge of the lower layer of the chip of Example 1 Figure 2 .

[0037] Figure 7 The local discharge of the lower layer of the chip of Example 1 Figure 3 .

[0038] Figure 8 This is a flow chart of the driving method of the centrifugal microfluidic chip of Example 2.

[0039] Fig. 9 It is a diagram of the droplet arrangement in the amplification detection area.

[0040] Fig.10 This is a schematic diagram of the preparation principle of the double network hydrogel material of Example 3.

[0041] Fig.11 This is a diagram showing the efficiency of nucleic acid extraction in a longitudinal comparison in Example 3.

[0042] Fig.12 This is a diagram comparing the efficiency of nucleic acid extraction in Example 3.

[0043] Figures 1 to 7The labels in the figure are: chip upper layer 1, chip lower layer 2, upper centrifugal shaft hole 101, sample loading hole 102, washing liquid loading hole 103, eluent loading hole 104, refueling hole 105, exhaust hole 106, disc body 201, lower centrifugal shaft hole 202, sample chamber 203, washing liquid chamber 204, eluent chamber 205, extraction channel 206, Coriolis valve 207, first channel 208, second channel 209, waste liquid chamber 210, collection chamber 211, amplification detection area 212, buffer chamber 213, distribution channel 214, isolation wall 215, third channel 216, sixth air valve 217, residual liquid chamber 218, rectangular trough 219, fourth air valve 208- a , the third siphon valve 208- b , Fifth air valve 208- c , first air valve 209- a , first siphon valve 209- b , second air valve 209- c , the second siphon valve 209- d , the third air valve 209- e , quantitative warehouse 212- a , Amplification reagent ball chamber 212- b 、The fourth channel 212- c , Amplification detection chamber 212- d 、Support column 212- e 、The fifth channel 212- f , bending portion 212- g 、Refueling port 212- h 、Exhaust port 212- i . DETAILED DESCRIPTION

[0044] The present invention will be further described below in conjunction with the accompanying drawings and embodiments.

[0045] Example 1 A centrifugal microfluidic chip, the chip is used for nucleic acid extraction, purification, amplification, and digital detection. Figure 1 and Figure 2 The centrifugal microfluidic chip shown comprises: an upper chip layer 1 and a lower chip layer 2 which are adapted to each other.

[0046] In this embodiment, the chip lower layer 2 is made of polymer material, and the polymer material at least includes: polycarbonate PC , or polymethyl methacrylate PMMA , or cycloolefin copolymer COC One or more of the following. The specific structure can be combined Figure 4The invention comprises: a disc body 201 with a predetermined thickness, a lower centrifugal shaft hole 202 is provided at the center of the disc body 201, and the lower centrifugal shaft hole 202 can be set to a hexagonal shape to facilitate driving the disc body 201. Further, corresponding cavities are provided on the disc body 201 according to requirements to form a sample chamber 203, a washing liquid chamber 204 and an eluent chamber 205. In combination with the above description, the sample chamber 203, the washing liquid chamber 204 and the eluent chamber 205 are arranged in an array around the lower centrifugal shaft hole 202.

[0047] An extraction channel 206 is arranged on the disc body 201 according to a predetermined path. In connection with the predetermined path, the front end of the extraction channel 206 is connected to the sample chamber 203, and the rear end is connected to the Coriolis valve 207; the radius of the extraction channel 206 gradually increases from the front end to the rear end, and its inner wall is modified with a double network hydrogel for interception and adsorption of nucleic acids. Furthermore, the radius of the extraction channel 206 gradually increases from the front end to the rear end so that the solution will gradually flow out of the extraction channel 206 under the drive of centrifugal force. Preferably, the predetermined path of this embodiment is to go around the disc body 201 for one circle, and the capture efficiency is increased by a longer path, and the gradual increase in radius does not affect the drive of the solution. It also includes: a first channel 208 and a second channel 209, whose input ends are respectively connected to the washing liquid chamber 204 and the elution liquid chamber 205, and whose output ends merge at the front end of the extraction channel 206; The waste liquid bin 210 and the collection bin 211 are symmetrically disposed on both sides of the Coriolis valve 207 and are connected to the Coriolis valve 207 ; the Coriolis valve 207 is used to realize the flow switching of the solution between the waste liquid bin 210 and the collection bin 211 .

[0048] A plurality of amplification detection areas 212 are evenly arranged in a ring shape at the edge of the disc body 201 ; the plurality of amplification detection areas 212 are connected to the collection chamber 211 through a distribution channel 214 , and each amplification detection area 212 is for quantitative detection.

[0049] In order to achieve sealing and facilitate detection at any time, such as Figure 3 The shape structure of the upper layer 1 of the chip shown corresponds to the shape structure of the lower layer 2 of the chip. The upper layer 1 of the chip is provided with: an upper centrifugal shaft hole 101, a liquid addition requirement hole, a plurality of amplification requirement holes and a positioning hole. Correspondingly, the upper centrifugal shaft hole 101 is a hexagon. It is worth noting that the upper layer 1 of the chip is processed by a pressure-sensitive adhesive film die-cutting process.

[0050] The liquid adding holes include a sample adding hole 102, a washing liquid adding hole 103 and an eluent adding hole 104 corresponding to the sample chamber 203, the washing liquid chamber 204 and the eluent chamber 205 respectively; and are used to realize the addition of sample, washing liquid and eluent respectively.

[0051] Correspondingly, each group of amplification requirement holes includes: a refueling hole 105 and an exhaust hole 106 .

[0052] In a further embodiment, Figure 5 As shown, the sample chamber 203, the washing liquid chamber 204 and the eluting liquid chamber 205 are all provided with a partition wall 215 at a position far from the outlet; each group of partition walls 215 is provided with a corresponding sixth air valve 217 through a third channel 216, wherein the third channel 216 is a capillary channel. Taking the sample chamber 203 as an example, after adding the sample from the sample loading hole, the droplets tend to exist in the form of droplets due to the tension of the interface, and the liquid will not flow to the inside of the partition wall 215. Therefore, the internal pressure is adjusted through the corresponding capillary channel and the sixth air valve 217 to prevent the internal pressure of the three sample loading layers from being too high due to the sample loading and thus failing to load the sample.

[0053] In order to control the flow speed and flow time of the sample, washing solution and eluent and supply the liquid according to demand, in a further embodiment, the lower layer 2 of the chip also includes: a cache chamber 213, which is connected to the eluent chamber 205 through the second channel 209.

[0054] The corresponding adjustment is as follows: the second channel 209 between the eluent bin 205 and the buffer bin 213 is provided with a first air valve 209- a , first siphon valve 209- b and the second air valve 209- c ; The second channel 209 between the buffer chamber 213 and the extraction channel 206 is provided with a second siphon valve 209- d and the third air valve 209- e .

[0055] The first channel 208 between the washing liquid tank 204 and the extraction channel 206 is provided with fourth air valves 208- a , the third siphon valve 208- b and the fifth air valve 208- c .

[0056] A fourth siphon valve is disposed between the collecting chamber 211 and the distribution channel 214 .

[0057] By staggered arrangement of the air valve and the siphon valve, it is ensured that the washing liquid and the eluent flow into the extraction channel 206 after the sample, and the washing liquid flows into the extraction channel 206 before the eluent.

[0058] In a further embodiment, the Coriolis valve 207 is symmetrically butterfly-shaped, having a head end and two sets of butterfly-shaped tail ends; the head end is used to connect to the extraction channel 206, and the two sets of butterfly-shaped tail ends are respectively connected to the waste liquid bin 210 and the collection bin 211. When the chip rotates counterclockwise around the center (i.e., in the direction of the waste liquid bin 210), the solution will be biased toward the waste liquid bin 210 due to the existence of the Euler force and the Coriolis force; when the chip rotates clockwise (i.e., in the direction of the collection bin 211), the solution will be biased toward the collection bin 211.

[0059] A rectangular groove 219 is provided between the Coriolis valve 207 and the waste liquid bin 210 and the collection bin 211. The depth of the rectangular groove 219 is at least three times the depth of the connecting channel. The purpose is to use the rectangular groove 219 as an air valve to prevent backflow contamination of the liquid. Figure 6 and Figure 7 The amplification detection area 212 includes: a quantitative chamber 212-1 having an input end connected to a distribution channel 214; a The quantitative bin 212- a The output end passes through the fourth channel 212- c Connected with amplification reagent ball chamber 212- b , the amplification reagent ball chamber 212- b At the same time, through the fifth channel 212- f Connected with amplification detection chamber 212- d Among them, the fourth channel 212- c and the fifth channel 212- f They are all capillary channels.

[0060] In this embodiment, the amplification reagent ball chamber 212- b Amplification reagent freeze-dried pellets are placed inside, such as PCR Amplification reagent freeze-dried balls, isothermal amplification reagent freeze-dried balls, etc. Amplification detection chamber 212- d A support column 212 is provided inside. e , and they are all in diamond shape, because the smallest unit of droplet arrangement is diamond, in order to ensure that there are no defects in the droplet arrangement, the external shape also needs to be consistent. d The two ends of the long side of the rhombus are respectively provided with a refueling port 212- h and exhaust port 212- i , the refueling port 212- h and exhaust port 212- i The refueling hole 105 and the exhaust hole 106 are respectively adapted to the chip upper layer 1 .

[0061] Before using the chip, first refuel from the refueling port 212- hAdd droplets to form the necessary fluorinated oil, and then seal it with pressure-sensitive adhesive to prevent leakage. d Initially, the amplification solution is immersed in fluorinated oil from the fifth channel 212- f Enter at a certain speed, use the force of rotation to pull out droplets one by one, and finally arrange them in the amplification detection chamber 212- d middle.

[0062] In order to prevent the amplification solution from entering the amplification detection chamber 212- d The speed in the amplification detection chamber 212- d No droplets can be formed, so the fifth channel 212- f In the amplification detection chamber 212- d The connection point is set to be a curved portion 212- g , combined with diagrams to understand.

[0063] In combination with the above-mentioned amplification detection area 212, in a further embodiment, the front end of the distribution channel 214 is connected to the collection chamber 211, and is sequentially connected to the amplification detection area 212 from the front end at predetermined intervals to the end; the inner diameter and outer diameter of the distribution channel 214 are gradually increased from the front end to the end. This allows the nucleic acid solution to enter the quantitative chamber 212 at a low flow rate. a , and will not enter the next level of the channel. a The quantified nucleic acid solution is then centrifuged at high speed and driven synchronously to the amplification reagent ball chamber 212- b , and finally connected to the amplification detection chamber 212- d .

[0064] In a further embodiment, it further includes: a residual liquid tank 218 connected to the end of the distribution channel 214 for collecting excess liquid.

[0065] The centrifugal microfluidic chip disclosed in this embodiment is applied to drug-resistant gene detection, low-abundance gene expression detection, gene mutation detection, copy number variation analysis and liquid biopsy.

[0066] Example 2 Based on the centrifugal microfluidic chip disclosed in Example 1, this embodiment discloses a driving method of the centrifugal microfluidic chip, such as Figure 8 Said, comprising the following steps: Step 1, adding samples, washing liquid and eluent into the sample chamber, washing liquid chamber and eluent chamber through the sample adding hole, washing liquid adding hole and eluent adding hole respectively; adding fluorinated oil into the amplification detection chamber through the filling hole, and the fluorinated oil and the vent hole are sealed with a pressure-sensitive adhesive film; Step 2: At a predetermined speed and booking duration The centrifugal microfluidic chip is rotated toward the waste liquid bin, and the sample enters the waste liquid bin through the extraction channel. Figure 8 The blue schematic part of step 2 in the figure; the first air valve and the fourth air valve corresponding to the eluent tank and the washing liquid tank are opened, the first siphon valve and the third siphon valve are closed, and the eluent and the washing liquid stop moving. Among them, the predetermined speed in this embodiment The value range is 1500~2500 rpm , scheduled duration 60~100 s , and combined with the direction towards the waste liquid bin in the figure, it is counterclockwise.

[0067] Step 3: Let stand for 20-60 seconds s , all samples flow into the waste liquid tank; the nucleic acid molecules are intercepted and adsorbed by the hydrogel in the extraction channel, the first siphon valve and the third siphon valve are opened, and the eluent and washing liquid are driven by capillary to flow to the second air valve and the fifth air valve respectively.

[0068] Step 4: At a predetermined speed and booking duration Rotate the centrifugal microfluidic chip toward the waste liquid bin, and open the second air valve and the fifth air valve. The eluent flows into the buffer bin, and the washing liquid flows into the waste liquid bin through the extraction channel. Figure 8 The red color in the diagram of step 4 changes, and the red color is the eluent, and it rotates in a counterclockwise direction. The value range is 2500~3500 rpm , scheduled duration 150~200 s .

[0069] Step 5: Let stand for 20-60 seconds s , all the washing liquid flows into the waste liquid tank, open the second siphon valve, and the eluent flows to the third air valve under capillary drive; Figure 8 The red part (eluent) and green part (washing solution) in the diagram of step 4 in Figure 1 are shown.

[0070] Step 6: At a predetermined speed and booking duration The centrifugal microfluidic chip is rotated in the direction of the collection chamber (clockwise) and the third air valve is opened, and the eluent flows into the collection chamber through the extraction channel, that is, the red part in the figure enters the collection chamber. The value range is 3500~4500 rpm , scheduled duration 250~350 s .

[0071] Step 7: Let stand for 20-60 seconds s Afterwards, the fourth siphon valve connected to the collection chamber is opened, and the eluent is driven to the front end of the distribution channel by capillary force.

[0072] Step 8-Step 9, at a predetermined speed and booking duration The centrifugal microfluidic chip is rotated toward the collection chamber, and the eluent enters the amplification detection area through the distribution channel, and the excess eluent enters the residual liquid chamber; wherein the predetermined rotation speed The value range is 400~1300 rpm , scheduled duration 400~600 s .

[0073] Step 10: At a predetermined speed and booking duration The centrifugal microfluidic chip is rotated in the direction of the collection chamber, and the eluent enters the amplification reagent ball chamber through the quantitative chamber and dissolves the amplification reagent freeze-dried balls in the amplification reagent ball chamber, and is left to stand for a predetermined time. ; Among them, the predetermined speed The value range is 800~2000 rpm , scheduled duration 100~150 s , let it stand for a predetermined period of time The value range is 60~120 s , to ensure sufficient dissolution.

[0074] Step 11: At a predetermined speed and booking duration The amplification reagents continue to rotate, and enter the amplification detection chamber, and stably exist in the amplification detection chamber in the form of "water-in-oil" droplets. The value range is 5000~7000 rpm , scheduled duration 350~450 s .

[0075] Step 12: Heat the centrifugal microfluidic chip at a predetermined temperature, and use an endpoint method to detect the fluorescent image of the droplet array.

[0076] In a further embodiment, the following steps are also included: The fluorescent signal of the droplets containing the target gene is defined as bright, and the number is recorded as , the fluorescence signal of the droplet without the target gene is dark, and the number is recorded as ; The copy number of the mutant was calculated using the following formula: : ; In the formula, is the total volume of amplification reagents, is the volume of the droplet.

[0077] The droplets prepared by this method were analyzed for regularity. Fig. 9 It can be seen that the droplet size in the amplification detection area is 200 microns, arranged in a uniform diamond structure, and the arrangement effect is significant.

[0078] Example 3 In order to realize the centrifugal microfluidic chip required in Example 1, this example also discloses a modification method of the double network hydrogel described in Example 1, such as Fig.10 The following steps are shown: Step 1: Using the layer-by-layer self-assembly method to modify the dense (3-glycidyl propoxy) trimethoxysilane ( GPTMS ), the modified process conditions are in the applicant's authorized invention patent CN 105755463 B It is mentioned in the article, so I won’t elaborate on it here. Step 2: In order to ensure the mechanical properties of the hydrogel in centrifugal microfluidics, a double network hydrogel was used to prepare the material for nucleic acid extraction. It should be noted that the principles for selecting gel materials are: (1) the gel material has abundant hydroxyl and amino groups, which are conducive to the adsorption of nucleic acids; (2) one of the gel materials must contain amino groups for the adsorption of nucleic acids. GPTMS The epoxy groups react and connect to the base material; (3) one material has high strength and the other has good stretchability. The double network hydrogel composed of the two in a certain proportion may form a hydrogel with high strength and good stretchability. Therefore, chitosan and polyacrylamide hydrogel are selected as nucleic acid extraction hydrogels in this patent; Step 3: Mix 1-3% chitosan and 8-15% acrylamide for 30 minutes, and add dropwise during stirring. N , N '-Methylenebisacrylamide ( MBA ),ensure MBA The final concentration is 0.01~0.1%; stir thoroughly for 1 hour, and add ammonium persulfate dropwise during the stirring process ( APS ),ensure APS The final concentration is 0.01~0.1%; stir thoroughly for 1 hour, and add tetramethylethylenediamine ( TEMED ),ensure TEMED The final concentration is 0.005~0.05%; Step 4: Pour the fully stirred hydrogel prepolymer onto the modified GPTMSThe chip was placed in the extraction channel and waited for more than 24 hours for full polymerization. The chip was soaked in deionized water for 24 hours to remove uncross-linked reactants. After being taken out and dried, the chip modification process was completed.

[0079] The performance of the double network hydrogel prepared by the above method was compared: before and after extraction DNA The absolute concentration of the nucleic acid extraction was 41.3% in the longitudinal comparison. Fig.11 Compared with the commercial column extraction kit, the efficiency of nucleic acid extraction was 82.4%. Fig.12 As shown, the extraction efficiency within the microfluidic chip is comparable to that of a mature commercial extraction kit outside the chip.

Claims

1. A centrifugal microfluidic chip, comprising: The chip upper layer and the chip lower layer; characterized in that the chip lower layer comprises: The disc body has a predetermined thickness; a lower centrifugal shaft hole is opened at the center of the disc body; The sample chamber, the washing liquid chamber and the eluent chamber are arranged in a circular array around the centrifugal shaft hole of the lower layer; An extraction channel is provided on the disc body according to a predetermined path; the front end of the extraction channel is connected to the sample chamber, and the rear end is connected to the Coriolis valve; the radius of the extraction channel gradually increases from the front end to the rear end and circles around the lower centrifugal shaft hole, and the inner wall thereof is modified with a double network hydrogel for interception and adsorption of nucleic acids; The first channel and the second channel have input ends connected to the washing liquid tank and the elution liquid tank respectively, and output ends merged at the front end of the extraction channel; A waste liquid tank and a collection tank are symmetrically arranged on both sides of the Coriolis valve and are connected to the Coriolis valve; A plurality of amplification detection areas are evenly arranged in a ring shape at the edge of the disc body; the plurality of amplification detection areas are connected to the collection chamber through a distribution channel, and each amplification detection area is quantitatively detected.

2. A centrifugal microfluidic chip according to claim 1, characterized in that: The shape structure of the upper layer of the chip corresponds to the shape structure of the lower layer of the chip, and the upper layer of the chip is provided with: an upper centrifugal shaft hole, a liquid addition requirement hole, a plurality of groups of amplification requirement holes and a positioning hole; Wherein, the liquid adding demand holes include a sample adding hole, a washing liquid adding hole and an eluent adding hole corresponding to the sample chamber, the washing liquid chamber and the eluent chamber respectively; Each set of expansion requirement holes includes: a refueling hole and an exhaust hole.

3. A centrifugal microfluidic chip according to claim 1, characterized in that: The lower layer of the chip further includes: a buffer chamber connected to the eluent chamber through a second channel; The second passage between the eluent bin and the buffer bin is provided with a first air valve, a first siphon valve and a second air valve in sequence; A second siphon valve and a third air valve are sequentially arranged on the second channel between the cache bin and the extraction channel.

4. A centrifugal microfluidic chip according to claim 1, characterized in that: The amplification detection zone comprises: A quantitative bin, the input end of which is connected to the distribution channel; An amplification reagent ball bin is connected to the quantitative bin through a fourth channel; freeze-dried amplification reagent balls are placed in the amplification reagent ball bin; an amplification detection chamber connected to the amplification reagent ball chamber via a fifth channel; A plurality of diamond-shaped support columns are arranged in the amplification detection chamber in a predetermined distribution pattern.

5. A centrifugal microfluidic chip according to claim 1, characterized in that: The double network hydrogel comprises: a hydrogel for nucleic acid extraction and a base material for fixing the hydrogel in a channel; wherein the base material is dense (3-glycidyl propoxy) trimethoxy silane; The hydrogel is a double network hydrogel formed by chitosan and polyacrylamide The chitosans have different molecular weights; The polyacrylamide is acrylamide, N , N It is prepared by mixing methylenebisacrylamide, ammonium persulfate and tetramethylethylenediamine.

6. A centrifugal microfluidic chip according to claim 1, characterized in that: A fourth air valve, a third siphon valve and a fifth air valve are sequentially arranged on the first channel between the washing liquid bin and the extraction channel.

7. A centrifugal microfluidic chip according to claim 1, characterized in that: A fourth siphon valve is arranged between the collecting bin and the distribution channel.

8. The centrifugal microfluidic chip according to claim 1, characterized in that: The sample chamber, the washing liquid chamber and the eluting liquid chamber are all provided with isolation walls at positions away from the outlet; each set of isolation walls is provided with a corresponding sixth air valve through the third channel.

9. A centrifugal microfluidic chip according to claim 4, characterized in that: The front end of the distribution channel is connected to the collection chamber, and is sequentially connected to the amplification detection area at predetermined intervals from the front end to the end; The inner diameter and the outer diameter of the distribution channel gradually increase from the front end to the rear end, and the channel width gradually increases.

10. A centrifugal microfluidic chip according to claim 9, characterized in that: Also includes: The residual liquid tank is connected to the end of the distribution channel.

11. The centrifugal microfluidic chip according to claim 1, characterized in that: The Coriolis valve is symmetrically butterfly-shaped, having a head end and two sets of butterfly-shaped tail ends; the head end is used to connect to the extraction channel, and the two sets of butterfly-shaped tail ends are respectively connected to the waste liquid bin and the collection bin.

12. The centrifugal microfluidic chip according to claim 1, characterized in that: A rectangular groove body is provided between the Coriolis valve and the waste liquid bin and the collection bin, and the depth of the rectangular groove body is at least 3 times the depth of the connecting channel.

13. A centrifugal microfluidic chip according to claim 4, characterized in that: The fifth channel is configured as a curved portion flowing toward the center of the circle at the connection of the amplification detection chamber.

14. A centrifugal microfluidic chip according to claim 4, characterized in that: A refueling port and an exhaust port are respectively provided at designated positions of the amplification detection chamber, and the refueling port and the exhaust port are respectively adapted to the refueling hole and the exhaust hole on the upper layer of the chip.

15. The centrifugal microfluidic chip according to claim 1, characterized in that: The upper layer of the chip is processed by a pressure-sensitive adhesive film die-cutting process.

16. The centrifugal microfluidic chip according to claim 1, characterized in that: The lower layer of the chip is made of polymer material, and the polymer material at least includes: polycarbonate PC , or polymethyl methacrylate PMMA , or cyclic olefin copolymer COC One or more of the following.

17. A driving method for a centrifugal microfluidic chip according to any one of claims 1 to 16, characterized in that: The following steps are involved: Add samples, washing liquid and eluent into the sample chamber, washing liquid chamber and eluent chamber through the sample adding hole, washing liquid adding hole and eluent adding hole respectively; add fluorinated oil into the amplification detection chamber through the filling hole, and the fluorinated oil and the vent hole are sealed with a pressure-sensitive adhesive film; At a predetermined speed and booking duration The centrifugal microfluidic chip is rotated toward the direction of the waste liquid bin, and the sample enters the waste liquid bin through the extraction channel; the first air valve and the fourth air valve corresponding to the eluent bin and the washing liquid bin are opened, the first siphon valve and the third siphon valve are closed, and the eluent and the washing liquid stop moving; After standing, all samples flow into the waste liquid tank; nucleic acid molecules are intercepted and adsorbed by the hydrogel in the extraction channel, the first siphon valve and the third siphon valve are opened, and the eluent and washing liquid are driven by capillaries to flow to the second air valve and the fifth air valve respectively; At a predetermined speed and booking duration Rotate the centrifugal microfluidic chip toward the waste liquid bin, and open the second air valve and the fifth air valve, so that the eluent flows into the buffer bin, and the washing liquid flows into the waste liquid bin through the extraction channel; Let it stand until all the washing liquid flows into the waste liquid tank, and open the second siphon valve to allow the eluent to flow to the third air valve under capillary drive; At a predetermined speed and booking duration Rotate the centrifugal microfluidic chip toward the collection chamber and open the third air valve, so that the eluent flows into the collection chamber through the extraction channel; After the mixture is left to stand, the fourth siphon valve connected to the collection chamber is opened, and the eluent is driven to the front end of the distribution channel by capillary force; At a predetermined speed and booking duration The centrifugal microfluidic chip is rotated toward the direction of the collection chamber, and the eluent enters the amplification detection area through the distribution channel, and the excess eluent enters the residual liquid chamber; At a predetermined speed and booking duration The centrifugal microfluidic chip is rotated in the direction of the collection chamber, and the eluent enters the amplification reagent ball chamber through the quantitative chamber and dissolves the amplification reagent freeze-dried balls in the amplification reagent ball chamber, and is left to stand for a predetermined time. ; At a predetermined speed and booking duration With continuous rotation, the amplification reagent enters the amplification detection chamber and stably exists in the amplification detection chamber in the form of "water-in-oil" droplets; The centrifugal microfluidic chip is heated to a predetermined temperature, and the fluorescence image of the droplet array is detected using an endpoint method.

18. The driving method of the centrifugal microfluidic chip according to claim 17, characterized in that: The following steps are also included: The fluorescent signal of the droplets containing the target gene is defined as bright, and the number is recorded as , the fluorescence signal of the droplet without the target gene is dark, and the number is recorded as ; The copy number of the mutant was calculated using the following formula: : ； In the formula, is the total volume of amplification reagents, is the volume of the droplet.

19. The use of the centrifugal microfluidic chip according to any one of claims 1 to 16, characterized in that: It is used for drug-resistant gene detection, low-abundance gene expression detection, gene mutation detection, copy number variation analysis and liquid biopsy.

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