Gene methylation micro-fluidic chip

Abstract

The invention provides a gene methylation micro-fluidic chip. The chip comprises: a substrate; the thin film is positioned at the bottom of the substrate; a binding area, a vulcanization binding area, a washing and eluting area, a PCR (Polymerase Chain Reaction) detection area and a waste liquid area are arranged in the substrate; a first pipeline, a second pipeline and a third pipeline which are used for enabling a sample to circulate among the areas are laid on the thin film; the first pipeline is communicated with the binding area and the vulcanization binding area, the second pipeline is communicated with the vulcanization binding area and the washing and eluting area, and the third pipeline is communicated with the washing and eluting area and the PCR detection area; air inlet holes are formed in the tops of the combination area, the vulcanization combination area and the washing and elution area; magnetic attraction positions are respectively arranged in a space between the binding area and the vulcanization binding area and a space between the vulcanization binding area and the washing and eluting area; heating positions are arranged around the vulcanization bonding area and the washing and eluting area; a plurality of liquid inlet holes are formed in the side walls of the combination area, the vulcanization combination area and the washing and elution area.

Description

Technical Field

[0001] The present application relates to the field of microfluidic chips, and in particular, to a gene methylation microfluidic chip. Background Art

[0002] Nucleic acid is a biological macromolecule composed of many nucleotides and is one of the most basic substances of life. Nucleic acid is widely present in all animal and plant cells and microorganisms. Nucleic acid in organisms often combines with proteins to form nucleoproteins. In recent years, with the rapid development of molecular biology, nucleic acid-based molecular diagnosis and detection technologies have increasingly highlighted their vital role in many fields; the existing nucleic acid purification has the advantages of short time consumption, high purification accuracy, and automated processing of large quantities of experimental samples.

[0003] Gene methylation refers to the process in which cytosine (C) in CpG dinucleosides on DNA molecules selectively adds methyl groups to form 5'-methylcytosine under the action of enzymes. CpG dinucleosides are often located near gene transcription regulatory regions, and their methylation can cause changes in chromatin structure, DNA conformation, DNA stability, etc., thereby regulating gene transcription and expression. Abnormal gene methylation is one of the most common epigenetic changes in tumorigenesis. The occurrence of tumors is manifested by a decrease in the overall methylation level of the genome (oncogenes) and an abnormal increase in the local methylation level of CpG islands (tumor suppressor genes).

[0004] Polymerase chain reaction (PCR) is a molecular biology technique used to amplify specific DNA fragments. It can be regarded as a special DNA replication outside the body. The biggest feature of PCR is that it can greatly increase trace amounts of DNA. PCR uses the fact that DNA denatures into single strands at a high temperature of 95°C in vitro. At low temperatures (usually around 60°C), primers and single strands combine according to the principle of base complementary pairing. The temperature is then adjusted to the optimal reaction temperature of DNA polymerase (around 72°C). DNA polymerase synthesizes complementary chains along the direction from phosphate to pentose (5'-3'). The PCR instrument manufactured based on polymerase is actually a temperature control device that can well control the denaturation temperature, annealing temperature, and extension temperature.

[0005] Microfluidics is a technology that precisely controls and manipulates microscale fluids, especially submicron structures. It is also called Lab-on-a-Chip or microfluidic chip technology. It integrates the basic operating units of biological, chemical, and medical analysis processes, such as sample preparation, reaction, separation, and detection, into a micron-scale chip to automatically complete the entire analysis process. Due to its huge potential in the fields of biology, chemistry, and medicine, it has developed into a new research field that intersects biology, chemistry, medicine, fluids, electronics, materials, and mechanics. Due to the micron-scale structure, fluids in microfluidic chips display and produce special properties that are different from those at the macroscale, thus developing unique analytical performance. At the same time, it also has the advantages of light size, small amount of samples and reagents, low energy consumption, fast reaction speed, large-scale parallel processing, and disposable. Summary of the invention

[0006] In order to solve the problems in the prior art of unstable test results and low ability of the equipment to recognize low values, while avoiding the excessive size of the immunochromatographic analysis device, the present application provides a compact immunochromatographic analyzer reading device using a photoelectric sensor.

[0007] The technical solution of this application is as follows:

[0008] 1. A gene methylation microfluidic chip, comprising:

[0009] A substrate for forming a main body of a microfluidic chip; and

[0010] a thin film located at the bottom of the substrate;

[0011] in,

[0012] The substrate is provided with a binding area, a sulfurization binding area, a washing and elution area, and a PCR detection area for performing sample pretreatment, methylation reaction, and finally PCR reaction;

[0013] A first pipeline, a second pipeline, and a third pipeline are laid on the film to allow the sample to flow between the above-mentioned areas.

[0014] Wherein, the first pipeline connects the bonding area and the vulcanization bonding area,

[0015] The second pipeline connects the vulcanization bonding area and the washing and elution area.

[0016] The third pipeline connects the washing and elution area with the PCR detection area.

[0017] 2. A chip according to claim 1, wherein:

[0018] The substrate also includes the following components:

[0019] Air inlet holes are arranged on the tops of the bonding area, the vulcanization bonding area and the washing and elution area;

[0020] Magnetic suction positions are respectively arranged in the space between the bonding area and the vulcanization bonding area and between the vulcanization bonding area and the washing and elution area;

[0021] Heating positions are arranged around the vulcanization bonding area and the washing and elution area;

[0022] A plurality of liquid inlet holes are arranged on the side walls of the binding area, the sulfurization binding area and the washing and elution area, and samples or other liquids are introduced into the binding area, the sulfurization binding area or the washing and elution area through the liquid inlet holes.

[0023] 3. The chip according to item 1 or 2, wherein the substrate further comprises:

[0024] The waste liquid area is connected to the bonding area, the vulcanization bonding area and the washing and elution area respectively through the waste liquid pipeline arranged on the film.

[0025] 4. The chip according to item 3, wherein the waste liquid pipeline comprises a first waste liquid pipeline, a second waste liquid pipeline, a third waste liquid pipeline and a three-way pipeline;

[0026] The first waste liquid pipeline connects the waste liquid outlet of the combined area with the first waste liquid area.

[0027] The second waste liquid pipeline is connected to the waste liquid outlet of the vulcanization bonding area and the three-way pipeline.

[0028] The third waste liquid pipeline is connected to the waste liquid outlet of the washing and elution zone and the three-way pipeline.

[0029] The three ends of the three-way pipeline lead to the vulcanization and bonding area, the washing and elution area and the waste liquid area respectively.

[0030] 5. The chip according to items 1 and 4, wherein the first pipeline, the second pipeline, the third pipeline, the first waste liquid pipeline, the second waste liquid pipeline and the third waste liquid pipeline are all provided with a check valve.

[0031] 6. The chip according to item 1, wherein the film is an organic high molecular polymer film, preferably a PE film.

[0032] 7. A chip according to items 1-4, wherein the cross-sections of all the channels are semicircular, semi-elliptical or rectangular, and are combined with the film to form a closed and complete channel.

[0033] 8. A chip according to item 1, wherein the substrate is rectangular.

[0034] 9. The chip according to item 8, wherein the substrate is (60-80) mm long, (22.5-32.5) mm wide, and (25-45) mm high.

[0035] 10. The chip according to item 1, wherein the substrate is made of an organic polymer, preferably polymethyl methacrylate (PMMA).

[0036] 11. The chip according to item 1, wherein the volume of the binding area is 2 to 15 ml, preferably 11 mm.

[0037] 12. The chip according to item 1, wherein the volume of the sulfurized bonding area is 0.5 to 2.5 ml, and the volume is preferably 2.2 ml.

[0038] 13. The chip according to item 1, wherein the volume of the washing and elution zone is 0.5 to 2.5 ml, and the volume is preferably 2.2 ml.

[0039] 14. The chip according to item 1, wherein the chip is docked with a liquid storage tank through a liquid inlet hole, and the liquid storage tank comprises a first liquid storage tank and a second liquid storage tank having different structures from each other.

[0040] 15. The chip according to item 14, wherein the first liquid storage tank comprises: a liquid storage tank rubber cover, a liquid storage tank aluminum film, a first liquid storage tank piston, a first liquid storage tank shell, and a first liquid storage tank screw cap.

[0041] 16. The chip according to item 15, wherein the liquid storage tank rubber cover is sleeved on one end of the first liquid storage tank piston; the liquid storage tank aluminum film is placed between the liquid storage tank rubber cover and the first liquid storage tank piston to ensure the sealing of the liquid storage tank;

[0042] One end of the first liquid storage tank piston is in communication with one end of the first liquid storage tank housing, and the other end is sleeved with a liquid storage tank rubber cover, and the first liquid storage tank piston can move inside the first liquid storage tank housing;

[0043] One end of the first liquid storage tank housing is connected to the first liquid storage tank piston, and the other end is a port with a smaller diameter, and the outside of the port is provided with a thread;

[0044] The first liquid storage bin screw cap is screwed on and fixed to a port at an end of the first liquid storage bin with a smaller diameter by means of threads.

[0045] 17. A chip according to item 14, wherein the second liquid storage tank comprises: a second upper liquid storage tank screw cap, a second liquid storage tank piston, a second liquid storage tank shell, and a second lower liquid storage tank screw cap.

[0046] 18. The chip according to item 16, wherein the screw cap of the second liquid storage tank is screwed and fixed to a port at one end of the second liquid storage tank with a larger diameter by means of a thread;

[0047] One end of the second liquid storage tank piston is in communication with one end of the second liquid storage tank housing, and the second liquid storage tank piston can move inside the second liquid storage tank housing;

[0048] The second liquid storage tank is of variable diameter, with a thread provided on the inner wall of the end with a larger diameter, and a port with a smaller diameter at the other end, with a thread provided on the outside of the port;

[0049] The second liquid storage bin screw cap is screwed on and fixed to a port at an end of the second liquid storage bin with a smaller diameter by means of threads.

[0050] 19. The chip according to item 14, wherein the second liquid storage tank piston of the second liquid storage tank is a hollow structure, and the inner wall of the second liquid storage tank piston is provided with threads, which are tightened and fixed to the smaller diameter port of the first liquid storage tank through the threads.

[0051] 20. The chip according to item 14, wherein both the first liquid storage tank and the second liquid storage tank can be screwed and fixed to the chip through threads in the liquid inlet hole of the chip.

[0052] The beneficial effects of this application are:

[0053] The present application provides a gene methylation microfluidic chip, which implements experimental processes such as manual nucleic acid extraction and PCR detection using microfluidic technology. At the same time, based on the existing technology, namely, PCR detection and analysis microfluidic chip, the present application realizes the application of microfluidic technology to gene methylation, and integrates manual nucleic acid extraction, PCR detection and gene methylation on the same microfluidic chip. While achieving the above functions, the film set at the bottom of the pipeline and the change in diameter of the pipeline overcome the problem of a large solution flow rate required in the front methylation experimental area, thereby maintaining the integrity of the function and the stability of the structure. BRIEF DESCRIPTION OF THE DRAWINGS

[0054] Figure 1 A cross-sectional view of the gene methylation microfluidic chip provided for this application;

[0055] Figure 2 The structure diagram of the gene methylation microfluidic chip provided in this application;

[0056] Figure 3 An exploded view of the first liquid storage tank for the gene methylation microfluidic chip provided in this application;

[0057] Figure 4 An exploded view of the second liquid storage tank for the gene methylation microfluidic chip provided in this application;

[0058] 10-binding area, 20-sulfurization binding area, 30-washing and elution area, 40-PCR detection area, 50-waste liquid area, 60-heating position, 101-first pipeline, 102-first waste liquid pipeline, 103-air inlet, 104-liquid inlet, 105-liquid inlet, 201-second pipeline, 202-second waste liquid pipeline, 203-air inlet, 204-liquid inlet, 301-third pipeline, 302-third waste liquid pipeline, 303-air inlet, 304-liquid inlet, 305-liquid inlet, 501-three-way pipeline, 701-first magnetic suction position, 702-second magnetic suction position. DETAILED DESCRIPTION

[0059] The specific embodiments of the present invention will be described in more detail below with reference to the accompanying drawings. Although the specific embodiments of the present invention are shown in the accompanying drawings, it should be understood that the present invention can be implemented in various forms and should not be limited by the embodiments set forth herein. On the contrary, these embodiments are provided in order to enable a more thorough understanding of the present invention and to enable the scope of the present invention to be fully communicated to those skilled in the art.

[0060] To facilitate the understanding of the specific solutions of the present invention, several specific embodiments will be further explained below with reference to the accompanying drawings, and each of the accompanying drawings does not constitute a limitation of the present invention.

[0061] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by technicians in the technical field of the present application. The terms used in the specification of the present application are only for the purpose of describing specific embodiments and are not intended to limit the present application. The term "and / or" used herein includes any and all combinations of one or more related listed items. In addition, the terms "first" and "second" are used for descriptive purposes only and cannot be understood as indicating or implying relative importance or implicitly indicating the number of technical features indicated. In the present application, unless otherwise clearly specified and limited, the terms "installed", "connected", "connected", "fixed" and the like should be understood in a broad sense, for example, it can be a fixed connection, a detachable connection, or an integral body; it can be a mechanical connection or an electrical connection; it can be directly connected or indirectly connected through an intermediate medium, it can be the internal connection of two elements or the interaction relationship between two elements, unless otherwise clearly defined. For ordinary technicians in the field, the specific meanings of the above terms in the present application can be understood according to the specific circumstances.

[0062] Microfluidics refers to the science and technology involved in systems that use microchannels (tens to hundreds of micrometers) to process or manipulate tiny fluids (microliters or even nanoliters in volume). It is an emerging cross-cutting technology involving chemistry, fluid physics, microelectronics, new materials, biology, and biomedical engineering. The system has the following five advantages: ① Integration and automation. Microfluidics can concentrate multiple steps of sample detection on a small chip, and integrate these operation steps through the combination of flow channel size and curvature, microvalves, and cavity design, ultimately making the entire detection integrated miniaturized and automated. ② High throughput. Since microfluidics can be designed as multiple channels, the sample to be tested can be diverted to multiple reaction units at the same time through the microchannel network, and the reaction units are isolated from each other so that each reaction does not interfere with each other. Therefore, multiple items can be tested in parallel on the same sample as needed. Compared with conventional item-by-item testing, it greatly shortens the detection time, improves the detection efficiency, and has the characteristics of high throughput. ③ Low consumption of detection reagents. Due to the miniaturization of integrated detection, the reaction unit cavity on the microfluidic chip is very small. Although the concentration of the reagent formula may be increased by a certain percentage, the amount of reagent used is much lower than that of conventional reagents, which greatly reduces the consumption of reagents. ④ Small sample volume requirement. Since the detection is only completed on a small chip, the amount of sample required to be detected is very small, often only microliters are required. In addition, whole blood can be used directly for testing, which makes it more convenient for infants, the elderly, and the disabled who have little blood and difficulty in venous collection. At the same time, for very precious and rare samples, it is possible to detect multiple indicators. ⑤ Less pollution. The integrated function of the microfluidic chip enables all operations that originally needed to be completed manually in the laboratory to be integrated into the chip automatically, and the pollution of the sample to the environment is reduced to a minimum.

[0063] In a specific embodiment of the present application, the chip includes at least a substrate and a film, and a binding area 10, a vulcanization binding area 20, a washing and elution area 30, and a PCR detection area 40 are constructed in the substrate. The material of the substrate is not specifically limited, and any material that a person skilled in the art considers suitable for making a microfluidic chip can be used, such as polymethyl methacrylate (PMMA), polycarbonate, polystyrene, acrylonitrile-butadiene-styrene copolymer (ABS), cyclic olefin copolymer, polydimethylsiloxane, glass, etc., preferably polymethyl methacrylate 0; the film material is selected from organic polymers, such as polyethylene, polyamide, polysulfone, polyethersulfone, polyvinylidene fluoride, polytetrafluoroethylene, polypropylene, polyimide, polymethyl methacrylate, etc.

[0064] In a specific embodiment of the present application, the bonding area 10 is connected to the sulfurization bonding area 20 through a first pipe 101; the sulfurization bonding area 20 is connected to the washing and elution area 30 through a second pipe 201; the washing and elution area 30 is connected to the PCR detection area 40 through a third pipe 301.

[0065] In a specific embodiment of the present application, a waste liquid area 60 is also constructed in the base 1.

[0066] In a specific embodiment of the present application, the shape of the substrate is not specifically limited, and one side of the substrate is completely hollowed out and completely sealed by a film.

[0067] In a specific embodiment of the present application, the substrate is preferably rectangular, and the length range of the substrate is 60 to 80 mm, for example, it can be 60mm, 61mm, 62mm, 63mm, 64mm, 65mm, 66mm, 67mm, 68mm, 69mm, 70mm, 71mm, 72mm, 73mm, 74mm, 75mm, 76mm, 77mm, 78mm, 79mm, 80mm.

[0068] In a specific embodiment of the present application, the substrate is preferably rectangular, and the width of the substrate is in the range of 22.5 to 32.5 mm, for example, 22.5 mm, 23 mm, 23.5 mm, 24 mm, 24.5 mm, 25 mm, 25.5 mm, 26 mm, 26.5 mm, 27 mm, 27.5 mm, 28 mm, 28.5 mm, 29 mm, 29.5 mm, 30 mm, 30.5 mm, 31 mm, 31.5 mm, 32 mm, 32.5 mm.

[0069] In a specific embodiment of the present application, the substrate is preferably rectangular, and the height range of the substrate is 25 to 45 mm, for example, it can be 25mm, 26mm, 27mm, 28mm, 29mm, 30mm, 31mm, 32mm, 33mm, 34mm, 35mm, 36mm, 37mm, 38mm, 39mm, 40mm, 41mm, 42mm, 43mm, 44mm, 45mm.

[0070] In a specific embodiment of the present application, the volume of the combining area 10 is 2-15 ml, for example, it can be 2 ml, 3 ml, 4 ml, 5 ml, 6 ml, 7 ml, 8 ml, 9 ml, 10 ml, 11 ml, 12 ml, 13 ml, 14 ml, 15 ml.

[0071] In a specific embodiment of the present application, the volume of the vulcanization bonding area 20 is 0.5-2.5 ml, for example, it can be 0.5 ml, 0.6 ml, 0.7 ml, 0.8 ml, 0.9 ml, 1.0 ml, 1.1 ml, 1.2 ml, 1.3 ml, 1.4 ml, 1.5 ml, 1.6 ml, 1.7 ml, 1.8 ml, 1.9 ml, 2.0 ml, 2.1 ml, 2.2 ml, 2.3 ml, 2.4 ml, 2.5 ml.

[0072] In a specific embodiment of the present application, the volume of the washing and elution zone 30 is 0.5-2.5 ml, for example, it can be 0.5 ml, 0.6 ml, 0.7 ml, 0.8 ml, 0.9 ml, 1.0 ml, 1.1 ml, 1.2 ml, 1.3 ml, 1.4 ml, 1.5 ml, 1.6 ml, 1.7 ml, 1.8 ml, 1.9 ml, 2.0 ml, 2.1 ml, 2.2 ml, 2.3 ml, 2.4 ml, 2.5 ml.

[0073] In a specific embodiment of the present application, the volume of the waste liquid area 50 is 3.5-20.5ml, for example, it can be 3.5ml, 4.0ml, 4.5ml, 5.0ml, 5.5ml, 6.0ml, 6.5ml, 7.0ml, 7.5ml, 8.0ml, 8.5ml, 9.0ml, 9.5ml, 10.0ml, 10.5ml, 11.0ml, 11.5ml, 12.0ml, 12.5ml, 13.0ml, 13.5ml, 14.0ml, 14.5ml, 15.0ml, 15.5ml, 16.0ml, 16.5ml, 17.0ml, 17.5ml, 18.0ml, 18.5ml, 19.0ml, 19.5ml, 20.0ml, 20.5ml.

[0074] In a specific embodiment of the present application, the combining area 10 is connected to the combining area 10 through a first waste liquid pipe 102; the waste liquid area 50 is connected to the vulcanization combining area 20 and the washing and elution area 30 through a waste liquid pipe, and the waste liquid pipe is a combination of a second waste liquid pipe 202, a third waste liquid pipe 302 and a three-way pipe 501.

[0075] In a specific embodiment of the present application, the first pipeline, the second pipeline, the third pipeline, the first waste liquid pipeline, the second waste liquid pipeline, and the third waste liquid pipeline are all provided with check valves. There is no specific limitation on the type and structure of the check valves, and all structures that are considered suitable for preventing liquid reflux by those skilled in the art can be used.

[0076] In a specific embodiment of the present application, the material of the first pipe, the second pipe, the third pipe, the first waste liquid pipe, the second waste liquid pipe, the third waste liquid pipe and the three-way pipe is selected from any one of glass, polymethyl methacrylate (PMMA), polycarbonate (PC), polystyrene (PS), and acrylonitrile-butadiene-styrene copolymer (ABS), and the preferred constituent material is polymethyl methacrylate.

[0077] In a specific embodiment of the present application, the cross-section of all the pipes is semicircular, semi-elliptical or rectangular, and is combined with the film to form a closed and complete pipe, and the cross-sectional area of ​​the pipe is 0.7-7mm 2 , for example, it can be 0.7mm 2 , 1.0mm 2 , 1.5mm 2 , 1.7mm 2 , 2.0mm 2 , 2.5mm 2 , 3.0mm 2 、3.7mm 2 , 4.0mm 2 , 4.5mm 2 , 4.7mm 2 , 5.0mm 2 , 5.5mm 2 、5.7mm 2 、6.0mm 2 、6.5mm 2 、6.7mm 2 、7.0mm 2 .

[0078] In a specific embodiment of the present application, the cross-sectional area of ​​the first pipeline 101 is the largest, and the cross-sectional areas of the second pipeline 201 and the third pipeline 301 are smaller than the cross-sectional area of ​​the first pipeline 101. The cross-sectional areas of the second pipeline 201 and the third pipeline 301 can be the same or different.

[0079] In a specific embodiment of the present application, the material of the first pipe 101, the second pipe 201, the third pipe 301, the first waste liquid pipe 102, the second waste liquid pipe 202, the third waste liquid pipe 302, and the three-way pipe 501 is composed of one of polyetheretherketone, polytetrafluoroethylene, fluorinated ethylene propylene, ethylene tetrafluoroethylene, polyethylene, and polypropylene.

[0080] In a specific embodiment of the present application, the chip is further provided with a heating position 60 , and the heating position 60 is arranged around the vulcanization bonding area 20 and the washing and elution area 30 .

[0081] In a specific embodiment of the present application, the chip is also provided with a magnetic attraction position, the first magnetic attraction position 701 is arranged in the space between the bonding area 10 and the vulcanization bonding area 20 in the substrate, and the second magnetic attraction position is arranged in the space between the vulcanization bonding area 20 and the washing and elution area 30 in the substrate.

[0082] Figure 3 , Figure 4 Different structures of liquid storage tanks are shown, among which, Figure 3 The storage capacity of a liquid storage tank shown is 0.5ml~6ml, for example, it can be 0.5ml, 1.0ml, 1.5ml, 2.0ml, 2.5ml, 3.0ml, 3.5ml, 4.0ml, 4.5ml, 5.0ml, 5.5ml, 6.0ml; preferably, it is 1ml~4ml, such as 1.0ml, 1.5ml, 2.0ml, 2.5ml, 3.0ml, 3.5ml, 4.0ml.

[0083] In a specific embodiment of the present application, the first liquid storage tank is composed of a liquid storage tank rubber cover 801, a liquid storage tank aluminum film 802, a first liquid storage tank piston 803, a first liquid storage tank screw cap 805 and a first liquid storage tank shell 804; the liquid storage tank rubber cover 801 is sleeved on one end of the first liquid storage tank piston 803; the liquid storage tank aluminum film 802 is placed between the liquid storage tank rubber cover 801 and the first liquid storage tank piston 803 to ensure the sealing of the liquid storage tank; the other end of the first liquid storage tank piston 803 is communicated with one end of the first liquid storage tank shell 804, and the first liquid storage tank piston 803 can move inside the first liquid storage tank shell 804; the first liquid storage tank screw cap 805 is tightened and fixed to the port of the first liquid storage tank end with a smaller diameter by means of threads; one end of the first liquid storage tank shell 804 is communicated with the first liquid storage tank piston 803, and the other end is a port with a smaller diameter, and the outside of the port is provided with threads.

[0084] like Figure 4 The storage capacity of a liquid storage tank shown is 180-220μl, for example, it can be 180μl, 185μl, 190μl, 195μl, 200μl, 205μl, 210μl, 215μl, 220μl; preferably it is 190-200μl, such as 190μl, 191μl, 192μl, 193μl, 194μl, 195μl, 196μl, 197μl, 198μl, 199μl, 200μl.

[0085] In a specific embodiment of the present application, the second liquid storage tank is composed of a second upper liquid storage tank screw cover 901, a second liquid storage tank piston 902, a second liquid storage tank shell 903, and a second lower liquid storage tank screw cover 904; the second upper liquid storage tank screw cover 901 is tightened and fixed to the port at the end with a larger diameter of the second liquid storage tank by means of threads; one end of the second liquid storage tank piston 902 is communicated with one end of the second liquid storage tank shell 903, and the second liquid storage tank piston 902 can move inside the second liquid storage tank shell 903 by means of external force; the second liquid storage tank is a variable diameter, and a thread is provided on the inner wall of the end with a larger diameter, and the other end is a port with a smaller diameter, and a thread is provided on the outside of the port; the second lower liquid storage tank screw cover 904 is tightened and fixed to the port at the end with a smaller diameter of the second liquid storage tank by means of threads.

[0086] In a specific embodiment of the present application, the second liquid storage tank piston 902 of the second liquid storage tank is a hollow structure, and the inner wall of the second liquid storage tank piston 902 is provided with threads. The first liquid storage tank shell 805 can replace the first liquid storage tank screw cap 901, and the first liquid storage tank and the second liquid storage tank are tightened and fixed by the threads. In this way, when the liquid in the second liquid storage tank is not completely drained, the residual liquid can be discharged with the help of the first liquid storage tank.

[0087] In a specific embodiment of the present application, the side walls of the sulfurization bonding area 20, the washing and elution area 30, the PCR detection area 40 and the waste liquid area 50 are all provided with liquid inlet holes, and the liquid to be used is introduced into the bonding area 10, the sulfurization bonding area 20 or the washing and elution area 30 through the liquid inlet holes.

[0088] In a specific embodiment of the present application, both the first liquid storage tank and the second liquid storage tank can be screwed and fixed to the chip through threads in the liquid inlet hole of the chip.

[0089] like Figure 1As shown in the figure, in a specific embodiment of the present application, the gene methylation microfluidic chip at least includes a substrate and a film, and a binding area 10, a sulfurized binding area 20, a washing and elution area 30, a PCR detection area 40 and a waste liquid area 50 are arranged in the substrate. The binding area 10 is the starting end of the process, and the sample (plasma) can be injected through the circular hole on the top of the binding area 10, and then the lysate is injected through the injection hole 105 on the side of the binding area 10, so that the sample is lysed in the binding area 10; then the magnetic beads and anhydrous ethanol are injected through the injection hole 105 on the side of the binding area; after the binding is completed, the magnetic suction operation is performed at the first magnetic suction position 701, the magnetic beads are adsorbed on the inner wall of the binding area, and the waste liquid is discharged into the waste liquid area 50 through the first waste liquid pipeline 102. The washing operation is performed by injecting washing liquid through the combining area 10, and the washing liquid and the magnetic beads are transferred to the vulcanization combining area 20 through the first pipe 101. At this time, the magnetic suction operation is performed at the second magnetic suction position 702, the magnetic beads are adsorbed on the side wall of the vulcanization combining area, and the waste liquid is passed through the second waste liquid pipe 202 and the three-way pipe 501 to the waste liquid area 50. The eluent is injected through the injection hole 204 on the side of the vulcanization bonding area 20 for elution, and then a magnetic suction operation is performed at the second magnetic suction position 702 to suck the magnetic beads to the top of the hole position of the vulcanization bonding area to separate them from the eluent, and then a protective liquid and sulfite are injected through the injection hole 204 on the side of the vulcanization bonding area, and the vulcanization bonding area is heated through the heating area 60; after a period of time, the magnetic beads and the washing solution are re-injected through the injection hole 204 on the side of the vulcanization bonding area 20 for a combination operation, and after the combination is completed, a magnetic suction operation is performed at the second magnetic suction position 702 to adsorb the magnetic beads on the inner wall of the vulcanization bonding area 20, and the waste liquid in the vulcanization bonding area 20 flows through the second waste liquid pipeline 202 through the three-way pipeline 501 to the waste liquid area 50. The washing liquid is re-injected into the injection hole 204 on the side of the vulcanization bonding area 20 for washing, and the washing liquid and the magnetic beads are transferred to the washing and elution area 30 through the second pipe 201; the magnetic attraction operation is performed at the second magnetic attraction position 702 to adsorb the magnetic beads on the inner wall of the washing and elution area 30, and the washing liquid flows through the third waste liquid pipe 302 through the three-way pipe 501 to the waste liquid area 50. The washing liquid is re-injected through the injection hole 304 on the side of the washing and elution area 30 for washing, and the magnetic attraction operation is performed at the second magnetic attraction position 702 to flow through the third waste liquid pipe 302 through the three-way pipe 501 to the waste liquid area 50, and then the above washing operation is repeated once. After washing is completed, a drying operation is performed, and then the eluent is injected through the injection hole 304 on the side of the washing and elution area 30 to perform the elution operation. After the elution is completed, a magnetic attraction operation is performed at the second magnetic attraction position 702 to adsorb the magnetic beads on the inner wall of the washing and elution area 30, and the eluent flows into the PCR detection area 40 through the third pipe 301 to perform the PCR detection operation.

[0090] Example

[0091] like Figure 1The illustrated gene methylation microfluidic chip has a substrate made of polymethyl methacrylate (PMMA) with a size of length × width × height = 72 mm × 27.5 mm × 35 mm, wherein the surface enclosed by the length and width is used as the bottom surface, and only a film is provided on the bottom surface for sealing, and the film is made of PE material. The substrate is provided with a binding area 10, a vulcanization binding area 20, a washing and elution area 30, a PCR detection area 40, and a waste liquid area 50, and each area is connected through a number of pipes. The volume of the binding area 10 is 11 mm, and a first pipe 101 and a first waste liquid pipe 102 extend from the binding area 10, respectively passing into the vulcanization binding area 20 and the waste liquid area 50. The cross-sections of the first pipe 101 and the first waste liquid pipe 102 are both semicircular, and form a closed and complete pipe with the film. The cross-sectional area of ​​the above two pipes is 4.5 square millimeters. The sulfide bonding area 20 is a cylinder with a diameter of 9 mm and a volume of 2.2 ml. A second pipe 201 extends from the sulfide bonding area 20 to the washing and elution area 30; the washing and elution area 30 is a cylinder with a diameter of 9 mm and a volume of 2.2 ml. A third pipe 301 extends from the washing and elution area 30 to the PCR detection area 40; the sulfide bonding area 20 and the washing and elution area 30 are also respectively provided with a second waste liquid pipe 202 and a third waste liquid pipe 302, which are combined with a three-way pipe 501 and then connected to the waste liquid area 50. The cross-sectional area of ​​the second pipe 201, the third pipe 301, the second waste liquid pipe 202, the third waste liquid pipe 302 and the three-way pipe are all 1.15 square millimeters, and the structure is consistent with the first pipe 101 and the first waste liquid pipe 102. The first magnetic attraction position 701 and the second magnetic attraction position 702 are respectively arranged between the binding area 10 and the vulcanization binding area 20, and between the vulcanization binding area 20 and the washing and elution area 30, and a heating area 60 is arranged around the vulcanization binding area 20 and the washing and elution area 30. The binding area 10, the vulcanization binding area 20, the washing and elution area 30 and the PCR detection area are all provided with different numbers of injection holes and air inlets, wherein the injection holes are used to inject samples, washing liquids and magnetic beads required for the detection process, and the air inlets provide power for the flow of liquid in the microfluidic chip, and all the above pipelines are provided with check valves to prevent liquid reflux.

[0092] The gene methylation microfluidic chip as described above is used for sample PCR detection. The binding area 10 of the chip is the starting end of the process. 3.5 ml of sample (plasma) is injected through the circular hole on the top of the binding area 10, and then 3.5 ml of lysis solution is injected through the injection hole 105 on the side of the binding area 10 to lyse the sample in the binding area 10; then 90 μl of magnetic beads and 2.5 ml of anhydrous ethanol are injected through the injection hole 105 on the side of the binding area; after the binding is completed, a magnetic suction operation is performed at the first magnetic suction position 701 to adsorb the magnetic beads on the inner wall of the binding area, and the waste liquid is discharged into the waste liquid area 50 through the first waste liquid pipeline 102. Through the binding area 10 and injecting 1.5 ml of washing solution A, a washing operation is performed, and the washing solution and magnetic beads are transferred to the vulcanization binding area 20 through the first pipe 101. At this time, a magnetic suction operation is performed at the second magnetic suction position 702 to adsorb the magnetic beads on the side wall of the vulcanization binding area, and the waste liquid is passed through the second waste liquid pipe 202 and the three-way pipe 501 to the waste liquid area 50. 120 μl of eluent is injected through the injection hole 204 on the side of the vulcanization bonding area 20 for elution, and then a magnetic suction operation is performed at the second magnetic suction position 702 to suck the magnetic beads to the top of the hole of the vulcanization bonding area to separate them from the eluent, and then 30 μl of protective solution and 180 μl of sulfite are injected through the injection hole 204 on the side of the vulcanization bonding area, and the vulcanization bonding area is heated through the heating area 60; after a period of time, 20 μl of magnetic beads and 1 ml of washing solution A are re-injected through the injection hole 204 on the side of the vulcanization bonding area 20 for a binding operation, and after the binding is completed, a magnetic suction operation is performed at the second magnetic suction position 702 to adsorb the magnetic beads on the inner wall of the vulcanization bonding area 20, and the waste liquid in the vulcanization bonding area 20 flows through the second waste liquid pipeline 202 through the three-way pipeline 501 to the waste liquid area 50. 800 μl of washing solution is re-injected into the injection hole 204 on the side of the vulcanization bonding area 20 for washing, and the washing solution and magnetic beads are transferred to the washing and elution area 30 through the second pipe 201; the magnetic suction operation is performed at the second magnetic suction position 702 to adsorb the magnetic beads on the inner wall of the washing and elution area 30, and the washing solution flows through the third waste liquid pipe 302 through the three-way pipe 501 to the waste liquid area 50. 800 μl of washing solution B is re-injected through the injection hole 304 on the side of the washing and elution area 30 for washing, and the magnetic suction operation is performed at the second magnetic suction position 702, and the washing solution flows through the third waste liquid pipe 302 through the three-way pipe 501 to the waste liquid area 50, and then 400 μl of washing solution B is re-injected through the injection hole 304 on the side of the washing and elution area 30 for washing, and the magnetic suction operation is performed at the second magnetic suction position 702, and the washing solution flows through the third waste liquid pipe 302 through the three-way pipe 501 to the waste liquid area 50. After washing is completed, a drying operation is performed, and then 60 μl of elution solution is injected through the injection hole 304 on the side of the washing and elution area 30 to perform an elution operation. After the elution is completed, a magnetic attraction operation is performed at the second magnetic attraction position 702 to adsorb the magnetic beads on the inner wall of the washing and elution area 30, and the elution solution flows into the PCR detection area 40 through the third pipe 301 to perform a PCR detection operation.The injection of the above-mentioned samples, washing solutions and magnetic beads is all carried out through the above-mentioned liquid storage tank as a carrier.

[0093] A technical solution and / or a combination of multiple technical solutions in the above embodiments of the present application have the following beneficial effects:

[0094] (1) Technical solution for nucleic acid extraction and methylation detection using microfluidic chips.

[0095] (2) By setting a thin film at the bottom of the substrate and changing the diameter of the pipe for buffering, the problem of large solution flow required in the front methylation experimental area was overcome, and the functional integrity and structural stability were maintained.

[0096] (3) The present application also provides a dedicated liquid storage tank as a carrier of the solution, and the liquid storage tanks of the two structures can be combined and connected.

[0097] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0098] The above-described embodiments only express several implementation methods of the present application, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the present application. It should be pointed out that, for a person of ordinary skill in the art, several variations and improvements can be made without departing from the concept of the present application, and these all belong to the protection scope of the present application. Therefore, the protection scope of the patent of the present application shall be subject to the attached claims.

Claims

1. A gene methylation microfluidic chip, characterized in that: include: A substrate for forming a main body of a microfluidic chip; as well as a thin film located at the bottom of the substrate; in, The substrate is provided with a binding area, a sulfurization binding area, a washing and elution area, and a PCR detection area for performing sample pretreatment, methylation reaction, and finally PCR reaction; A first pipeline, a second pipeline, and a third pipeline are laid on the film to allow the sample to flow between the above-mentioned areas. Wherein, the first pipeline connects the bonding area and the vulcanization bonding area, The second pipeline connects the vulcanization bonding area and the washing and elution area. The third pipeline connects the washing and elution area with the PCR detection area.

2. The chip according to claim 1, wherein: The substrate also includes the following components: Air inlet holes are arranged on the tops of the bonding area, the vulcanization bonding area and the washing and elution area; Magnetic suction positions are respectively arranged in the space between the bonding area and the vulcanization bonding area and between the vulcanization bonding area and the washing and elution area; Heating positions are arranged around the vulcanization bonding area and the washing and elution area; A plurality of liquid inlet holes are arranged on the side walls of the binding area, the sulfurization binding area and the washing and elution area, and samples or other liquids are introduced into the binding area, the sulfurization binding area or the washing and elution area through the liquid inlet holes.

3. The chip according to claim 1 or 2, wherein: Also included in the substrate are: The waste liquid area is connected to the bonding area, the vulcanization bonding area and the washing and elution area respectively through the waste liquid pipeline arranged on the film.

4. The chip according to claim 3, wherein: The waste liquid pipeline includes a first waste liquid pipeline, a second waste liquid pipeline, a third waste liquid pipeline and a three-way pipeline; The first waste liquid pipeline connects the waste liquid outlet of the combined area with the first waste liquid area. The second waste liquid pipeline is connected to the waste liquid outlet of the vulcanization bonding area and the three-way pipeline. The third waste liquid pipeline is connected to the waste liquid outlet of the washing and elution zone and the three-way pipeline. The three ends of the three-way pipeline lead to the vulcanization and bonding area, the washing and elution area and the waste liquid area respectively.

5. The chip according to claim 1 and 4, wherein: The first pipeline, the second pipeline, the third pipeline, the first waste liquid pipeline, the second waste liquid pipeline and the third waste liquid pipeline are all provided with a check valve.

6. The chip according to claim 1, wherein: The substrate is rectangular.

7. The chip according to claim 6, wherein: The substrate is (60-80) mm long, (22.5-32.5) mm wide and (25-45) mm high.

8. The chip according to claim 1, wherein: The volume of the binding area is 2-15 ml, preferably 11 mm.

9. The chip according to claim 1, wherein: The volume of the vulcanization bonding area is 0.5-2.5 ml, and the volume is preferably 2.2 ml.

10. The chip according to claim 1, wherein: The volume of the washing and elution zone is 0.5-2.5 ml, and the volume is preferably 2.2 ml.

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