Digital full-automatic molecular diagnostic microfluidic chip and use method thereof

Abstract

The application discloses a kind of digital full-automatic molecular diagnostic microfluidic chip and its use method, the microfluidic chip includes lower cover and upper cover, lower cover and upper cover sealing fixed connection, sample processing module and amplification reaction module are equipped on lower cover, wherein, sample processing module includes sequentially communicating sample chamber, mixed liquid chamber, first waste liquid pool, and with mixed liquid chamber and first waste liquid pool respectively communicating absolute quantitative chamber, and first flow channel switch is equipped between mixed liquid chamber and first waste liquid pool;Amplification reaction module includes sequentially communicating relative quantitative chamber, reagent mixing pool, reaction detection pool and second waste liquid pool, wherein relative quantitative chamber is communicated with mixed liquid chamber and first waste liquid pool respectively, and second flow channel switch is equipped between relative quantitative chamber and mixed liquid chamber, and reaction detection pool is integrally formed by upper surface microreaction unit with lower cover injection molding and is formed by superconductive heat insert.The upper cover is equipped with several openings, respectively with sample chamber, mixed liquid chamber, reagent mixing pool, waste liquid pool and so on corresponding communication.This microfluidic chip has the advantages of low cost, simple operation, rapid and accurate detection, and is conducive to the popularization and application of scale, commercialization in molecular diagnostic field.

Description

Technical Field

[0001] The present invention belongs to the technical field of molecular diagnosis, and in particular relates to a digital fully automatic molecular diagnosis microfluidic chip and a method for using the same. Background Art

[0002] In recent years, digital PCR (dPCR) technology has been widely used in prenatal diagnosis, tumor screening, liquid biopsy and other disease fields. It is a very promising digital molecular diagnostic technology. Unlike traditional fluorescent quantitative PCR, dPCR does not rely on standard curves and reference genes, but directly detects the copy number of the target sequence. The detection limit can reach a single copy. It is an absolute quantitative technology for nucleic acid molecules with the advantages of high sensitivity, absolute quantification, high stability and repeatability. The detection process of dPCR mainly includes nucleic acid extraction, dispersion of reaction mixture, amplification reaction and signal reading. Traditional dPCR must rely on special and expensive instruments and equipment in each of the above steps. The entire experimental process needs to be completed manually in a clean environment. Not only are the steps cumbersome and the experiment time-consuming, but because it is a manual operation, the sample is easily contaminated and operational errors occur, resulting in poor accuracy of the final detection data.

[0003] Therefore, it is necessary to develop a dPCR detection platform that can integrate sample preparation, reaction solution dispersion, reaction amplification and fluorescence detection, so as to reduce detection costs, speed up detection, simplify the workflow, and enable the entire operation process to be carried out in a closed space, which neither pollutes the environment nor the samples to be tested are contaminated by the environment, thereby making the dPCR detection results more credible and facilitating the large-scale and commercial operation of this technology route. It is a long-awaited expansion platform in the field of molecular diagnosis and a problem that urgently needs to be solved in this field.

[0004] The rapid development of microfluidics technology has made it possible to address these challenges. Microfluidics integrates essential laboratory functions in biology and chemistry, such as sample preparation, dispersion, reaction, and detection, onto a microfluidic chip measuring just a few square centimeters. With the help of auxiliary equipment, the analysis process can be fully automated. Microfluidic chips offer advantages such as low sample consumption, rapid detection, ease of operation, multifunctional integration, compact size, and portability. These chips enable the dPCR process, previously performed in specialized laboratories, to be performed anywhere. Currently, a representative example is Bio-Rad's QX-200 Digital PCR System, but its integration is limited, requiring droplets to be transferred between instruments. Its subsequent all-in-one digital PCR system, the QX ONE, integrates both droplet preparation and reaction steps into a single system, but lacks nucleic acid extraction capabilities and is very expensive. Another representative example is Thermo Fisher Scientific's QuantStudio 3D Digital PCR System, which similarly lacks nucleic acid extraction and reaction mixture preparation integrated into the system chip. Summary of the Invention

[0005] To solve the above problems, the purpose of the present invention is to provide a digital fully automatic molecular diagnostic microfluidic chip and its use method that utilizes microfluidic technology to integrate nucleic acid extraction, preparation of reaction mixture, dispersion of reaction mixture, amplification of target sequence, and reading of fluorescence signal.

[0006] In order to achieve the above object, the technical solution adopted by the present invention is as follows:

[0007] On one hand, the present invention provides a digital fully automatic molecular diagnostic microfluidic chip, comprising a lower cover and an upper cover, wherein the upper cover is tightly attached to the surface of the lower cover and is sealed and fixedly connected to the upper cover. The lower cover is provided with a sample processing module and an amplification reaction module, wherein the sample processing module comprises a sample chamber, a mixing chamber, an absolute quantification chamber, and a first waste liquid tank, wherein the sample chamber, the mixing chamber, and the first waste liquid tank are sequentially connected, and a first flow channel switch is provided between the mixing chamber and the first waste liquid tank, and the absolute quantification chamber is respectively connected to the mixing chamber and the first waste liquid tank;

[0008] The amplification reaction module includes a relative quantitative chamber, a reagent mixing tank, a reaction detection tank, and a second waste liquid tank, which are connected in sequence. The relative quantitative chamber is connected to the mixing chamber and the first waste liquid tank respectively, and a second flow channel switch is provided between the relative quantitative chamber and the mixing chamber. The reaction detection tank is formed by injection molding a superconducting heat-conducting insert with a micro-reaction unit on the upper surface and the lower cover as a whole.

[0009] The upper cover is provided with openings, the openings include a sample liquid adding port, a lysis liquid adding port, a pressurizing port, a washing liquid adding port, a magnetic bead adding port, an eluent adding port, a mixed liquid chamber pressurizing exhaust port, a mixing pool pressurizing exhaust port, a first waste liquid pool exhaust port and a second waste liquid pool exhaust port, wherein the sample liquid adding port, the lysis liquid adding port and the pressurizing port are communicated with the sample chamber respectively, the washing liquid adding port, the magnetic bead adding port, the eluent adding port and the mixed liquid chamber pressurizing exhaust port are communicated with the mixed liquid chamber respectively, the eluent adding port is also communicated with the absolute quantification chamber, and the absolute quantification chamber is located between the mixed liquid chamber and the eluent adding port, and the mixing pool pressurizing exhaust port, the first waste liquid pool exhaust port and the second waste liquid pool exhaust port are communicated with the reagent mixing pool, the first waste liquid pool and the second waste liquid pool respectively.

[0010] A transparent window is further arranged on the upper cover corresponding to the position of the reaction detection pool, for reading the signal after the amplification reaction is completed.

[0011] In a preferred embodiment of the present application, the thermal conductivity of the superconducting thermal insert is greater than 5, and the upper surface of the superconducting thermal insert is hydrophilic or hydrophobic.

[0012] In a preferred embodiment of the present application, the sample chamber is provided with a lysis and absorption element, which is used for absorbing and releasing the sample to be tested and the lysis liquid to make them fully contact and lysis.

[0013] In a preferred embodiment of the present application, the materials of the upper cover and the lower cover are both high molecular materials, which are selected from one or a combination of polycarbonate, polymethyl methacrylate and acrylonitrile-butadiene-styrene plastic, and the upper cover is transparent.

[0014] In a preferred embodiment of the present application, the upper cover includes an upper cover plate and an intermediate sealing element, the intermediate sealing element is installed between the upper cover plate and the lower cover, the intermediate sealing element is provided with through holes corresponding to the openings and window hole positions corresponding to the positions of the reaction detection pool and the transparent window, and is further provided with a first flow channel switch and a second flow channel switch matched with the corresponding positions of the lower cover.

[0015] In a preferred embodiment of the present application, the intermediate sealing element is a soft rubber film, and the material of the soft rubber film is a soft material.

[0016] In a preferred embodiment of the present application, the micro-reaction units formed on the upper surface of the superconducting thermal insert are honeycomb-shaped micro-holes, the number of the micro-holes is greater than 8000, the depth of the micro-holes is greater than 0.01 μm, the volume of the micro-holes is less than 0.15 nL, and the shape of the micro-holes is hexagonal, square, circular or triangular.

[0017] In a preferred embodiment of the present invention, the micro-reaction units formed on the upper surface of the superconducting thermal insert are micro-droplets formed by droplet generation technology, the number of the micro-droplets is greater than 10,000, and the volume of the micro-droplets is less than 0.15 nL.

[0018] In a preferred embodiment of the present invention, flow channel plugs are respectively provided between the reaction detection pool and the reagent mixing pool and the second waste liquid pool.

[0019] Another aspect of the present invention further provides a method for using the microfluidic chip, comprising the following steps:

[0020] Step 1: Add the sample to be tested and the lysis solution into the sample chamber to lyse the sample to be tested;

[0021] Step 2: The lysed sample to be tested is hydraulically injected into the mixing chamber, and the target fragment is extracted from the sample using a magnetic bead method;

[0022] Step 3: Based on the preliminary experiment, the volume of the eluent for eluting the target fragment is controlled by the absolute quantitative chamber to obtain a test liquid containing the target fragment with a known concentration;

[0023] Step 4: Press the test liquid containing the target fragment into the relative quantification chamber and the reagent mixing tank in sequence, and mix it with the diagnostic reagent in the reagent mixing tank to obtain a reaction mixture;

[0024] Step 5: The reaction mixture in the reagent mixing pool is fluidized into the micro-reaction unit of the superconducting thermal insert of the reaction detection pool and a heating amplification reaction is performed. After the reaction is completed, the fluorescence signal is read through the transparent window for statistical analysis.

[0025] Compared with the prior art, the present invention has the following beneficial effects:

[0026] (1) The digital method fully automatic molecular diagnostic microfluidic chip provided by the present invention integrates the entire operation process of dPCR detection, including nucleic acid extraction, reaction mixture preparation, reaction mixture dispersion, target sequence amplification, and fluorescence signal reading, on a chip. The technician only needs to add the sample to be tested to the chip and then put the chip into the auxiliary equipment. The subsequent work process is all completed automatically by the auxiliary equipment, without the need for tedious and challenging experimental operations. Using the technology of the present invention, first, time can be saved and test results can be obtained quickly; second, experimental errors caused by human operation can be avoided, making the test results more accurate; third, the entire detection process is completed in a closed chip, and the sample is not easily contaminated, nor will it pollute the environment. The detection process that originally needed to be completed in a special laboratory can be completed anywhere; fourth, the demand for samples and reagents is small. Due to the small size of the microfluidic chip, the required sample volume and reagent volume are also greatly reduced. On the one hand, it is especially important for precious or difficult-to-obtain samples, and on the other hand, the reduction in the amount of reagents reduces the detection cost.

[0027] (2) Since the principle of digital molecular diagnosis is to divide the test agent into several tiny reaction units, each of which contains or does not contain the target sequence, thereby achieving absolute quantification of the target sequence, the more micro-reaction units there are, the higher the sensitivity and accuracy of the detection. The digital fully automatic molecular diagnostic microfluidic chip provided by the present invention can contain up to tens of thousands of micro-reaction units in its reaction detection pool, further improving the sensitivity and accuracy of the detection.

[0028] (3) The present invention is the first to use the embedded combined injection molding process of superconductors and polymer materials to generate various forms of micro-reaction units (honeycomb-shaped micropores, micro-droplets) on a digital fully automatic molecular diagnostic microfluidic chip. The superconductor can quickly transfer the temperature to the reaction mixture, which saves amplification time on the one hand and ensures the accuracy of the amplification temperature on the other hand, thereby further improving the accuracy of the detection results.

[0029] (4) The digital fully automatic molecular diagnostic microfluidic chip provided by the present invention can be mass-produced through an assembly line, and its auxiliary equipment does not need to rely on expensive imported equipment, which can greatly reduce the detection cost. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 Schematic diagram of the microfluidic chip structure of Example 1;

[0031] Figure 2 This is a front view of the microfluidic chip of Example 1;

[0032] Figure 3 This is a side view of the microfluidic chip of Example 1;

[0033] Figure 4 Schematic diagram of the assembly structure of the microfluidic chip of Example 1;

[0034] Figure 5 Schematic diagram of the microfluidic chip structure of Example 2.

[0035] In the figure: 1. Lower cover; 11. Sample chamber; 12. Mixing chamber; 13. First waste liquid tank; 14. Absolute quantification chamber; 15. Relative quantification chamber; 16. Reagent mixing tank; 17. Reaction detection tank; 170. Superconducting thermal insert; 171. Micropore; 172. Microdroplet; 18. Second waste liquid tank; 19. Sensor; 2. Upper cover; 201. Upper cover plate; 202. Middle seal; 21. Window hole; 22. Sample Liquid addition port; 22, lysis solution addition port; 23, pressurization port; 24, washing solution addition port; 25, magnetic beads addition port; 26, elution solution addition port; 27, mixing chamber pressurization exhaust port; 28, mixing tank pressurization exhaust port; 29, first waste liquid tank exhaust port; 210, second waste liquid tank exhaust port; 211, transparent window; 212, air avoidance hole; 301, first flow channel switch; 302, second flow channel switch; 303, flow channel plug. DETAILED DESCRIPTION

[0036] In order to make the purpose and technical solution of the present invention clearer and more complete, the present invention is further described in detail below in conjunction with the embodiments. Obviously, the embodiments described are only part of the embodiments of the present invention, rather than all the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention. Those skilled in the art can make modifications or equivalent substitutions based on the understanding of the technical solution of the present invention without departing from the spirit and scope of the technical solution of the present invention, and all belong to the scope of protection of the present invention.

[0037] It should be understood that, in the description of the present invention, the terms "upper", "lower", "front", "back", "inside", "outside", etc., indicating the orientation or position relationship, are based on the orientation or position relationship shown in the accompanying drawings, and are only for the convenience of describing the present invention and simplifying the description, rather than indicating or implying that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and therefore cannot be understood as a limitation on the present invention.

[0038] It should be noted that, in the description of the present invention, unless otherwise clearly specified and limited, the terms "installed", "connected" and "connected" should be understood in a broad sense. For example, it can be a fixed connection, an integral connection, or a detachable connection; it can be the internal connection of two components; it can be a direct connection or an indirect connection through an intermediate medium. For ordinary technicians in this field, the specific meanings of the above terms in the present invention can be understood according to specific circumstances.

[0039] If no specific techniques or conditions are specified in the following examples, the experiments were carried out according to the techniques or conditions described in the literature in the field or according to the product instructions. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0040] Example 1

[0041] This embodiment provides a digital fully automatic molecular diagnosis microfluidic chip, the structure of which is as follows: Figures 1-4 As shown, the device comprises a lower cover 1 and an upper cover 2, with the upper cover 2 being tightly attached to the surface of the lower cover 1. The lower cover 1 and the upper cover 2 are sealed and fixedly connected, specifically by conventional bonding, welding, bonding, or interlocking methods in the art. Both the lower cover 1 and the upper cover 2 are made of polymer materials, such as polycarbonate (PC), polymethyl methacrylate (PMMA), acrylonitrile butadiene styrene (ABS), or a combination thereof. It should be noted that in order to achieve fluorescence detection, the upper cover 2 must be made of a highly transparent material, while the lower cover 1 is not particularly limited.

[0042] like Figure 1 As shown, in this embodiment, a sample processing module and an amplification reaction module are provided on the lower cover 1. The functions of the sample processing module and the amplification reaction module require the cooperation of the lower cover 1 and the upper cover 2. The sample processing module includes a sample chamber 11, a mixing chamber 12, an absolute quantification chamber 14, and a first waste liquid tank 13, which are provided on the lower cover 1. The sample chamber 11, the mixing chamber 12, and the first waste liquid tank 13 are connected in sequence through microfluidic channels, and a first flow channel switch 301 is provided on the microfluidic channel between the mixing chamber 12 and the first waste liquid tank 13. One end of the absolute quantification chamber 14 is connected to the mixing chamber 12 through a microfluidic channel, and the other end is connected to the first waste liquid tank 13 through a microfluidic channel.

[0043] The amplification reaction module includes a relative quantitative chamber 15, a reagent mixing pool 16, a reaction detection pool 17 and a second waste liquid pool 18 opened on the lower cover 1. The relative quantitative chamber 15, the reagent mixing pool 16, the reaction detection pool 17 and the second waste liquid pool 18 are connected in sequence through microchannels, wherein the reaction detection pool 17 is injection molded as one piece with the lower cover 1 through a superconducting heat-conducting insert 170 whose upper surface can form a microreaction unit. One end of the relative quantitative chamber 15 is connected to the mixing chamber 12 through a microchannel, and the other end is connected to the first waste liquid pool 13 through a microchannel, and a second flow channel switch 302 is provided on the microchannel between the relative quantitative chamber 15 and the mixing chamber 12. It should be noted that there is no special limit on the number of reaction detection pools 17, which is at least one. The specific number can be adjusted according to the detection needs and the chip size, such as two, three, four, eight, etc. Correspondingly, the number of reagent mixing pools 16 and relative quantitative chambers 15 is adjusted according to the reaction detection pool 17 and kept consistent therewith. In the chip structure of this embodiment, four reaction detection pools 17, reagent mixing pools 16 and relative quantitative chambers 15 are respectively provided. In addition, the above-mentioned microchannels are all fine pores opened on the surface of the lower cover 1, and the fine pores are covered by the upper cover 2 to form a sealed microchannel space. It should be pointed out that the specific position of each structure can be adjusted according to actual needs and there is no special limit.

[0044] like Figures 1-2 As shown, a number of openings are provided on the upper cover 2, including a sample liquid addition port 21, a lysate addition port 22, a pressurization port 23, a washing liquid addition port 24, a magnetic bead addition port 25, an eluent addition port 26, a mixing chamber pressurization exhaust port 27, a mixing pool pressurization exhaust port 28, a first waste liquid pool exhaust port 29, and a second waste liquid pool exhaust port 210, wherein the sample liquid addition port 21, the lysate addition port 22, and the pressurization port 23 are respectively connected to the sample chamber 11; the washing liquid addition port 25 is connected to the sample chamber 11; The inlet 24, the magnetic bead addition port 25, the eluent addition port 26 and the mixing chamber pressurization exhaust port 27 are respectively connected to the mixing chamber 12, the eluent addition port 26 is also connected to the absolute quantitative chamber 14, and the absolute quantitative chamber 14 is located between the mixing chamber 12 and the eluent addition port 26; the mixing tank pressurization exhaust port 28, the first waste liquid tank exhaust port 29 and the second waste liquid tank exhaust port 210 are respectively connected to the reagent mixing tank 16, the first waste liquid tank 13 and the second waste liquid tank 18. The specific number and position of the above-mentioned openings can be adjusted as needed, and their functions can also be adjusted to a liquid addition port, a pressurization port, an exhaust port or a combination of several functions as needed. In addition, a transparent window 211 is also provided on the upper cover 2 corresponding to the position of the reaction detection tank 17 of the lower cover 1, so as to be used to read the signal after the reaction is completed.

[0045] It is worth noting that, combined with Figures 1-4In the embodiment, the upper cover 2 is composed of an upper cover plate 201 and an intermediate sealing member 202, and the intermediate sealing member 202 is used to seal the grooves (such as the sample chamber 11, the mixing chamber 12, the first waste liquid pool 13, etc.) and the micro flow channel on the lower cover 1, so as to isolate them from the outside air. The intermediate sealing member 202 is installed between the upper cover plate 201 and the lower cover 1, and has through holes corresponding to the openings (such as the sample liquid adding port 21, the lysis liquid adding port 22, the pressurizing port 23, etc.) on the upper cover plate 201, and a window hole position 2021 corresponding to the positions of the reaction detection pool 17 of the lower cover 1 and the transparent window 211 of the upper cover plate 201. It should be noted that the first flow channel switch 301 and the second flow channel switch 302 are both composed of the lower cover 1 and the intermediate sealing member 202, as shown in Figure 1 and Figure 4 , the micro flow channel on the lower cover 1 is provided with a breaking point at the corresponding position, and the intermediate sealing member 202 is provided with a switch structure matched with the breaking point at the corresponding position, so as to constitute a switch element for controlling the micro flow channel. In addition, the upper cover plate 201 is also provided with a clearance hole 212 corresponding to the positions of the first flow channel switch 301 and the second flow channel switch 302. In the embodiment, the intermediate sealing member 202 is a soft rubber film, and the material is a soft material, not limited to silicone. In other embodiments, the upper cover plate 201 and the intermediate sealing member 202 can be an integrated structure that cannot be separated, and at this time, the upper cover 2 is provided with a soft material such as silicone at the corresponding positions of the grooves and the micro flow channel of the lower cover 1, and the purpose is to form a sealed space and block the connection with the outside.

[0046] Specifically, the sample chamber 11 is used to collect and lyse the sample to be tested, and the sample to be tested can be a whole blood sample, a throat swab, etc. The sample chamber 11 is provided with a lysis and release cotton, which has a hydrophilic effect and is used to absorb the sample to be tested and the lysis liquid, so that the sample to be tested and the lysis liquid are in full contact and lysis. As shown in Figure 1 and Figure 2 , the upper cover 2 corresponding to the position of the sample chamber 11 is provided with a sample liquid adding port 21, through which the sample to be tested can be added to the sample chamber 11. The upper cover 2 is also provided with a lysis liquid adding port 22 and a pressurizing port 23, which are respectively connected with the sample chamber 11 through the micro flow channel. The pressurizing port 23 is used to drive the sample liquid in the sample chamber 11 into the mixing chamber 12 through pressure, so as to perform the next purpose fragment extraction work.

[0047] Further, the mixing chamber 12 is connected with the sample chamber 11 through the micro flow channel, and the mixing chamber 12 is used to further lyse the sample to be tested and extract the purpose fragment from the lysed sample liquid. In the embodiment, the magnetic bead method is used to extract the purpose fragment from the sample to be tested in the mixing chamber 12, in combination with Figure 1 , Figure 2 and Figure 4, a washing liquid addition port 24, a magnetic bead addition port 25 and an eluent addition port 26 are provided on the upper cover 2, which are connected to the mixing chamber 12, and these three addition ports are connected to the same microchannel, wherein the eluent addition port 26 is located at the upstream end, and there is no special requirement for the position of the other two addition ports. At the same time, the eluent addition port 26 is a multifunctional hole, which can be used for adding liquid and can also be switched to a pressurization port or an exhaust port. In some other embodiments, the washing liquid addition port 24 and the magnetic bead addition port 25 can also be connected to the mixing chamber 12 separately. In addition, an electromagnet is also provided in the mixing chamber 12, which can adsorb the magnetic beads when energized, so as to facilitate subsequent washing and elution operations. The electromagnet can be set at the outer bottom of the mixing chamber 12, or on the outer side wall, and there is no special requirement for the position. As Figures 1-2 As shown, a mixing chamber pressurization exhaust port 27 is further provided on the upper cover 2, and its function is to pressurize the mixing chamber 12 and drive the flow of liquid through pressure.

[0048] It is important to understand that if Figure 1 As shown, absolute quantification chamber 14 is positioned between mixing chamber 12 and eluent inlet 26. The purpose of absolute quantification chamber 14 is to precisely control the volume of eluent used to elute the target fragments adsorbed on the magnetic beads based on preliminary experiments, thereby providing a known concentration of the target fragments in the test liquid. Therefore, the volume of absolute quantification chamber 14 is predetermined. Furthermore, absolute quantification chamber 14 is connected to first waste liquid reservoir 13 via a microchannel, allowing excess eluent to be discharged into the first waste liquid reservoir 13.

[0049] Furthermore, if Figure 1 As shown, the first waste liquid pool 13 is connected to the mixing chamber 12 via a microchannel, and a first channel switch 301 is provided on the microchannel to control the connection or shutoff between the two. The first waste liquid pool 13 is used to collect waste liquid generated during the extraction of the target fragment in the mixing chamber 12. It is worth noting that in this embodiment, the first waste liquid pool 13 is designed in a multi-step form, such as Figure 4 As shown, the first waste liquid tank 13 is composed of three sequentially connected primary waste liquid tanks, a secondary waste liquid tank, and a tertiary waste liquid tank. Concave communication notches are provided on both sides of the secondary waste liquid tank to prevent waste liquid from overflowing from the exhaust port during operation. In other embodiments, only the primary and secondary waste liquid tanks may be retained as needed.

[0050] Since different diagnostic reagents require different amounts of nucleic acid templates, a relative quantitative chamber 15 is provided between the mixing chamber 12 and the reagent mixing pool 16. The volume of the relative quantitative chamber 15 is set according to the requirements of the diagnostic reagent and the concentration of the target fragment in the liquid to be tested. Figure 1As shown, the four relative quantitative chambers 15 in this embodiment have the same structure and size, are connected in sequence, and the relative quantitative chamber 15 located at one end is connected to the mixing chamber 12 through a microchannel. A second channel switch 302 is provided on the microchannel to control the connection or closing between the two. The relative quantitative chamber 15 located at the other end is connected to the first waste liquid pool 13 through a microchannel. This structure allows the test liquid containing the target fragment to flow from the mixing chamber 12 into the relative quantitative chamber 15 in sequence until the last relative quantitative chamber 15 is filled, and the excess liquid is discharged into the first waste liquid pool 13.

[0051] Furthermore, if Figure 1 As shown, in this embodiment, the reagent mixing pool 16 located between the relative quantitative chamber 15 and the reaction detection pool 17 is used to mix the test liquid containing the target fragment and the diagnostic reagent before the amplification test. A mixing pool pressurization and exhaust port 28 is provided on the upper cover 2 corresponding to the position of the reagent mixing pool 16. It is also a multifunctional hole that can be used for adding liquid, pressurizing and exhausting. The diagnostic reagent in this embodiment is a freeze-dried ball pre-buried in the reagent mixing pool 16. The diagnostic reagent can also be added through the mixing pool pressurization and exhaust port 28 during the detection process.

[0052] Furthermore, the reaction detection pool 17 is used for amplification of target sequences and reading of fluorescence signals, such as Figure 1 As shown, in this embodiment, the reaction detection pool 17 is connected to the reagent mixing pool 16 through the corresponding microchannel, and the reaction mixture obtained by mixing the test liquid containing the target fragment and the diagnostic reagent into the corresponding reaction detection pool 17 is pressurized by the mixing pool pressurization exhaust port 28. Figure 1 and Figure 4 In this embodiment, the micro-reaction units formed on the upper surface of the superconducting thermal insert 170 are honeycomb-shaped micropores 171, numbering greater than 8,000 and having a depth greater than 0.01 μm. The volume of each micropore 171 is less than 0.15 nL. The shape of micropores 171 is not limited and can be hexagonal, square, circular, triangular, or other shapes. During amplification, the superconducting thermal insert 170 can quickly transfer temperature to the reaction mixture, improving amplification speed and accuracy. Preferably, the thermal conductivity of the superconducting thermal insert 170 is greater than 5.

[0053] It should be noted that because the honeycomb-shaped micropores 171 have been hydrophilically modified, when the reaction mixture flows from the reagent mixing pool 16 through the microfluidic channel into the reaction detection pool 17, each micropore 171 can be filled, and the excess reaction mixture finally flows through the microfluidic channel into the second waste liquid pool 18. Each reaction detection pool 17 can be equipped with a waste liquid pool. In other embodiments, multiple reaction detection pools 17 can also share a waste liquid pool.

[0054] The workflow and principle of the digital fully automatic molecular diagnostic microfluidic chip of this embodiment are as follows:

[0055] (1) The sample liquid to be tested is dripped onto the lysis-absorbing cotton in the sample chamber 11 from the sample liquid addition port 21. Subsequently, the chip is inserted into the chip port of the auxiliary device. The chip is provided with a sensor 19 to help determine whether the chip is properly placed. The reagent mixing pool 16 of the chip is pre-embedded with a diagnostic reagent freeze-dried ball. The auxiliary device is covered and operated. All subsequent procedures are automatically carried out under the control of the auxiliary device;

[0056] (2) A prescribed volume of lysate is added from the lysate inlet 22. After the auxiliary device provides a vibration function for about 2 seconds, the pressure port 23 is pressurized to force the lysed sample into the mixing chamber 12. During this process, the lysate first flows onto the lysing absorbent cotton in the sample chamber 11 and contacts the sample. The vibration function allows the lysate to fully lyse the sample.

[0057] (3) Add a specified amount of magnetic bead liquid from the magnetic bead adding port 25, and after the auxiliary device provides a vibration function for about 2 seconds, energize the electromagnet located at the bottom of the mixing chamber 12, turn on the electromagnet function for about 2 seconds, and then turn off the vibration function; during this process, the magnetic bead liquid flows into the mixing chamber 12, the vibration function enables the magnetic beads to fully absorb the target fragments in the lysed sample liquid, and the electromagnet function absorbs the magnetic beads to the bottom of the mixing chamber 12;

[0058] (4) Open the first flow channel switch 301 and the first waste liquid pool exhaust port 29, keep the second flow channel switch 302 in the closed state, pressurize the pressurizing port 23, and pressurize the waste liquid in the mixing chamber 12 into the first waste liquid pool 13 through the waste liquid dedicated microchannel. After the waste liquid is drained, turn off the electromagnet function and release the magnetic beads;

[0059] (5) Add a specified volume of washing liquid from the washing liquid inlet 24, and the auxiliary device provides a vibration function for about 2 seconds, then turns on the electromagnet function, and then turns off the vibration function again after about 2 seconds; during this process, the washing liquid flows into the mixing chamber 12, and the vibration function allows the washing liquid to fully wash the magnetic beads with the target fragments, and the electromagnet function adsorbs the magnetic beads to the bottom of the mixing chamber 12 again;

[0060] (6) Pressurizing the pressure port 23, the waste liquid in the mixing chamber 12 is again pressed into the first waste liquid pool 13 through the waste liquid dedicated microchannel. After the waste liquid is drained, the electromagnet function is turned off to release the magnetic beads, and the first flow channel switch 301 and the first waste liquid pool exhaust port 29 are closed at the same time;

[0061] (7) Open the exhaust valve of the pressurized exhaust port 27 of the mixing chamber, then switch the eluent addition port 26 to the gas addition state, pressurize the microchannel through the eluent addition port 26, and remove the residual liquid in the microchannel into the mixing chamber 12; then, turn on the electromagnet function, close the exhaust valve of the pressurized exhaust port 27 of the mixing chamber, then open the first flow channel switch 301 and the first waste liquid pool exhaust port 29, pressurize the pressurized port 23, and discharge the residual liquid into the first waste liquid pool 13 through the waste liquid-dedicated microchannel, and turn off the electromagnet function;

[0062] (8) Close the first flow channel switch 301, the exhaust port 29 of the first waste liquid pool is still in the exhaust state, and the eluent addition port 26 is switched to the liquid addition state. Add eluent greater than the specified amount to the absolute quantitative chamber 14, and the excess eluent flows into the first waste liquid pool 13 through the microchannel above the absolute quantitative chamber 14;

[0063] (9) Switch the eluent addition port 26 to the gas addition state, pressurize the microchannel through the eluent addition port 26, and pressurize all the residual liquid in the microchannel into the first waste liquid pool 13;

[0064] (10) Open the exhaust valve of the pressurized exhaust port 27 of the mixing chamber, close the exhaust port 29 of the first waste liquid pool, and pressurize the eluent addition port 26 to pressurize the eluent in the absolute quantitative chamber 14 into the mixing chamber 12. After the vibration function of the device is turned on for about 2 seconds, the electromagnet function is turned on, and the vibration function is turned off after about 2 seconds. During this process, the vibration function allows the target fragments adsorbed by the magnetic beads to be fully eluted by the eluent, and the electromagnet function adsorbs the magnetic beads to the bottom of the mixing chamber 12.

[0065] (11) Open the first waste liquid pool exhaust port 29 and the second flow channel switch 302, switch the mixing chamber pressurization exhaust port 27 to the gas pressurization state, and allow the eluted test liquid containing the target fragment to flow through the microchannel dedicated to the purification liquid and the four relative quantitative chambers 15 into the first waste liquid pool 13, and completely remove any residual liquid in the microchannel;

[0066] (12) Close the first waste liquid pool exhaust port 29, first open the mixing pool pressurized exhaust port 28 on the reagent mixing pool 16 which is opposite to the one end of the quantitative chamber 15 and the mixing liquid chamber 12, pressurize the mixing liquid chamber pressurized exhaust port 27 to press all the test liquid containing the target fragment in the opposite quantitative chamber 15 into the corresponding reagent mixing pool 16; then, close the mixing pool pressurized exhaust port 28, open the next mixing pool pressurized exhaust port 28 in turn to press all the test liquid containing the target fragment in the corresponding opposite quantitative chamber 15 into the reagent mixing pool 16, operate in turn until all the test liquid containing the target fragment in all the opposite quantitative chamber 15 is pressed into the corresponding reagent mixing pool 16, then close all the mixing pool pressurized exhaust port 28 and the second flow channel switch 302; open the vibration mode to make the chip vibrate to make the target fragment and the diagnostic reagent fully mixed to form a reaction mixture;

[0067] (13) Open the second waste liquid pool exhaust port 210, switch the mixing pool pressurized exhaust port 28 to the gas pressurized state, pressurize the reagent mixing pool 16, and make the reaction mixture in the reagent mixing pool 16 flow into the corresponding reaction detection pool 17 along the micro flow channel. Since the honeycomb-shaped micro holes 171 on the reaction detection pool 17 are modified by hydrophilicity, the reaction mixture can fill each micro hole 171 after flowing through the reaction detection pool 17, and the excess reaction mixture is discharged into the second waste liquid pool 18 through the micro flow channel;

[0068] (14) The auxiliary equipment provides the temperature conditions required for the cyclic amplification reaction, completes all the cyclic amplification reactions, and then the auxiliary equipment reads the fluorescence information in the reaction detection pool 17 through the transparent window 211 for analysis and processing; the number of cycles required for the reaction is set according to the requirements of the diagnostic reagent, and after the reaction is completed, the chip is automatically ejected.

[0069] Example 2

[0070] As Figures 1-5As shown, the structure of the digital fully automatic molecular diagnostic microfluidic chip provided in this embodiment is basically the same as that in Example 1, except that the upper surface of the superconducting heat-conducting insert 170 of the reaction detection pool 17 in this embodiment is a smooth mirror surface and is hydrophobically modified, and the micro-reaction unit thereon is a micro-droplet 172 generated by the step emulsification technology (Assembled Step Emulsification Device for Multiplex Droplet Digital PCR[J].Analytical Chemistry,2019,91(3).DOI:10.1021 / acs.analchem.8b04313.), the number of micro-droplets 172 in each reaction detection pool 17 is greater than 10,000, and the volume of a single micro-droplet 172 is less than 0.15nL. Specifically, a structure for forming micro-droplets is provided around the superconducting thermal insert 170 of the reaction detection tank 17. When the aqueous reaction mixture in the reagent mixing tank 16 flows into the oil phase on the upper surface of the superconducting thermal insert 170 of the reaction detection tank 17, independent oil-in-water micro-droplets 172 are continuously generated. Therefore, the oil phase needs to be pre-placed on the upper surface of the superconducting thermal insert 170 of the reaction detection tank 17 during chip packaging. At the same time, the excess aqueous reaction mixture is discharged into the second waste liquid tank 18 through the microchannel. It should be noted that the upper surface of the superconducting thermal insert 170 cooperates with the upper cover 2 to form a cavity. The spatial distance of the cavity (between the upper surface of the superconducting thermal insert 170 and the lower surface of the upper cover 2) must be no greater than 1 / 5 of the diameter of the micro-droplets 172 formed. The purpose is to prevent the micro-droplets 172 from stacking during the formation process, causing detection errors. In addition, the width of the microchannel between the reaction and detection reservoir 17 and the second waste liquid reservoir 18 should also be less than 1 / 3 of the diameter of the microdroplets 172 to prevent the loss of a large amount of microdroplets 172. Furthermore, to prevent the oil phase from overflowing during storage and transportation of the chip, flow channel plugs 303 are installed on the microchannels at both ends of the reaction and detection reservoir 17.

[0071] Accordingly, the workflow and principle of the digital fully automatic molecular diagnostic microfluidic chip of this embodiment are basically the same as those of Example 1, except that the dispersion method of the reaction mixture is different, that is, the auxiliary equipment first opens the flow channel plug 303, and when the reaction mixture is pressed into the reaction detection pool 17 through the microchannel, the step emulsification technology is used to form tens of thousands of micro droplets 172 containing one or no target fragment, and the excess aqueous phase reaction mixture is discharged into the second waste liquid pool 18, and then the amplification reaction and fluorescence signal reading are carried out.

Claims

1. A digital fully automatic molecular diagnostic microfluidic chip, comprising a lower cover (1) and an upper cover (2), wherein the upper cover (2) is tightly attached to the surface of the lower cover (1), and the lower cover (1) and the upper cover (2) are sealed and fixedly connected, characterized in that: The lower cover (1) is provided with a sample processing module and an amplification reaction module, wherein the sample processing module comprises a sample chamber (11), a mixing chamber (12), an absolute quantitative chamber (14) and a first waste liquid pool (13), wherein the sample chamber (11), the mixing chamber (12) and the first waste liquid pool (13) are sequentially connected, and a first flow channel switch (301) is provided between the mixing chamber (12) and the first waste liquid pool (13), and the absolute quantitative chamber (14) is respectively connected to the mixing chamber (12) and the first waste liquid pool (13); The amplification reaction module comprises a relative quantitative chamber (15), a reagent mixing pool (16), a reaction detection pool (17) and a second waste liquid pool (18) which are connected in sequence, wherein the relative quantitative chamber (15) is connected to the mixing liquid chamber (12) and the first waste liquid pool (13) respectively, and a second flow channel switch (302) is provided between the relative quantitative chamber (15) and the mixing liquid chamber (12), and the reaction detection pool (17) is formed as a whole by injection molding a superconducting heat-conducting insert (170) whose upper surface can form a micro-reaction unit with the lower cover (1); The upper cover (2) is provided with openings, which include a sample liquid addition port (21), a lysis liquid addition port (22), a pressurization port (23), a washing liquid addition port (24), a magnetic bead addition port (25), an eluent addition port (26), a mixing chamber pressurization exhaust port (27), a mixing pool pressurization exhaust port (28), a first waste liquid pool exhaust port (29) and a second waste liquid pool exhaust port (210), wherein the sample liquid addition port (21), the lysis liquid addition port (22) and the pressurization port (23) are respectively connected to the sample chamber (11), and the washing liquid addition port (24) is connected to the sample chamber (11). ), the magnetic bead addition port (25), the eluent addition port (26) and the mixing chamber pressurization exhaust port (27) are respectively connected to the mixing chamber (12), the eluent addition port (26) is also connected to the absolute quantitative chamber (14), and the absolute quantitative chamber (14) is located between the mixing chamber (12) and the eluent addition port (26), the mixing tank pressurization exhaust port (28), the first waste liquid tank exhaust port (29), and the second waste liquid tank exhaust port (210) are respectively connected to the reagent mixing tank (16), the first waste liquid tank (13), and the second waste liquid tank (18); A transparent window (211) is also provided on the upper cover (2) corresponding to the position of the reaction detection pool (17) for reading the signal after the amplification reaction is completed; The thermal conductivity of the superconducting thermal insert (170) is greater than 5, and the upper surface of the superconducting thermal insert (170) is subjected to hydrophilic or hydrophobic modification. The micro-reaction units formed on the upper surface of the superconducting thermal insert (170) are honeycomb-shaped micropores (171), and the volume of the micropores (171) is less than 0.15 nL.

2. The microfluidic chip according to claim 1, characterized in that A lysis absorption and release component is provided in the sample chamber (11), and the lysis absorption and release component is used to absorb and release the sample to be tested and the lysis solution so that the two are fully contacted and lysed.

3. The microfluidic chip according to claim 2, characterized in that: The upper cover (2) and the lower cover (1) are both made of polymer materials, and the polymer materials are selected from one or a combination of polycarbonate, polymethyl methacrylate, and acrylonitrile-butadiene-styrene plastics, and the upper cover (2) is transparent.

4. The microfluidic chip according to claim 3, characterized in that The upper cover (2) comprises an upper cover plate (201) and an intermediate sealing member (202), wherein the intermediate sealing member (202) is installed between the upper cover plate (201) and the lower cover (1), and the intermediate sealing member (202) is provided with a through hole corresponding to the opening and a window hole position (2021) corresponding to the position of the reaction detection pool (17) and the transparent window (211), and is further provided with a first flow channel switch (301) and a second flow channel switch (302) that cooperate with corresponding positions of the lower cover (1).

5. The microfluidic chip according to claim 4, characterized in that: The intermediate sealing component (202) is a soft adhesive film, and the material of the soft adhesive film is a soft material.

6. The microfluidic chip according to any one of claims 1 to 5, characterized in that: The number of the micropores (171) is greater than 8000, the depth of the micropores (171) is greater than 0.01 µm, and the shape of the micropores (171) is hexagonal, square, circular or triangular.

7. The microfluidic chip according to claim 1, characterized in that Flow channel plugs (303) are respectively provided between the reaction detection pool (17), the reagent mixing pool (16), and the second waste liquid pool (18).

8. A method for using the microfluidic chip according to any one of claims 1 to 7, characterized in that: The following steps are involved: Step 1, adding a sample to be tested and a lysis solution into the sample chamber (11), and lysing the sample to be tested; Step 2, the lysed sample to be tested is hydraulically injected into the mixing chamber (12), and the target fragment is extracted from the sample to be tested using a magnetic bead method; Step 3, based on the preliminary experiment, the volume of the eluent for eluting the target fragment is controlled by the absolute quantitative chamber (14) to obtain a test liquid containing the target fragment with a known concentration; Step 4, the test liquid containing the target fragment is sequentially pressed into the relative quantitative chamber (15) and the reagent mixing tank (16), and mixed with the diagnostic reagent in the reagent mixing tank (16) to obtain a reaction mixture; In step 5, the reaction mixture in the reagent mixing tank (16) is hydraulically injected into the micro-reaction unit of the superconducting heat insert (170) of the reaction detection tank (17) and a heating amplification reaction is performed. After the reaction is completed, the fluorescence signal is read through the transparent window (211) and statistical analysis is performed.

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