Integrated closed chip

Abstract

The integrated closed chip comprises a substrate, the substrate comprises an upper piece and a lower piece, and a PCR amplification area, a CRISPR reaction area, a nucleic acid extraction area, a magnetic bead channel, a magnetic bead fixing area and an automatic buffer area are integrated in the upper piece. In the application, five parallel cylindrical reaction chambers are designed in a CRISPR reaction area as restrictive reaction spaces for CRISPR specific recognition of virus nucleic acid sequences, each cylinder of the reaction chamber is provided with a vent hole connected with air, once liquid flows to a waste liquid port II, the chambers are filled with the liquid at the same time, cross contamination of fluid flowing is avoided, and the chip integrates nucleic acid extraction, so that the detection accuracy is improved. According to the present invention, the single-base mutated multi-target nuclear loss sequence detection chip has characteristics of rapid, sensitive and ready-to-detect single-base mutated multi-target nuclear loss sequence, isothermal amplification, and CRISPR reaction detection, and further has the multi-site multi-channel fluorescence detection ability through the operation mode conversion;

Description

Technical Field

[0001] The present invention relates to the technical field of closed chips, and in particular to an integrated closed chip. Background Art

[0002] The shortcomings of current PCR platforms at home and abroad mainly include expensive instruments and reagents, complex operations, long detection time, high costs, and a narrow detection range. The chips used do not have the function of extracting nucleic acids within the chip.

[0003] The chip is only used for sample preparation and final reaction liquid storage. Some POCT products use nucleic acid-free extraction, which is generally at least one order of magnitude different from the full extraction of purified nucleic acid. Therefore, purification-free extraction can be roughly understood as follows: in order to more easily realize the full automation of nucleic acid detection, the detection sensitivity is sacrificed to simplify the nucleic acid extraction steps. Traditional POCT IVD manufacturers focus on the development of products such as colloidal gold test strips and immunofluorescence test strips, which have low accuracy and sensitivity and cannot meet the purpose of accurate diagnosis. Therefore, it is urgent to propose corresponding integrated closed chips to solve the above problems. Summary of the invention

[0004] The purpose of the present invention is to propose an integrated closed chip in order to solve the above-mentioned problem.

[0005] In order to achieve the above object, the present invention adopts the following technical solutions:

[0006] An integrated closed chip comprises a substrate, wherein the substrate comprises an upper piece and a lower piece, wherein the upper piece is internally integrated with a PCR amplification area, a CRISPR reaction area, a nucleic acid extraction area, a magnetic bead channel, a magnetic bead fixing area, and an automatic buffer zone, wherein the magnetic bead mixing area is connected to the magnetic bead fixing area, wherein the magnetic bead fixing area is connected to the automatic buffer zone, and wherein the upper piece is internally integrated with a waste discharge component for discharging waste from the magnetic bead fixing area and the CRISPR reaction area.

[0007] Preferably, the substrate is formed by bonding an upper sheet and a lower sheet, and low-viscosity dimethylsiloxane is integrated inside the upper sheet.

[0008] Preferably, the PCR amplification zone consists of a disc-shaped pipeline in an array state, and the magnetic bead fixation zone and the CRISPR reaction zone are connected through the PCR amplification zone.

[0009] Preferably, the nucleic acid extraction area is composed of a vertically arranged sample channel, a magnetic bead channel, a washing liquid channel, an eluent channel, a water channel and an oil channel, the sample channel and the magnetic bead channel are both connected to the magnetic bead mixing area, the washing liquid channel, the eluent channel, the water channel and the oil channel are connected in series through channel one, and the channel is connected to the magnetic bead fixing area.

[0010] Preferably, the CRISPR reaction area includes five groups of cylindrical reaction chambers connected in series through channel two phases, the middle three groups of the five groups of cylindrical reaction chambers are detection reagent channel reaction areas, the other two groups of the five groups of cylindrical reaction chambers are negative control areas and positive control areas, respectively, and the five groups of cylindrical reaction chambers are all connected with vents.

[0011] Preferably, the waste discharge component comprises a waste liquid port 1 and a waste liquid port 2, the waste liquid port 1 is connected to the magnetic bead fixing area, and the waste liquid port 2 is connected to the channel 2.

[0012] Preferably, the magnetic bead mixing zone and the magnetic bead fixing zone are connected via channel three, and the channel three is arranged in a coiled array.

[0013] Preferably, the method for using the integrated closed chip comprises the following steps:

[0014] S1, the sample is added with lysis buffer and injected into the chip, and then magnetic beads are injected to extract nucleic acid;

[0015] S2, mixing the extracted nucleic acid with the RPA reagent for amplification reaction;

[0016] S3. The CRISPR detection reagents in the detection chamber are mixed and the detection results are read out.

[0017] In summary, due to the adoption of the above technical solution, the beneficial effects of the present invention are:

[0018] 1. In this application, first, the sample is added with lysis solution and injected into the chip, and then magnetic beads are injected to extract nucleic acid, and then the extracted nucleic acid is mixed with RPA reagent for amplification reaction. Finally, the test results are read out after mixing with the CRISPR detection reagent in the detection chamber. In the nucleic acid extraction area, a nucleic acid extraction reagent based on magnetic beads is used, combined with the efficient triangular microcolumn array structure in the serpentine channel, to extract nucleic acid from the input sample. In the PCR amplification area, the RPA buffer is mixed with the nucleic acid solution for amplification reaction, and the amplification time is controlled by adjusting the reaction channel flow rate. In the CRISPR reaction area, 5 parallel cylindrical reaction chambers are designed as a restrictive reaction space for CRISPR to specifically identify viral nucleic acid sequences. Each cylinder in the reaction chamber has a vent to connect the air. Once the liquid flows to the waste liquid port 2, the liquid will fill the chamber at the same time to avoid cross-contamination of fluid flow. The chip integrates nucleic acid extraction, isothermal amplification, and CRISPR reaction detection, so that the chip has the ability to quickly and sensitively detect multi-target nuclear damage sequences of single-base mutations, and also has the ability of multi-site multi-channel fluorescence detection by changing the operation mode.

[0019] 2. In the present application, the upper sheet is mainly composed of microfluidic channels, inlet and outlet holes and vents, and the lower sheet is a thin sheet. The upper sheet and the lower sheet are bonded together to form a substrate, thereby forming a closed microfluidic. At the same time, low-viscosity dimethylsiloxane is added to the substrate to form a physical barrier, thereby forming an independent compartment to prevent cross-contamination due to free diffusion. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 It shows a schematic diagram of the overall structure provided by an embodiment of the present invention;

[0021] Figure 2 A schematic diagram of the CRISPR mode action flow provided according to an embodiment of the present invention is shown;

[0022] Figure 3 A three-dimensional schematic diagram provided according to an embodiment of the present invention is shown;

[0023] Figure 4 A schematic diagram of a top sheet provided according to an embodiment of the present invention is shown;

[0024] Figure 5 A schematic diagram of the structure of a lower sheet provided in an embodiment of the present invention is shown;

[0025] Figure 6 A schematic diagram of the structure of a magnetic bead mixing zone provided according to an embodiment of the present invention is shown.

[0026] Legend:

[0027] 1. Substrate; 101. Upper plate; 102. Lower plate; 2. PCR amplification area; 3. CRISPR reaction area; 4. Nucleic acid extraction area; 5. Sample channel; 6. Magnetic bead channel; 7. Washing liquid channel; 8. Eluent channel; 9. Water channel; 10. Oil channel; 11. Monitoring reagent channel reaction area; 12. Negative control area; 13. Positive control area; 14. Vent; 15. Waste liquid port 2; 16. Magnetic bead mixing area; 17. Magnetic bead fixing area; 18. Waste liquid port 1; 19. Automatic buffer. DETAILED DESCRIPTION

[0028] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0029] See also Figure 1-6 , the present invention provides a technical solution:

[0030] An integrated closed chip includes a substrate 1, the substrate 1 includes an upper chip 101 and a lower chip 102, the upper chip 101 is internally integrated with a PCR amplification area 2, a CRISPR reaction area 3, a nucleic acid extraction area 4, a magnetic bead channel 16, a magnetic bead fixing area 17, and an automatic buffer 19, the magnetic bead mixing area 16 is connected to the magnetic bead fixing area 17, the magnetic bead fixing area 17 is connected to the automatic buffer 19, and the upper chip 101 is internally integrated with a waste discharge component for discharging waste from the magnetic bead fixing area 17 and the CRISPR reaction area;

[0031] The method for using the integrated closed chip comprises the following steps:

[0032] S1, the sample is added with lysis buffer and injected into the chip, and then magnetic beads are injected to extract nucleic acid;

[0033] S2, mixing the extracted nucleic acid with the RPA reagent for amplification reaction;

[0034] S3, the CRISPR detection reagents in the detection chamber are mixed and the detection results are read out;

[0035] In the nucleic acid extraction area 4, a nucleic acid extraction reagent based on magnetic beads is used, combined with a high-efficiency triangular micro-column array structure in the serpentine channel, to extract nucleic acids from the input sample. In the PCR amplification area 2, the RPA buffer is mixed with the nucleic acid solution for amplification reaction, and the amplification time is controlled by adjusting the flow rate of the reaction channel. In the CRISPR reaction area 3, 5 parallel cylindrical reaction chambers are designed as a restrictive reaction space for CRISPR to specifically identify viral nucleic acid sequences. Each cylinder in the reaction chamber has a vent 14 to connect to the air. Once the liquid flows to the waste liquid port 15, the liquid will fill the chamber at the same time to avoid cross contamination of fluid flow. The chip integrates nucleic acid extraction, isothermal amplification, and CRISPR reaction detection, so that the chip has the ability to quickly and sensitively detect multi-target nuclear damage sequences of single-base mutations, and also has the ability of multi-site multi-channel fluorescence detection by changing the operation mode;

[0036] The efficiency of nucleic acid extraction from samples plays a vital role in both amplification and detection results. Generally speaking, the more thoroughly the nucleic acid is mixed with the magnetic beads in the microchannel, the higher the corresponding nucleic acid extraction efficiency. However, van der Waals forces, hydrogen bonds, chemical bonds, and charge transfer between nanoparticles can cause magnetic beads to aggregate, thereby reducing the probability of collision between nucleic acids and magnetic beads. Split-recombination and chaotic propulsion mechanisms are two effective methods to improve mixing efficiency. Therefore, a triangular (or rhombus) micro-column array in the droplet chamber is used to disperse the magnetic beads to increase the probability of collision with nucleic acids and improve mixing efficiency. At the same time, the downstream serpentine channel further extends the mixing path of the liquid, ensuring that the nucleic acid and the magnetic beads are fully mixed. When the magnetic beads enter the chamber, they collide with the micro-columns, resulting in a broken agglomeration state. After being separated by the micro-columns, part of the magnetic beads are reassembled, while the other part is impacted by the lateral flow and continues to collide with the micro-columns around them and the chamber wall. Finally, the magnetic beads at the chamber outlet reconverge and enter the serpentine microchannel. When the fluid periodically passes through the corner of the microchannel, the fluid is deflected toward the channel wall by the centrifugal force, causing the flow layer state (friction) near the channel wall to be destroyed. This periodic destruction is very beneficial to fluid mixing. When the fluid enters the serpentine microchannel, after passing through the two channel corners, uniform mixing has been achieved.

[0037] It should be noted that; taking the CRISPR mode as an example here, the chip is used together with a syringe pump, a magnet, a PCR heating plate and a fluorescence detector to achieve "single sample input and five results output". Among these components, the syringe pump is used to control the fluid flow rate, the magnet separates the magnetic beads from the sample liquid to achieve nucleic acid extraction, the PCR heating plate is used to provide the temperature environment required for nucleic acid extraction, RPA and CRISPR reactions, and the fluorescence detector is used to read the detection signal in the cylindrical chamber.

[0038] Specifically, Figure 2 and Figure 4 As shown, the substrate 1 is formed by bonding an upper sheet 101 and a lower sheet 102, and low-viscosity dimethylsiloxane is integrated inside the upper sheet 101. The upper sheet 101 is mainly a microfluidic channel, an inlet and outlet hole, and a vent 15. The lower sheet 102 is a thin sheet. The upper sheet 101 and the lower sheet 102 are bonded to form a substrate 1 as a whole, thereby forming a closed microfluidic. At the same time, low-viscosity dimethylsiloxane is added to the substrate 1 to form a physical barrier, thereby forming an independent compartment to prevent cross contamination due to free diffusion.

[0039] Specifically, Figure 2 and Figure 3As shown, the PCR amplification zone 2 is composed of a disc-shaped pipeline in an array state, the magnetic bead fixing zone 17 is connected to the CRISPR reaction zone 3 through the PCR amplification zone 2, the RPA buffer is mixed with the nucleic acid solution for amplification reaction, and the amplification time is controlled by adjusting the flow rate of the reaction channel. The nucleic acid extraction zone 4 is composed of a vertically arranged sample channel 5, a magnetic bead channel 6, a washing liquid channel 7, an eluent channel 8, a water channel 9 and an oil channel 10, the sample channel 5 and the magnetic bead channel 6 are both connected to the magnetic bead mixing zone 16, the washing liquid channel 7, the eluent channel 8, the water channel 9 and the oil channel 10 are connected in series through channel one, and the channel is connected to the magnetic bead fixing zone 17, a magnetic bead-based nucleic acid extraction reagent is used, combined with the efficient triangular micro-column array structure in the serpentine channel, to extract nucleic acids from the input sample, and the CRISPR reaction zone 3 includes a nucleic acid extraction reagent connected in series through channel two. There are five groups of cylindrical reaction chambers, the three groups in the middle of the five groups of cylindrical reaction chambers are detection reagent channel reaction areas, the other two groups of the five groups of cylindrical reaction chambers are negative control areas 12 and positive control areas 13, and the five groups of cylindrical reaction chambers are all connected with vents 14, the waste discharge component includes a waste liquid port 18 and a waste liquid port 2 15, the waste liquid port 18 is connected to the magnetic bead fixing area 17, and the waste liquid port 2 15 is connected to the channel 2. Five parallel cylindrical reaction chambers are designed as a restrictive reaction space for CRISPR to specifically identify viral nucleic acid sequences. Each cylinder of the reaction chamber has a vent 14 to connect to the air. Once the liquid flows to the waste liquid port 2 15, the liquid will fill the chamber at the same time to avoid cross contamination of fluid flow. The magnetic bead mixing area 16 is connected to the magnetic bead fixing area 17 through the channel 3, and the channel 3 is arranged in an array and coiled to ensure sufficient reaction between the magnetic beads and the sample.

[0040] Working principle: First, the sample is added with lysis solution and injected into the chip, and then magnetic beads are injected to extract nucleic acid. Then, the extracted nucleic acid is mixed with RPA reagent for amplification reaction. Finally, it is mixed with CRISPR detection reagent in the detection chamber and the detection result is read out. In nucleic acid extraction area 4, a nucleic acid extraction reagent based on magnetic beads is used, combined with the efficient triangular micro-column array structure in the serpentine channel, to extract nucleic acid from the input sample. In PCR amplification area 2, RPA buffer is mixed with nucleic acid solution for amplification reaction, and the amplification time is controlled by adjusting the flow rate of the reaction channel. In CRISPR reaction area 3, 5 parallel cylindrical reaction chambers are designed as the restrictive reaction space for CRISPR to specifically identify viral nucleic acid sequences. Each cylinder in the reaction chamber has The vent 14 is connected to the air. Once the liquid flows to the waste liquid port 15, the liquid will fill the chamber at the same time to avoid cross contamination of fluid flow. The chip integrates nucleic acid extraction, isothermal amplification, and CRISPR reaction detection, so that the chip has the ability to quickly and sensitively detect multi-target nuclear damage sequences of single-base mutations, and also has the ability of multi-site multi-channel fluorescence detection by changing the operation mode. The upper sheet 101 is mainly a microfluidic channel and inlet and outlet holes and a vent 15, and the lower sheet 102 is a thin sheet. The upper sheet 101 and the lower sheet 102 are bonded to form a substrate 1 to form a closed microfluidic. At the same time, low-viscosity dimethylsiloxane is added to the substrate 1 to form a physical barrier, thereby forming an independent compartment to prevent cross contamination due to free diffusion.

[0041] The above description of the embodiments enables those skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but will conform to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. An integrated closed chip, comprising a substrate (1), characterized in that: The substrate (1) comprises an upper plate (101) and a lower plate (102); the upper plate (101) is internally integrated with a PCR amplification area (2), a CRISPR reaction area (3), a nucleic acid extraction area (4), a magnetic bead channel (16), a magnetic bead fixing area (17), and an automatic buffer zone (19); the magnetic bead mixing area (16) is connected to the magnetic bead fixing area (17); the magnetic bead fixing area (17) is connected to the automatic buffer zone (19); and the upper plate (101) is internally integrated with a waste discharge component for discharging waste from the magnetic bead fixing area (17) and the CRISPR reaction area.

2. The integrated enclosed chip according to claim 1, characterized in that: The substrate (1) is formed by bonding an upper sheet (101) and a lower sheet (102), and low-viscosity dimethylsiloxane is integrated inside the upper sheet (101).

3. The integrated enclosed chip according to claim 2, characterized in that: The PCR amplification area (2) is composed of a disc-shaped pipeline in an array state, and the magnetic bead fixing area (17) and the CRISPR reaction area (3) are connected through the PCR amplification area (2).

4. The integrated enclosed chip according to claim 3, characterized in that: The nucleic acid extraction area (4) is composed of a sample channel (5), a magnetic bead channel (6), a washing liquid channel (7), an eluent channel (8), a water channel (9) and an oil channel (10) arranged vertically. The sample channel (5) and the magnetic bead channel (6) are both connected to the magnetic bead mixing area (16). The washing liquid channel (7), the eluent channel (8), the water channel (9) and the oil channel (10) are connected in series through a channel 1, and the channel is connected to the magnetic bead fixing area (17).

5. The integrated enclosed chip according to claim 4, characterized in that: The CRISPR reaction area (3) comprises five groups of cylindrical reaction chambers connected in series through channel two phases, the middle three groups of the five groups of cylindrical reaction chambers are detection reagent channel reaction areas, the other two groups of the five groups of cylindrical reaction chambers are negative control areas (12) and positive control areas (13), and the five groups of cylindrical reaction chambers are all connected with ventilation holes (14).

6. The integrated enclosed chip according to claim 5, characterized in that: The waste discharge assembly comprises a waste liquid port 1 (18) and a waste liquid port 2 (15), wherein the waste liquid port 1 (18) is connected to the magnetic bead fixing area (17), and the waste liquid port 2 (15) is connected to the channel 2.

7. The integrated enclosed chip according to claim 6, characterized in that: The magnetic bead mixing area (16) and the magnetic bead fixing area (17) are connected via a channel three, and the channel three is arranged in a coiled array.

8. A method for using an integrated enclosed chip, based on the integrated enclosed chip as claimed in claim 1, characterized in that: The following steps are involved: S1, the sample is added with lysis buffer and injected into the chip, and then magnetic beads are injected to extract nucleic acid; S2, mixing the extracted nucleic acid with the RPA reagent for amplification reaction; S3. The CRISPR detection reagents in the detection chamber are mixed and the detection results are read out.

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