A LAMP-coupled CRISPR microfluidic chip

Abstract

The present invention discloses a LAMP-coupled CRISPR microfluidic chip, and relates to the field of microfluidic technology. The microfluidic chip is fan-shaped, and the center of the fan-shaped chip is a rotation positioning port. The microfluidic chip is divided into a LAMP reaction area and a CRISPR reaction area. The LAMP reaction area and the CRISPR reaction area are separated by a plurality of strip holes. The LAMP reaction area is arranged near the rotation positioning port. A LAMP reaction chamber and a plurality of temperature control chambers are arranged in the LAMP reaction area; an arc-shaped diversion channel, a waste liquid chamber and a plurality of CRISPR reaction units are arranged in the CRISPR reaction area. The present invention proposes a closed centrifugal microfluidic chip of LAMP-coupled CRISPR, which integrates LAMP reaction and CRISPR reaction into different areas of the same microfluidic chip, does not cause aerosol pollution, saves time and improves the accuracy of detection.

Description

Technical Field

[0001] The present invention relates to the field of microfluidics, and in particular to a LAMP-coupled CRISPR microfluidics chip. Background Art

[0002] Nucleic acid amplification technology is a technology that rapidly and specifically amplifies target nucleic acid fragments in vitro through specific biochemical reactions. Due to its high sensitivity and high specificity, this technology has been widely used in the diagnosis of infectious diseases, diagnosis and evaluation of tumors, diagnosis and screening of genetic diseases, and clinical research such as pharmacogenomics. However, due to its high testing cost, easy contamination of samples, the need for professional operation, and reliance on temperature control equipment with high temperature control accuracy and high cost, its use scenarios are relatively limited. Existing nucleic acid detection methods based on polymerase chain reaction technology (PCR) face major challenges.

[0003] With the advancement of technology, isothermal amplification technologies such as LAMP, RPA, HDA, and RCA have emerged. Among them, LAMP has the characteristics of high amplification efficiency, low detection cost, and constant reaction temperature, which is suitable for on-site nucleic acid detection. Although isothermal amplification technology solves the problem of PCR requiring variable temperature and precise temperature control, there are still problems such as insufficient sensitivity and false positives. Therefore, it is of great significance to establish a highly specific, low-cost, and easy-to-operate nucleic acid detection method. Currently, many studies combine isothermal amplification technology (such as LAMP loop-mediated isothermal amplification technology) with gene editing technology (CRISPR / Cas system). The CRISPR / Cas system has a mild reaction temperature and strong signal amplification ability. The specific recognition and signal amplification strategy of this technology are used to achieve accurate and rapid nucleic acid detection.

[0004] The above technology is usually divided into two steps. The first step is the first step, which is to amplify the target nucleic acid fragment in the sample to be tested by isothermal amplification technology, such as LAMP loop-mediated isothermal amplification reaction. At a constant temperature of 65°C, the number of target nucleic acid fragments in the sample to be tested is increased exponentially by Bst DNA polymerase, and the amplification is completed within the conventional reaction time of 40 minutes. The second step is the end-point fluorescence detection of the LAMP reaction product mediated by the CRISPR / Cas system (such as CRISPR / Cas12a). After the LAMP reaction is completed, take an appropriate volume of the reaction solution and add it to a test tube containing the premixed CRISPR / Cas12a system. At a temperature of about 37°C, the Cas12a protein can specifically identify and cut the target double-stranded DNA in the added reaction solution under the guidance of crRNA. At the same time, its trans-cutting activity will be activated, and the single-stranded DNA probe with a fluorescent / quenching group will be non-specifically cut. The broken single-stranded DNA probe releases a specific wavelength of fluorescence signal collected by the instrument, thereby detecting the target nucleic acid fragment in the sample to be tested.

[0005] However, in conventional operations, the two reactions need to be performed separately, which leads to some inevitable problems, such as: 1. After the LAMP reaction is completed, the lid needs to be opened to add the reaction solution to the CRISPR / Cas system. This operation will cause serious aerosol contamination and lead to false positive results; 2. The repeated pipetting operation is more complicated, which prolongs the experimental time and the professional requirements of the operation. Summary of the invention

[0006] The present invention aims to solve the problem that the LAMP reaction and CRISPR reaction are easily caused by aerosol pollution when they are carried out separately, and to provide support for the development of integrated and automated detection equipment. A closed centrifugal microfluidic chip of LAMP coupled with CRISPR is proposed, which embeds the LAMP reaction and the CRISPR reaction into different areas of the same microfluidic chip, realizes fully enclosed integrated detection of the chip, does not cause aerosol pollution, and the whole process can be automated, which simplifies manual professional operation, saves time and improves the accuracy of detection.

[0007] In order to achieve the above object, the present invention adopts the following technical solutions:

[0008] A LAMP-coupled CRISPR microfluidic chip, the microfluidic chip is composed of a top plate, a middle plate and a bottom plate bonded together from top to bottom, the microfluidic chip is fan-shaped, the center of the fan-shaped chip is a rotation positioning port, the microfluidic chip is divided into a LAMP reaction area and a CRISPR reaction area, the LAMP reaction area and the CRISPR reaction area are separated by a plurality of strip holes, and the LAMP reaction area is arranged close to the rotation positioning port;

[0009] The LAMP reaction area is provided with a LAMP reaction chamber and a plurality of temperature control chambers;

[0010] The CRISPR reaction area is provided with an arc-shaped diversion channel, a waste liquid chamber and a plurality of CRISPR reaction units; one end of the diversion channel is connected to the LAMP reaction chamber through a microfluidic channel, and the other end is connected to the waste liquid chamber through a drainage channel; each CRISPR reaction unit includes a liquid separation chamber and a CRISPR reaction chamber, the liquid separation chamber is located on the side of the diversion channel away from the rotation positioning port, the liquid separation chamber is U-shaped, and the opening side of the U-shape is connected to the outer wall of the diversion channel; the CRISPR reaction chamber is located on the side of the corresponding liquid separation chamber away from the rotation positioning port, and the bottom of the liquid separation chamber is connected to the corresponding CRISPR reaction chamber through a liquid suction channel; the CRISPR reaction chamber corresponds to the temperature control chamber one by one, and the CRISPR reaction chamber is connected to the corresponding temperature control chamber through an air pressure balance channel, and a buffer preset chamber is provided on the air pressure balance channel near the CRISPR reaction chamber.

[0011] Preferably, the LAMP reaction chamber, temperature control chamber, waste liquid chamber and CRISPR reaction chamber are arranged through the middle plate from top to bottom, the microfluidic channel, guide channel, liquid separation chamber and drainage channel are arranged on the upper surface of the middle plate, and the liquid suction channel, air pressure balance channel and buffer solution pre-setting chamber are arranged on the lower surface of the middle plate.

[0012] Preferably, the shortest distance between the liquid suction channel and the rotation positioning port is smaller than the shortest distance between the flow guide channel and the rotation positioning port.

[0013] Preferably, the arc center of the guide channel coincides with the center of the rotation positioning opening.

[0014] Preferably, the buffer pre-setting cavity is in the form of an elongated strip, with both ends in the length direction connected to the air pressure balance channel.

[0015] Preferably, the liquid outlet of the LAMP reaction chamber is located at a side of the LAMP reaction chamber away from the rotation positioning port and the liquid outlet of the LAMP reaction chamber is in a constricted trumpet shape.

[0016] Preferably, the docking interface between the CRISPR reaction chamber and the liquid aspiration channel and the docking interface between the CRISPR reaction chamber and the air pressure balance channel are both located on the side of the CRISPR reaction chamber facing the rotation positioning port.

[0017] Preferably, the top plate is provided with a liquid injection hole, the inner side of the liquid injection hole is communicated with the LAMP reaction chamber, and a hole plug is provided at the outer opening of the liquid injection hole.

[0018] The present invention has the following beneficial effects: 1. LAMP reaction and CRISPR reaction are integrated in one microfluidic chip, and the reaction liquid is transferred in the middle without contacting the external air, and aerosol pollution is not caused; 2. When the LAMP constant temperature amplification reaction process is carried out at 65°C, the temperature control chamber can automatically transport the buffer solution in the buffer pre-setting chamber to the CRISPR reaction chamber through the principle of thermal expansion and contraction of air, and mix and dissolve the buffer solution with the freeze-dried powder in the CRISPR reaction chamber into a premixed liquid, so as to prepare for the CRISPR reaction in advance; 3. After the LAMP reaction is completed, the microfluidic chip is rotated by a centrifuge, and the reaction liquid can be evenly distributed into the liquid separation chamber by centrifugal force, and during the air cooling process in the temperature control chamber, the reaction liquid in the liquid separation chamber can be automatically siphoned into the CRISPR reaction chamber for CRISPR reaction. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 It is a structural schematic diagram of the present invention.

[0020] Figure 2 It is a perspective view of the present invention.

[0021] Figure 3 yes Figure 2 Enlarged view of point a in the middle.

[0022] Figure 4 It is a top view of the channel on the upper surface of the middle plate of the present invention.

[0023] Figure 5 It is a top view of the channel on the lower surface of the middle plate of the present invention.

[0024] 1: LAMP reaction area; 2: CRISPR reaction area; 3: strip hole; 4: rotation positioning port; 5: LAMP reaction chamber; 6: temperature control chamber; 7: diversion channel; 8: waste liquid chamber; 9: microfluidic channel; 10: drainage channel; 11: liquid separation chamber; 12: CRISPR reaction chamber; 13: suction channel; 14: air pressure balance channel; 15: buffer pre-setting chamber; 16: injection hole. DETAILED DESCRIPTION

[0025] The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments.

[0026] A LAMP-coupled CRISPR microfluidic chip, see Figures 1 to 5The microfluidic chip is composed of three layers of plates, namely, a top plate, a middle plate and a bottom plate, which are bonded from top to bottom. The top plate and the bottom plate can be replaced by a pressure-sensitive film. The microfluidic chip is fan-shaped, and the arc center angle of the fan is 180°, that is, a semicircle. Two chips with 180° rotational symmetry can form a circular chip. The center position of the fan is a rotation positioning port 4. The microfluidic chip is divided into a LAMP reaction area 1 and a CRISPR reaction area 2. The LAMP reaction area 1 and the CRISPR reaction area 2 are separated by a plurality of strip holes 3. The plurality of strip holes 3 are located on the same arc with the center of the rotation positioning port 4 as the center. The LAMP reaction area 1 is arranged close to the rotation positioning port 4, and the CRISPR reaction area 2 is located on the side of the dividing line of the strip holes 3 away from the rotation positioning port 4.

[0027] The LAMP reaction area 1 is provided with a LAMP reaction chamber 5 and five temperature control chambers 6;

[0028] The CRISPR reaction area 2 is provided with an arc-shaped guide channel 7, a waste liquid chamber 8 and five CRISPR reaction units; the arc center of the guide channel 7 coincides with the center of the rotation positioning port 4, one end of the guide channel 7 is connected with the LAMP reaction chamber 5 through the microfluidic channel 9, the liquid outlet of the LAMP reaction chamber 5 is located on the side of the LAMP reaction chamber 5 away from the rotation positioning port 4 and the liquid outlet of the LAMP reaction chamber 5 is in a contracted trumpet shape, the other end of the guide channel 7 is connected with the waste liquid chamber 8 through the drainage channel 10, the waste liquid chamber 8 can be used to collect excess reagents, and can also be used to cooperate with the air in the temperature control chamber 6 to better realize air flow when thermal expansion and contraction occur.

[0029] Each CRISPR reaction unit includes a liquid separation chamber 11 and a CRISPR reaction chamber 12, wherein the liquid separation chamber 11 is located on the side of the guide channel 7 away from the rotation positioning port 4, and the liquid separation chamber 11 is U-shaped, and the opening side of the U-shape is connected to the outer wall of the guide channel 7; the CRISPR reaction chamber 12 is located on the side of the corresponding liquid separation chamber 11 away from the rotation positioning port 4, and the bottom of the liquid separation chamber 11 is connected to the corresponding CRISPR reaction chamber 12 through a liquid suction channel 13, and the closest distance between the liquid suction channel 13 and the rotation positioning port 4 is smaller than the closest distance between the guide channel 7 and the rotation positioning port 4. This arrangement can prevent the reagent in the guide channel 7 from entering the CRISPR reaction chamber 12 in advance when the rotating microfluidic chip uses centrifugal force to separate liquid, so that the liquid separation in each CRISPR reaction unit is more uniform, and thus the final test result is more accurate. The CRISPR reaction chamber 12 corresponds to the temperature control chamber 6 one by one, and the CRISPR reaction chamber 12 is connected to the corresponding temperature control chamber 6 through an air pressure balance channel 14. A buffer pre-setting chamber 15 is provided on the air pressure balance channel 14 near the CRISPR reaction chamber 12. The buffer pre-setting chamber 15 is in a long strip structure, and both ends in the length direction are connected to the air pressure balance channel 14. The long strip-shaped buffer pre-setting chamber 15 enables the buffer pre-stored inside to be more fully transported to the CRISPR reaction chamber 12 when subjected to air pressure, and the docking interface between the CRISPR reaction chamber 12 and the liquid aspiration channel 13 and the docking interface between the CRISPR reaction chamber 12 and the air pressure balance channel 14 are both located on the side of the CRISPR reaction chamber 12 facing the rotation positioning port 4.

[0030] The LAMP reaction chamber 5, temperature control chamber 6, waste liquid chamber 8 and CRISPR reaction chamber 12 are arranged vertically through the middle plate, the microfluidic channel 9, flow guide channel 7, liquid separation chamber 11 and liquid discharge channel 10 are arranged on the upper surface of the middle plate, and the liquid suction channel 13, air pressure balance channel 14 and buffer pre-setting chamber 15 are arranged on the lower surface of the middle plate. Some channels are arranged in layers to avoid conflicts between channels.

[0031] The top plate is provided with a liquid injection hole 16 , the inner side of the liquid injection hole 16 is communicated with the LAMP reaction chamber 5 , and a hole plug is provided at the outer opening of the liquid injection hole 16 .

[0032] Before leaving the factory, the present invention can pre-embed the required CRISPR / Cas12a reaction solution in the form of lyophilized powder in the CRISPR reaction chamber 12 according to customer needs, and pre-store the buffer in the buffer pre-setting chamber 15.

[0033] When the nucleic acid amplification detection test is performed using the present invention, the specific steps are as follows:

[0034] 1. Mix the sample to be tested with the enzyme of the reaction to prepare the reaction solution of LAMP reaction, inject the reaction solution into the LAMP reaction chamber 5 through the injection hole 16, and cover the hole with a plug.

[0035] 2. Place the LAMP reaction area 1 of the microfluidic chip on a heater and heat it at a constant temperature of 65° C. for about 40 minutes to perform a LAMP isothermal amplification reaction to amplify the number of target nucleic acid fragments.

[0036] During the constant temperature heating process of the LAMP reaction area 1 at 65°C, the temperature in the temperature control chamber 6 will also rise, and the air in the temperature control chamber 6 will expand due to the heat, and the pressure will increase. In order to balance the air pressure, the high-pressure gas in the temperature control chamber 6 will pass through the air pressure balance channel 14, the buffer preset chamber 15, the CRISPR reaction chamber 12, the liquid aspiration channel 13, the liquid separation chamber 11, the diversion channel 7, and enter the waste liquid chamber 8 in sequence, and finally make the air pressure in all connected chambers and channels reach a stable value (the stable value is higher than the initial air pressure value when not heated). When the high-pressure gas passes through the buffer preset chamber 15, the buffer in the buffer preset chamber 15 can be pushed into the CRISPR reaction chamber 12, so that the buffer is mixed and dissolved with the lyophilized powder pre-buried in the CRISPR reaction chamber 12 to generate the premix required for the CRISPR reaction. This setting not only solves the problem of pre-embedded premixed liquid, which is easy to deteriorate over time and affect the test accuracy, but also solves the problem of temporary configuration of premixed liquid injection, which is easy to cause aerosol contamination. In addition, during the LAMP reaction, the premixed liquid required for the CRISPR reaction can be automatically configured, which effectively improves the convenience of the experiment.

[0037] 3. After the LAMP reaction is completed, the microfluidic chip is transferred from the heater to the centrifuge for rotation. Through the action of centrifugal force, the reaction liquid in the LAMP reaction chamber 5 will flow into the diversion channel 7 through the microfluidic channel 9. When the reaction liquid in the diversion channel 7 flows through the liquid separation chamber 11, it will fill each liquid separation chamber 11, and the remaining reaction liquid will eventually flow into the waste liquid chamber 8, and the liquid separation process time is relatively short. Since the closest distance between the liquid suction channel 13 and the rotation positioning port 4 is smaller than the closest distance between the diversion channel 7 and the rotation positioning port 4, therefore, during the liquid separation process under the action of centrifugal force, the reaction liquid in the liquid separation chamber 11 will not enter the CRISPR reaction chamber 12 through the liquid suction channel 13, so that the reaction liquid in each liquid separation chamber 11 during the liquid separation process is as equal as possible, thereby ensuring the consistency of the subsequent experiments in each CRISPR reaction chamber 12.

[0038] Then, the microfluidic chip continues to rotate, and finally the temperature in the temperature control chamber 6 is reduced to room temperature. During the temperature reduction process in the temperature control chamber 6, the air pressure in the temperature control chamber 6 decreases, and a pressure difference is formed with the waste liquid chamber 8 again. At this time, the gas flow direction is the waste liquid chamber 8, the diversion channel 7, the liquid separation chamber 11, the liquid suction channel 13, the CRISPR reaction chamber 12, the buffer preset chamber 15, the air pressure balance channel 14 and the temperature control chamber 6 in sequence. It can also be understood that the temperature reduction in each temperature control chamber 6 generates suction, and during this gas flow, each temperature control chamber 6 will absorb the LAMP reaction solution in the corresponding liquid separation chamber 11 into the corresponding CRISPR reaction chamber 12, and mix it with the premixed solution in the CRISPR reaction chamber 12 to form the reaction solution required for the CRISPR reaction. Since the docking interface between the CRISPR reaction chamber 12 and the liquid aspiration channel 13 and the docking interface between the CRISPR reaction chamber 12 and the air pressure balance channel 14 are both located on the side of the CRISPR reaction chamber 12 facing the annular center of the CRISPR reaction area 2, during the centrifugal rotation process, the reaction liquid in the CRISPR reaction chamber 12 will no longer flow out from the air pressure balance channel 14.

[0039] 4. Remove the microfluidic chip from the centrifuge, place it on the heater, and perform the CRISPR reaction during constant temperature heating at 37°C. This constant temperature heating can be for CRISPR reaction area 2 or the entire microfluidic chip.

[0040] 5. After the CRISPR reaction is completed, the final detection result can be obtained by simply observing the fluorescence intensity of the reaction solution in each CRISPR reaction chamber 12 through a CCD fluorescence camera.

Claims

1. A LAMP-coupled CRISPR microfluidic chip, the microfluidic chip is composed of a top plate, a middle plate and a bottom plate bonded together from top to bottom, characterized in that: The microfluidic chip is fan-shaped, and the center of the fan-shaped chip is a rotation positioning port (4). The microfluidic chip is divided into a LAMP reaction area (1) and a CRISPR reaction area (2). The LAMP reaction area (1) and the CRISPR reaction area (2) are separated by a plurality of strip-shaped holes (3). The LAMP reaction area (1) is arranged close to the rotation positioning port (4). The LAMP reaction area (1) is provided with a LAMP reaction chamber (5) and a plurality of temperature control chambers (6); The CRISPR reaction area (2) is provided with an arc-shaped flow diversion channel (7), a waste liquid chamber (8) and a plurality of CRISPR reaction units; one end of the flow diversion channel (7) is connected to the LAMP reaction chamber (5) via a microfluidic channel (9), and the other end is connected to the waste liquid chamber (8) via a drainage channel (10); each CRISPR reaction unit comprises a liquid separation chamber (11) and a CRISPR reaction chamber (12); the liquid separation chamber (11) is located on a side of the flow diversion channel (7) away from the rotation positioning port (4); the liquid separation chamber (11) is U-shaped, and the opening side of the U-shape is connected to the flow diversion channel (5). The CRISPR reaction chamber (12) is located on a side of the corresponding liquid separation chamber (11) away from the rotation positioning port (4), and the bottom of the liquid separation chamber (11) is connected to the corresponding CRISPR reaction chamber (12) via a liquid suction channel (13); the CRISPR reaction chamber (12) corresponds to the temperature control chamber (6) one by one, and the CRISPR reaction chamber (12) is connected to the corresponding temperature control chamber (6) via an air pressure balance channel (14), and a buffer pre-setting chamber (15) is provided on the air pressure balance channel (14) near the CRISPR reaction chamber (12); The LAMP reaction chamber (5), the temperature control chamber (6), the waste liquid chamber (8) and the CRISPR reaction chamber (12) are arranged to pass through the middle plate from top to bottom; the microfluidic channel (9), the flow guide channel (7), the liquid separation chamber (11) and the liquid discharge channel (10) are arranged on the upper surface of the middle plate; and the liquid suction channel (13), the air pressure balance channel (14) and the buffer pre-setting chamber (15) are arranged on the lower surface of the middle plate; The shortest distance between the liquid suction channel (13) and the rotation positioning opening (4) is smaller than the shortest distance between the flow guide channel (7) and the rotation positioning opening (4).

2. A LAMP-coupled CRISPR microfluidic chip according to claim 1, characterized in that: The arc center of the guide channel (7) coincides with the center of the rotation positioning opening (4).

3. A LAMP-coupled CRISPR microfluidic chip according to claim 1, characterized in that: The buffer pre-setting chamber (15) is in the form of a long strip, with both ends in the length direction connected to the air pressure balance channel (14).

4. A LAMP-coupled CRISPR microfluidic chip according to claim 1, characterized in that: The liquid outlet of the LAMP reaction chamber (5) is located at a side of the LAMP reaction chamber (5) away from the rotation positioning port (4), and the liquid outlet of the LAMP reaction chamber (5) is in the shape of a shrunken trumpet.

5. The LAMP-coupled CRISPR microfluidic chip according to claim 1, characterized in that: The docking interface between the CRISPR reaction chamber (12) and the liquid aspiration channel (13) and the docking interface between the CRISPR reaction chamber (12) and the air pressure balance channel (14) are both located on the side of the CRISPR reaction chamber (12) facing the rotation positioning port (4).

6. A LAMP-coupled CRISPR microfluidic chip according to claim 1, characterized in that: The top plate is provided with a liquid injection hole (16), the inner side of the liquid injection hole (16) is connected to the LAMP reaction chamber (5), and a hole plug is provided at the outer opening of the liquid injection hole (16).

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