LAMP-coupled CRISPR microfluidic chip

Abstract

By embedding LAMP and CRISPR reactions in different regions of the same microfluidic chip, we achieved fully sealed, integrated testing of the chip, automating the entire process without causing aerosol contamination. [Solution] The present invention discloses a LAMP-coupled CRISPR microfluidic chip relating to the microfluidic technology field, wherein the microfluidic chip is fan-shaped, the center of the fan is a rotational positioning port, the microfluidic chip is divided into a LAMP reaction region and a CRISPR reaction region, the LAMP reaction region and the CRISPR reaction region are separated by several strip-shaped holes, the LAMP reaction region is provided close to the rotational positioning port, the LAMP reaction region is provided with one LAMP reaction chamber and several temperature control chambers, the CRISPR reaction region is provided with one arc-shaped guide channel, one waste liquid chamber and a plurality of CRISPR reaction units.

Description

Technical Field

[0001] The present invention relates to the field of microfluidic technology, and particularly to a microfluidic chip for LAMP-coupled CRISPR.

Background Art

[0002] Nucleic acid amplification technology is a technology that rapidly and specifically amplifies a target nucleic acid fragment in vitro by a specific biochemical reaction. Because of its characteristics of high sensitivity and high specificity, this technology is widely used in clinical research such as the diagnosis of infectious diseases, the diagnosis and evaluation of tumors, the diagnostic screening of genetic diseases, and pharmacogenomics. However, there are problems such as high detection costs, easy sample contamination, the need for specialized operations, dependence on a temperature control device with high temperature control accuracy and relatively high cost, etc., so the applicable scenarios are limited. Therefore, the conventional nucleic acid detection method mainly based on the polymerase chain reaction technology (PCR) is facing major challenges.

[0003] With the progress of technology, isothermal amplification technologies such as LAMP, RPA, HDA, and RCA have emerged. Among them, LAMP has characteristics such as high amplification efficiency, low detection cost, and a constant reaction temperature, and is applied to on-site nucleic acid testing. The isothermal amplification technology has solved the problems of PCR that require temperature changes and accurate temperature control, but there are still problems such as insufficient sensitivity and false positives. Therefore, it is of great significance to establish a nucleic acid detection method with high specificity, low cost, and simple operation. In many current studies, an isothermal amplification technology (such as the LAMP loop-mediated isothermal amplification technology) and a gene editing technology (CRISPR / Cas system) are used in combination. The CRISPR / Cas system has a mild reaction temperature and high signal amplification ability, and realizes accurate and rapid nucleic acid detection by utilizing the specific recognition and signal amplification strategy of this technology.

[0004] The above technique is usually divided into two steps. First, the first step involves amplifying the target nucleic acid fragment in the sample to be measured using isothermal amplification techniques. For example, the LAMP loop-mediated isothermal amplification reaction exponentially increases the number of target nucleic acid fragments in the sample to be measured by BstDNA polymerase at a constant temperature of 65°C, completing the amplification within the usual reaction time of 40 minutes. Next, the second step involves the detection of the endpoint fluorescence of the LAMP reaction product induced by the CRISPR / Cas system (e.g., CRISPR / Cas12a). After the LAMP reaction is complete, an appropriate amount of this reaction solution is taken and added to a test tube containing the CRISPR / Cas12a system premix. At a temperature of around 37°C, under the induction of crRNA, the Cas12a protein specifically recognizes and cleaves the target double-stranded DNA in the reaction solution, and its trans-cleavage activity is activated, nonspecifically cleaving single-stranded DNA probes containing fluorescent / quench groups. The cleaved single-stranded DNA probe emits a fluorescent signal of a specific wavelength and is collected by the instrument, allowing for the detection of the target nucleic acid fragment in the sample being measured.

[0005] However, in normal operation, the two reactions must be carried out separately, which leads to several unavoidable problems. For example: 1) After the LAMP reaction is complete, the lid must be opened and the reaction mixture added to the CRISPR / Cas system, which can cause serious aerosol contamination and lead to false positive results. 2) Repeated pipetting is required, which increases the experimental time, makes the procedure relatively complex, and requires expertise. [Overview of the Initiative]

[0006] This invention aims to solve the problem of aerosol contamination that can easily occur when LAMP and CRISPR reactions are performed separately, and to support the development of an integrated, automated detection device. We propose a closed-loop centrifugal microfluidic chip for LAMP-coupled CRISPR, embedding the LAMP and CRISPR reactions in different regions of the same microfluidic chip. This enables fully sealed, integrated testing of the chip, automating the entire process without causing aerosol contamination. Furthermore, it simplifies specialized manual operation, saving time while simultaneously improving the accuracy of the test.

[0007] To achieve the above objective, the present invention employs the following technical solutions. The microfluidic chip is LAMP-coupled CRISPR microfluidic chip, which is constructed by joining three layers of plates from top to bottom: a top plate, an intermediate plate, and a bottom plate. The microfluidic chip is fan-shaped, and the center of the fan shape is a rotational positioning port. The microfluidic chip is divided into a LAMP reaction region and a CRISPR reaction region, separated from the LAMP reaction region by a plurality of strip-shaped holes, and the LAMP reaction region is located close to the rotational positioning port. Within the aforementioned LAMP reaction region, one LAMP reaction chamber and multiple temperature control chambers are provided. Within the CRISPR reaction region, there is a single arc-shaped conduit, a waste liquid chamber, and multiple CRISPR reaction units. One end of the conduit communicates with the LAMP reaction chamber via a microchannel, and the other end communicates with the waste liquid chamber via a drainage passage. Each CRISPR reaction unit comprises a separatory chamber and a CRISPR reaction chamber. The separatory chamber is located on the side of the conduit away from the rotational positioning port, and is U-shaped, with the opening of the U-shape communicating with the outer wall of the conduit. The CRISPR reaction chamber is located on the side of the corresponding separatory chamber away from the rotational positioning port, and the bottom of the separatory chamber communicates with the corresponding CRISPR reaction chamber via an absorption passage. There is a one-to-one correspondence between the CRISPR reaction chambers and temperature control chambers, and the CRISPR reaction chambers communicate with the corresponding temperature control chambers via a pressure equilibrium passage, the pressure equilibrium passage having a buffer reserve chamber near the CRISPR reaction chamber.

[0008] Preferably, the LAMP reaction chamber, temperature control chamber, waste liquid chamber, and CRISPR reaction chamber are provided penetrating the intermediate plate vertically, the microchannel, guide channel, separatory chamber, and drainage passage are provided on the upper surface of the intermediate plate, and the absorption passage, pressure equilibrium passage, and buffer reserve chamber are provided on the lower surface of the intermediate plate.

[0009] Preferably, the closest distance between the liquid absorption passage and the rotational positioning port is smaller than the closest distance between the guide channel and the rotational positioning port.

[0010] Preferably, the center of the arc of the guide channel coincides with the center of the rotation positioning port.

[0011] Preferably, the buffer solution reserve chamber has an elongated structure, with both ends in the longitudinal direction connected to a pressure equilibrium passage.

[0012] Preferably, the drain port of the LAMP reaction chamber is located on the side away from the rotation positioning port in the LAMP reaction chamber, and the drain port of the LAMP reaction chamber is formed in a trumpet shape that narrows.

[0013] Preferably, the interface between the CRISPR reaction chamber and the liquid absorption passage, and the interface between the CRISPR reaction chamber and the pressure equilibrium passage, are both located on the side of the CRISPR reaction chamber facing the rotational positioning port.

[0014] Preferably, the top plate is provided with one liquid injection hole, the inside of the liquid injection hole is in communication with the LAMP reaction chamber, and a plug is provided at the outer opening of the liquid injection hole.

[0015] The present invention has the following beneficial effects: 1. The LAMP reaction and CRISPR reaction are integrated into a single microfluidic chip, and the reaction solution does not come into contact with the outside air during its movement, thus preventing aerosol contamination. 2. During the LAMP constant-temperature amplification reaction at 65°C, the temperature control chamber automatically transports the buffer solution from the buffer preparation chamber to the CRISPR reaction chamber using the principle of thermal expansion and contraction of air, and can mix it with the freeze-dried powder in the CRISPR reaction chamber to dissolve it in the premix solution, thus preparing the area for the CRISPR reaction in advance. 3. After the LAMP reaction is complete, the microfluidic chip is rotated by a centrifuge, and the reaction solution can be uniformly distributed into the separatory chamber using centrifugal force. During the air cooling process in the temperature control chamber, the reaction solution in the separatory chamber is automatically drawn into the CRISPR reaction chamber by siphon, allowing the CRISPR reaction to proceed. [Brief explanation of the drawing]

[0016] [Figure 1] This is a schematic diagram of the present invention. [Figure 2] This is a perspective view of the present invention. [Figure 3] This is an enlarged view of Figure 2a. [Figure 4] This is a top view of the upper passage of the intermediate plate of the present invention. [Figure 5] This is a top view of the lower passage of the intermediate plate of the present invention. [Modes for carrying out the invention]

[0017] The present invention will be further described below with reference to the accompanying drawings and specific embodiments. The LAMP-coupled CRISPR microfluidic chip, referring to Figures 1 to 5, is constructed by joining three layers of plates from top to bottom: a top plate, an intermediate plate, and a bottom plate. Instead of the top plate and bottom plate, a pressure-sensitive film may be used to sandwich the intermediate plate from above and below. The microfluidic chip is fan-shaped, and the central angle of the arc of the fan is 180°, i.e., a semicircle. Two chips with 180° rotational symmetry can form a circular chip, and the center position of the fan is a rotational positioning port 4. The microfluidic chip is divided into a LAMP reaction region 1 and a CRISPR reaction region 2, and the LAMP reaction region 1 and the CRISPR reaction region 2 are separated by a plurality of strip-shaped holes 3. The plurality of strip-shaped holes 3 are located on the same arc with the center of the rotational positioning port 4 as the center. The LAMP reaction region 1 is provided close to the rotational positioning port 4, and the CRISPR reaction region 2 is located on the side of the dividing line of the strip-shaped holes 3 away from the rotational positioning port 4.

[0018] Inside the LAMP reaction region 1, there is one LAMP reaction chamber 5 and five temperature control chambers 6. Within the CRISPR reaction region 2, there is one arc-shaped conduit 7, one waste liquid chamber 8, and five CRISPR reaction units. The center of the arc of the conduit 7 coincides with the center of the rotation positioning port 4, one end of the conduit 7 communicates with the LAMP reaction chamber 5 via a microchannel 9, the drain port of the LAMP reaction chamber 5 is located away from the rotation positioning port 4, and the drain port of the LAMP reaction chamber 5 is formed in a constricting trumpet shape, and the other end of the conduit 7 communicates with the waste liquid chamber 8 via a drain passage 10. The waste liquid chamber 8 may be used to recover excess reagents, or it may be used to improve the airflow when the air in the temperature control chamber 6 expands and contracts due to heat and cold.

[0019] Each CRISPR reaction unit comprises a separatory chamber 11 located away from the rotational positioning port 4 of the conduit 7, and a CRISPR reaction chamber 12. The separatory chamber 11 is U-shaped, with the U-shaped opening communicating with the outer wall of the conduit 7. The CRISPR reaction chamber 12 is located away from the rotational positioning port 4 of the corresponding separatory chamber 11, and is connected to the bottom of the separatory chamber 11 by a single suction passage 13. The closest distance between the suction passage 13 and the rotational positioning port 4 is smaller than the closest distance between the conduit 7 and the rotational positioning port 4. This configuration prevents reagents in the conduit 7 from entering the CRISPR reaction chamber 12 prematurely when the microfluidic tip is rotated and centrifugal force is used for liquid separation. This allows for more uniform liquid separation within each CRISPR reaction unit, and further enables more accurate final test results. The CRISPR reaction chamber 12 and the temperature control chamber 6 are in one-to-one correspondence, and the CRISPR reaction chamber 12 and the corresponding temperature control chamber 6 are connected via a pressure equilibrium passage 14. A buffer reserve chamber 15 is provided near the CRISPR reaction chamber 12 on the pressure equilibrium passage 14, and the buffer reserve chamber 15 has a long structure, with both ends in the length direction connected to the pressure equilibrium passage 14. The long buffer reserve chamber 15 allows the buffer reserve stored inside to be transported more sufficiently into the CRISPR reaction chamber 12 when subjected to air pressure. Furthermore, the interface between the CRISPR reaction chamber 12 and the liquid absorption passage 13, and the interface between the CRISPR reaction chamber 12 and the pressure equilibrium passage 14 are both located on the side of the CRISPR reaction chamber 12 facing the rotation positioning port 4.

[0020] The LAMP reaction chamber 5, temperature control chamber 6, waste liquid chamber 8, and CRISPR reaction chamber 12 are provided vertically through an intermediate plate, the microchannel 9, guide channel 7, separatory chamber 11, and drainage passage 10 are provided on the upper surface of the intermediate plate, and the absorption passage 13, pressure equilibrium passage 14, and buffer reserve chamber 15 are provided on the lower surface of the intermediate plate. Some of the passages are arranged hierarchically to avoid collisions between them.

[0021] On the top plate, a liquid injection hole 16 whose inside communicates with the LAMP reaction chamber 5 is provided, and a hole plug is provided at the outer opening of the liquid injection hole 16.

[0022] In the present invention, before shipment, according to the customer's requirements, a desired CRISPR / Cas12a reaction solution can be pre-embedded as a lyophilized powder into the CRISPR reaction chamber 12, and a buffer solution can be pre-stored in the buffer solution reserve chamber 15.

[0023] When performing a nucleic acid amplification detection test using the present invention, the specific procedure is as follows. 1: Mix the sample to be detected and its reaction enzyme to prepare a reaction solution for the LAMP reaction. Inject this reaction solution into the LAMP reaction chamber 5 through the liquid injection hole 16, and cover it with the hole plug. 2: Place the LAMP reaction region 1 of the microfluidic chip on the heater and heat it to react at a constant temperature of 65°C for about 40 minutes to perform the LAMP isothermal amplification reaction and increase the number of target nucleic acid fragments. When the LAMP reaction region 1 is heated at a constant temperature of 65°C, the temperature in the temperature control chamber 6 also rises. The air in the temperature control chamber 6 receives heat and expands, and the pressure increases. In order to balance the air pressure, the high-pressure gas in the temperature control chamber 6 sequentially passes through the air pressure balance passage 14, the buffer solution reserve chamber 15, the CRISPR reaction chamber 12, the liquid absorption passage 13, the liquid separation chamber 11, and the flow guide passage 7, and enters the waste liquid chamber 8. Finally, the air pressure in all the communication chambers and passages reaches a stable value (the stable value is higher than the air pressure value when not heated initially). When the high-pressure gas passes through the buffer solution reserve chamber 15, the buffer solution in the buffer solution reserve chamber 15 can be pushed into the CRISPR reaction chamber 12. As a result, the buffer solution is mixed and dissolved with the lyophilized powder pre-embedded in the CRISPR reaction chamber 12, and a premix solution required for the CRISPR reaction can be generated. Such a setting solves the problem that the premix solution is pre-embedded and is likely to deteriorate over time, affecting the test accuracy, and solves the problem that the premix solution injection is temporarily prepared and is likely to cause aerosol contamination. It can automatically prepare the premix solution required for the CRISPR reaction during the LAMP reaction, and effectively improve the convenience of the test. 3. After the LAMP reaction is complete, the microfluidic chip is moved from the heater to the centrifuge and rotated. Due to centrifugal force, the reaction liquid in the LAMP reaction chamber 5 flows through the microchannel 9 into the conduit channel 7, and then flows from the conduit channel 7 into the separatory chambers 11, filling each of the separatory chambers 11. Any remaining reaction liquid that does not enter the separatory chambers 11 eventually flows into the waste liquid chamber 8. Furthermore, the time taken for this separation process is relatively short. Since the closest distance between the suction passage 13 and the rotation positioning port 4 is smaller than the closest distance between the conduit channel 7 and the rotation positioning port 4, in the separation process due to centrifugal force, the reaction liquid in the separatory chambers 11 does not enter the CRISPR reaction chambers 12 through the suction passage 13. By making the amount of reaction liquid in each separatory chamber 11 as equal as possible during the separation process, consistency in the subsequent tests in each CRISPR reaction chamber 12 is ensured. Next, the microfluidic chip is continuously rotated until the temperature inside the temperature control chamber 6 is reduced to room temperature. During the temperature reduction process inside the temperature control chamber 6, the air pressure inside the temperature control chamber 6 decreases, and a pressure difference is again formed with the waste liquid chamber 8. At this time, it can be understood that the gas flow direction is such that an suction force is generated in the following order as the temperature decreases inside each temperature control chamber 6: waste liquid chamber 8, conduit 7, separatory chamber 11, suction passage 13, CRISPR reaction chamber 12, buffer reserve chamber 15, pressure equilibrium passage 14, and then temperature control chamber 6. Furthermore, in this gas flow direction, each temperature control chamber 6 draws the LAMP reaction solution from the corresponding separatory chamber 11 into the corresponding CRISPR reaction chamber 12, where it mixes with the premix solution in the CRISPR reaction chamber 12 to form the reaction solution necessary for the CRISPR reaction. Since both the interface between the CRISPR reaction chamber 12 and the liquid absorption passage 13, and the interface between the CRISPR reaction chamber 12 and the pressure equilibrium passage 14 are located on the side of the CRISPR reaction chamber 12 facing the annular center of the CRISPR reaction region 2, the reaction solution in the CRISPR reaction chamber 12 does not flow out of the pressure equilibrium passage 14 during centrifugal rotation. 4. Remove the microfluidic chip from the centrifuge and place it on a heater to carry out the CRISPR reaction in a constant temperature heating process at 37°C. This constant temperature heating may be applied to either the CRISPR reaction region 2 or the entire microfluidic chip. 5. After the CRISPR reaction is complete, the final detection result can be obtained simply by observing the fluorescence intensity of the reaction solution in each CRISPR reaction chamber 12 with a CCD fluorescence camera. [Explanation of Symbols]

[0024] 1: LAMP reaction area, 2: CRISPR reaction area, 3: Strip-shaped pore, 4: Rotating positioning port, 5: LAMP reaction chamber, 6: Temperature control chamber, 7: Conduit channel, 8: Waste liquid chamber, 9: Microchannel, 10: Drainage passage, 11: Separation chamber, 12: CRISPR reaction chamber, 13: Absorption passage, 14: Pressure equilibrium passage, 15: Buffer reserve chamber, 16: Liquid injection port

Claims

1. A LAMP-coupled CRISPR microfluidic chip, wherein the microfluidic chip is constructed by joining three layers of plates, a top plate, an intermediate plate, and a bottom plate, in that order from top to bottom. The microfluidic chip is fan-shaped, the center of the fan is a rotational positioning port (4), the microfluidic chip is divided into a LAMP reaction region (1) and a CRISPR reaction region (2), the LAMP reaction region (1) and the CRISPR reaction region (2) are separated by a plurality of strip-shaped holes (3), the LAMP reaction region (1) is provided in close proximity to the rotational positioning port (4), Within the aforementioned LAMP reaction region (1), one LAMP reaction chamber (5) and multiple temperature control chambers (6) are provided. Within the CRISPR reaction region (2), there is one arc-shaped conduit (7), one waste liquid chamber (8), and a plurality of CRISPR reaction units. One end of the conduit (7) communicates with the LAMP reaction chamber (5) via a microchannel (9), and the other end communicates with the waste liquid chamber (8) via a drainage passage (10). Each CRISPR reaction unit comprises one separatory chamber (11) and one CRISPR reaction chamber (12). The separatory chamber (11) is located on the side of the conduit (7) away from the rotational positioning port (4). The separatory chamber (11) is U-shaped, and the U-shaped opening communicates with the outer wall of the conduit (7). A microfluidic chip for LAMP-coupled CRISPR, characterized in that the CRISPR reaction chamber (12) is located away from the rotation positioning port (4) of the corresponding separatory chamber (11), the bottom of the separatory chamber (11) and the corresponding CRISPR reaction chamber (12) are connected via a single absorption passage (13), the CRISPR reaction chamber (12) and the temperature control chamber (6) are in a one-to-one correspondence, the CRISPR reaction chamber (12) and the corresponding temperature control chamber (6) are connected via a pressure equilibrium passage (14), and a buffer reserve chamber (15) is provided in the vicinity of the CRISPR reaction chamber (12) in the pressure equilibrium passage (14).

2. The LAMP-coupled CRISPR microfluidic chip according to claim 1, characterized in that the LAMP reaction chamber (5), temperature control chamber (6), waste liquid chamber (8), and CRISPR reaction chamber (12) are provided penetrating the intermediate plate vertically, the microchannel (9), guide channel (7), separatory chamber (11), and drainage passage (10) are provided on the upper surface of the intermediate plate, and the liquid absorption passage (13), pressure equilibrium passage (14), and buffer reserve chamber (15) are provided on the lower surface of the intermediate plate.

3. The microfluidic chip of a LAMP-coupled CRISPR according to claim 1 or 2, characterized in that the closest proximity distance between the liquid absorption passage (13) and the rotational positioning port (4) is smaller than the closest proximity distance between the guide channel (7) and the rotational positioning port (4).

4. The microfluidic chip of a LAMP-coupled CRISPR according to claim 1 or 2, characterized in that the center of the arc of the guide channel (7) coincides with the center of the rotational positioning port (4).

5. The microfluidic chip of a LAMP-coupled CRISPR according to claim 1 or 2, characterized in that the buffer reserve chamber (15) has an elongated structure and both ends in the longitudinal direction are connected to a pressure equilibrium passage (14).

6. The microfluidic chip of a LAMP-coupled CRISPR according to claim 1 or 2, characterized in that the drain port of the LAMP reaction chamber (5) is located on the side away from the rotation positioning port (4) in the LAMP reaction chamber (5), and the drain port of the LAMP reaction chamber (5) is formed in a trumpet shape that narrows.

7. The microfluidic chip of a LAMP-coupled CRISPR according to claim 1 or 2, characterized in that the interface between the CRISPR reaction chamber (12) and the liquid absorption passage (13), and the interface between the CRISPR reaction chamber (12) and the pressure equilibrium passage (14) are both located on the side facing the rotational positioning port (4) in the CRISPR reaction chamber (12).

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

Images