Integrated nucleic acid detection chip and reagent adding method thereof

Abstract

The invention provides an integrated nucleic acid detection chip and a reagent adding method thereof.The chip comprises a splitting cavity, a first cavity, a first cleaning cavity, a second cavity, a reaction cavity, a sample adding opening, an elution liquid adding opening and a first cleaning liquid adding opening which are sequentially communicated from top to bottom, the sample adding opening is communicated with the splitting cavity, the first cleaning liquid adding opening is communicated with the first cleaning cavity, and the second cleaning cavity is communicated with the second cavity. The elution liquid adding opening is communicated with the reaction cavity; the micro-fluidic chip further comprises a storage cavity, a first filling port, a second filling port, a third filling port, a storage port and a storage cover; the storage cover covers the storage port of the storage cavity; the first cavity is filled with a first isolator; and the second cavity is filled with a second isolator. The storage cavity is located above the reaction cavity and communicated with the reaction cavity, and the storage cavity and the reaction cavity are separated through a third isolator; the first filling port is communicated with the first cavity; the second filling port is communicated with the second cavity; and the third filling port is communicated with the storage cavity and the reaction cavity. The chip is convenient to operate, good in sealing effect and small in size.

Description

Technical Field

[0001] The present invention relates to the technical field of microfluidics, and particularly to an integrated nucleic acid detection chip and a method for adding reagents thereto. Background Art

[0002] When performing nucleic acid analysis and detection in a laboratory environment, there are problems such as long detection time and the need for professional operation, which are difficult to meet the on-site, rapid, and portable detection requirements for additives. The microfluidic chip technology developed in recent years can not only greatly shorten the detection time, but also has the advantages of large detection throughput, high automation degree, and being used by non-professional personnel, and is expected to be widely applied in the field of nucleic acid analysis and detection.

[0003] A complete nucleic acid extraction and detection process generally requires four nucleic acid extraction steps: lysis, binding, washing, and elution, plus subsequent detection steps such as nucleic acid molecular hybridization, polymerase chain reaction (PCR), and biochips. In terms of the transfer of active ingredients in each step, manual transfer is mostly used in the prior art, which is not only cumbersome and time-consuming, but also difficult to transfer samples fully and efficiently, and manual operation is extremely likely to lead to unstable results.

[0004] An existing microfluidic chip for biological sample processing includes a plurality of reagent storage areas, a first functional area, a waste liquid area, and a microchannel switch. The reagent storage area is used to store reagents for processing biological samples, the first functional area is an area for processing biological samples with the reagents stored in the reagent storage area, and a reagent microchannel is respectively arranged between each reagent storage area and the sample area; the microchannel switch is used to switch the opening and closing of each reagent microchannel, and the waste liquid area is used to receive the waste liquid discharged from the first functional area.

[0005] However, in this solution, after each reaction with a reagent, the waste liquid needs to be discharged to the waste liquid area before mixing with the next reaction reagent. The operation is complex, the large number of chambers provided results in a large volume of the chip, and the structure is complex, with high production costs, and the mechanical valves provided have poor sealing effects. Summary of the Invention

[0006] In order to solve the problems existing in the above-mentioned prior art, the first object of the present invention is to provide an integrated nucleic acid detection chip with a simple structure, convenient operation, good sealing effect, and small volume.

[0007] The second object of the present invention is to provide a method for adding reagents to the above-mentioned integrated microfluidic chip.

[0008] To achieve the above first object, the integrated nucleic acid detection chip provided by the present invention includes a lysis chamber, a first cavity, a first washing chamber, a second cavity, and a reaction chamber that are connected in sequence from top to bottom; the microfluidic chip further includes a sample addition port, an elution liquid addition port, and a first washing liquid addition port. The sample addition port is connected to the lysis chamber, the first washing liquid addition port is connected to the first washing chamber, and the elution liquid addition port is connected to the reaction chamber; the microfluidic chip further includes a storage chamber, a first filling port, a second filling port, a third filling port, a storage opening, and a storage cover. The storage opening is connected to the storage chamber, and the storage cover is closed at the storage opening; the first cavity is filled with a first isolating substance; the second cavity is filled with a second isolating substance; the storage chamber is connected to the reaction chamber, the storage chamber is located above the reaction chamber, and the storage chamber and the reaction chamber are separated by a third isolating substance; the first isolating substance, the second isolating substance, and the third isolating substance are all hydrophobic isolating substances; the first filling port is connected to the first cavity; the second filling port is connected to the second cavity; the third filling port is located between the storage chamber and the reaction chamber, and the third filling port is respectively connected to the storage chamber and the reaction chamber.

[0009] As can be seen from the above, the microfluidic chip provided by the present invention includes a lysis chamber, a washing chamber, a reaction chamber, and a storage chamber, without the need to provide an elution chamber, a waste liquid chamber, etc. The structure is simple, and the volume of the chip is greatly reduced. Moreover, the provided storage chamber and the storage opening connected to the storage chamber can facilitate the user to add reaction reagents according to different detection requirements, and through the provided hydrophobic first isolating substance and second isolating substance, it can effectively prevent the reaction reagents between different chambers from flowing and disturbing during transportation and storage, and the sealing effect is good.

[0010] A further solution is that the microfluidic chip is provided with a first positioning blind hole for assisting the positioning of the ultrasonic device, and the first positioning blind hole is correspondingly arranged with the lysis chamber; and / or the microfluidic chip is further provided with a second positioning blind hole for assisting the positioning of the ultrasonic device, and the second positioning blind hole is correspondingly arranged with the first washing chamber.

[0011] As can be seen from the above, through the provided first positioning blind hole and second positioning blind hole, it is convenient for the ultrasonic device of the amplification detection device to quickly adjust to the corresponding position and perform ultrasonic mixing operation.

[0012] A further solution is that the microfluidic chip further includes a second washing chamber, a second washing liquid addition port, a fourth filling port, and a third cavity. The third cavity is located between the first washing chamber and the second washing chamber, and the second washing chamber is located between the third cavity and the second cavity; the second washing liquid addition port is connected to the second washing chamber; the fourth filling port is connected to the third cavity; the third cavity is filled with a hydrophobic fourth isolating substance.

[0013] As can be seen from the above, through the provided first washing chamber and second washing chamber, the washing effect can be effectively improved.

[0014] A further solution is that a third positioning blind hole for assisting in positioning the ultrasonic device is also provided on the microfluidic chip, and the third positioning blind hole is correspondingly arranged with the second cleaning chamber.

[0015] A further solution is that the second cavity includes a first cavity and a second cavity that are sequentially connected from top to bottom. Along the thickness direction of the microfluidic chip, the thickness of the second cavity is smaller than that of the first cavity, and the second cavity is located between the first cavity and the reaction chamber; the second isolating material includes silicone oil, and both the first cavity and the second cavity are filled with silicone oil; alternatively, the second isolating material includes paraffin and silicone oil, the second cavity is filled with paraffin, and the first cavity is filled with silicone oil; the second filling port is communicated with the first cavity.

[0016] As can be seen from the above, when the silicone oil flows from the first cavity downward to the second cavity, since the thickness of the first cavity is greater than that of the second cavity, the channel becomes narrower. When the silicone oil passes through, the viscosity of the channel wall to the liquid increases, making it difficult for the silicone oil to flow, and preventing the silicone oil from entering the reaction chamber during the PCR reaction process.

[0017] A further solution is that the first cavity includes a third cavity and a fourth cavity that are sequentially connected from top to bottom. Along the thickness direction of the microfluidic chip, the thickness of the third cavity is greater than that of the fourth cavity; the number of the first filling ports is two, one first filling port is communicated with the third cavity, and the other first filling port is communicated with the fourth cavity; the first isolating material includes paraffin and silicone oil, the third cavity is filled with paraffin, and the fourth cavity is filled with silicone oil.

[0018] To achieve the above second object, the method for adding reagents provided by the present invention is applied to any one of the above integrated nucleic acid detection chips, and the method includes the following steps:

[0019] S1: Add eluent to the eluent filling port until the reaction chamber is filled with the eluent;

[0020] S2: Add a third isolating material to the third filling port to separate the reaction chamber from the storage chamber;

[0021] S3: Add a second isolating material to the eluent filling port, the second isolating material fills the second cavity, and then add the second isolating material to the second filling port, the second isolating material fills the first cavity, to separate the first cleaning chamber from the reaction chamber;

[0022] S4: Add the first cleaning liquid to the first cleaning liquid filling port until the first cleaning chamber is filled with the first cleaning liquid;

[0023] S5: Add a first isolating material to the first filling port to isolate the lysis chamber from the first cleaning chamber.

[0024] As can be seen above, the cavities of the chip are arranged in sequence from top to bottom, and the reagent is added from bottom to top, making it not easy to leave bubbles during the reagent addition process and preventing the generated bubbles from affecting the reading of fluorescence detection data.

[0025] A further solution is that the second cavity includes a first cavity and a second cavity that communicate with each other, and the second cavity is located between the first cavity and the reaction cavity; the second isolation material includes paraffin and silicone oil, and step S3 further includes: adding paraffin to the elution liquid inlet until the second cavity is filled with paraffin, and then adding silicone oil to the second filling port until the first cavity is filled with silicone oil, separating the first cleaning cavity from the reaction cavity.

[0026] A further solution is that the first cavity includes a third cavity and a fourth cavity, and the fourth cavity is located between the third cavity and the first cleaning cavity; the number of the first filling ports is two, one first filling port communicates with the third cavity, and the other first filling port communicates with the fourth cavity; the first isolation material includes paraffin and silicone oil; step S5 further includes: adding silicone oil to one first filling port until the fourth cavity is filled with silicone oil, and then adding paraffin to the other first filling port until the third cavity is filled with paraffin.

[0027] A further solution is that the microfluidic chip further includes a second cleaning cavity, a fourth filling port and a third cavity, the third cavity is located between the first cleaning cavity and the second cleaning cavity, and the second cleaning cavity is located between the third cavity and the second cavity; the fourth filling port communicates with the third cavity; the third cavity is filled with a hydrophobic fourth isolation material, and the fourth isolation material includes silicone oil; step S3 further includes: adding the second isolation material to the elution liquid inlet, filling the second cavity with the second isolation material, then adding the second isolation material to the second filling port, filling the first cavity with the second isolation material, separating the first cleaning cavity from the reaction cavity, then adding the second cleaning liquid to the second cleaning liquid inlet until the second cleaning cavity is filled with the second cleaning liquid, and finally adding silicone oil to the fourth filling port until the third cavity is filled with silicone oil.

[0028] In summary, the integrated nucleic acid detection chip provided by the present invention has a simple structure, convenient operation, good sealing effect, small volume, can effectively reduce the risk of generating bubbles during reagent filling, and is convenient for observation. Description of the Drawings

[0029] Figure 1 It is a structural diagram of an embodiment of the integrated nucleic acid detection chip of the present invention.

[0030] Figure 2 It is an internal structural diagram of removing the back shell of an embodiment of the integrated nucleic acid detection chip of the present invention.

[0031] Figure 3 It is a cross-sectional view of an embodiment of the integrated nucleic acid detection chip of the present invention. Detailed Embodiments

[0032] See Figures 1 to 3 Figures 1 to 3 , the integrated nucleic acid detection chip provided in this embodiment includes a lysis chamber 1, a first cavity 2, a first cleaning chamber 3, a third cavity 4, a second cleaning chamber 5, a second cavity 6, a reaction chamber 7, a storage chamber 8, a storage port 9, a first filling port 100, a first filling port 101, a second filling port 11, a third filling port 12, a fourth filling port 13, an elution liquid adding port 30, a first cleaning liquid adding port 31, a second cleaning liquid adding port 32, and a sample adding port 14. The sample adding port 14 is communicated with the lysis chamber 1, the elution liquid adding port 30 is communicated with the reaction chamber 7, the first cleaning liquid adding port 31 is communicated with the first cleaning chamber 3 and is arranged near the bottom of the first cleaning chamber 3, and the second cleaning liquid adding port 32 is communicated with the second cleaning chamber 5 and is arranged near the bottom of the second cleaning chamber 5. The fourth filling port 13 is communicated with the third cavity 4 and is arranged near the bottom of the third cavity 4. The third filling port 12 is located between the storage chamber 8 and the reaction chamber 7 and is communicated with the storage chamber 8 and the reaction chamber 7. A sample adding cover 15 is covered at the sample adding port 14.

[0033] The lysis chamber 1, the first cavity 2, the first cleaning chamber 3, the third cavity 4, the second cleaning chamber 5, the second cavity 6, and the reaction chamber 7 are communicated with each other in sequence from top to bottom. The storage chamber 8 is located above the reaction chamber 7 and is communicated with the reaction chamber 7. A third isolation material (not shown in the figure) for spacing is filled between the storage chamber 8 and the reaction chamber 7. The storage chamber 8 is communicated with the storage port 9, and a storage cover 19 is covered at the storage port 9. The user can open the storage cover 19 to add the required reaction reagent into the storage port 9, or the reaction reagent is pre-stored in the storage chamber 8 in advance. A first isolation material (not shown in the figure) and a second isolation material (not shown in the figure) are respectively filled in the first cavity 2 and the second cavity 6. A fourth isolation material (not shown in the figure) is filled in the third cavity 4. The first isolation material, the second isolation material, the third isolation material, and the fourth isolation material are all hydrophobic.

[0034] On the microfluidic chip, there are provided a first positioning blind hole 16, a second positioning blind hole 17, and a third positioning blind hole 18 for assisting in the positioning of the ultrasonic device. The first positioning blind hole 16 is correspondingly arranged with the lysis chamber 1, the second positioning blind hole 17 is correspondingly arranged with the first cleaning chamber 3, and the third positioning blind hole 18 is correspondingly arranged with the second cleaning chamber 5.

[0035] The first cavity 2 includes a third cavity 20 and a fourth cavity 21 that are communicated with each other in sequence from top to bottom. Along the thickness direction of the microfluidic chip, the thickness of the third cavity 20 is greater than that of the fourth cavity 21. The third cavity 20 is communicated with the lysis chamber 1, the fourth cavity 21 is communicated with the first cleaning chamber 3, and the fourth cavity 21 is located between the third cavity 20 and the first cleaning chamber 3. The first filling port 100 is communicated with the third cavity 20 and is arranged near the bottom of the third cavity 20, and the first filling port 101 is communicated with the fourth cavity 21 and is arranged near the bottom of the fourth cavity 21.

[0036] The second cavity 6 includes a first cavity 60 and a second cavity 61 that are connected in sequence from top to bottom. The first cavity 60 is connected to the second cleaning cavity 5, and the second cavity 61 is connected to the reaction cavity 7. The second cavity 61 is located between the first cavity 60 and the reaction cavity 7. Along the thickness direction of the microfluidic chip, the thickness of the second cavity 61 is less than that of the first cavity 60. The second filling port 110 is connected to the first cavity 60 and is disposed near the bottom of the first cavity 60, and the elution liquid adding port 30 is connected to the second cavity 61 and is disposed near the bottom of the second cavity 61.

[0037] This embodiment also provides a method for adding reagents to the above microfluidic chip, and the method further includes the following steps:

[0038] S1: Add eluent to the elution liquid adding port 30 until the reaction cavity 7 is filled with the eluent;

[0039] S2: Add a third isolation substance to the third filling port 12. The third isolation substance includes paraffin,

[0040] to separate the reaction cavity 7 from the storage cavity 8;

[0041] S3: The second isolation substance includes silicone oil. Add silicone oil to the elution liquid adding port 30 until the second cavity 61 is filled with the silicone oil, then add silicone oil to the second filling port 11 until the first cavity 60 is filled with the silicone oil. Then add the second cleaning liquid to the second cleaning liquid adding port 32 until the second cleaning cavity 5 is filled with the second cleaning liquid. Finally, add silicone oil to the fourth filling port 13 until the third cavity 4 is filled with the silicone oil;

[0042] S4: Add the first cleaning liquid to the first cleaning liquid adding port 31 until the first cleaning cavity 3 is filled with the first cleaning liquid;

[0043] S5: Add a part of silicone oil to one of the first filling ports 101, and then add paraffin to the other first filling port 100 until the third cavity 20 is filled with the paraffin.

[0044] Optionally, the second isolation substance includes paraffin and silicone oil,

[0045] Step S3 includes: adding paraffin to the elution liquid adding port 30 until the second cavity 61 is filled with the paraffin; then adding silicone oil to the second filling port 11 until the first cavity 60 is filled with the silicone oil, separating the second cleaning cavity 5 from the reaction cavity 7. Then add the second cleaning liquid to the second cleaning liquid adding port 32 until the second cleaning cavity 5 is filled with the second cleaning liquid. Finally, add silicone oil to the fourth filling port 13 until the third cavity 4 is filled with the silicone oil.

[0046] When the microfluidic chip is in use, open the sampling cover 15. After adding magnetic bead reagent and the sample to be tested into the sampling port 14, add the lysis reagent. After the sampling is completed, cover the sampling cover 15, and place the microfluidic chip vertically into the detection device with the lysis chamber 1 on the top and the reaction chamber 7 on the bottom. The ultrasonic mechanism in the device closely contacts the first positioning blind hole 16 and disperses the magnetic beads. After the sample to be tested in the lysis chamber 1 is fully lysed, the magnetic bead device in the detection device adsorbs and aggregates the magnetic beads. The detection device heats the third cavity 20 to melt the paraffin into a liquid phase. The magnetic bead device drives the aggregated magnetic beads to enter the first cleaning chamber 3 successively through the paraffin and silicone oil in the first cavity 2. The ultrasonic mechanism closely contacts the second positioning blind hole 17 and disperses the magnetic beads in the first cleaning chamber 3. After the sample to be tested is fully washed in the first cleaning chamber 3, the magnetic bead device adsorbs and aggregates the magnetic beads. The magnetic bead device controls the aggregated magnetic beads to pass through the silicone oil in the third cavity 4 and enter the second cleaning chamber 5. The ultrasonic mechanism closely contacts the third positioning blind hole 18 and disperses the magnetic beads. After the sample to be tested is fully washed, the magnetic bead device adsorbs and aggregates the magnetic beads. The magnetic bead device controls the aggregated magnetic beads to pass through the silicone oil in the second cavity 6 and enter the reaction chamber 7 successively. Through the magnetic bead device and heating device of the detection device, the magnetic beads and the sample in the reaction chamber 7 are fully heated and mixed with the eluent, so that the magnetic beads are fully eluted and the purified nucleic acid is released. Then, the heating device of the detection device heats the third isolation layer between the storage chamber 8 and the reaction chamber 7 to melt it into a liquid phase. The magnetic bead device drags the dried reaction reagent (including magnetic bead and other magnetic carriers) in the storage chamber 8 downward into the reaction chamber 7, and repeatedly shakes the reaction reagent up and down to make it fully contact and redissolve with the liquid in the reaction chamber 7. After standing for a period of time, the magnetic bead device brings the magnetic carrier back up to the storage chamber 8, and waits for the third isolation layer to solidify. The magnetic bead device keeps adsorbing the magnetic carrier until the PCR reaction ends to ensure that the magnetic carrier will not fall naturally due to gravity during the reaction process. Finally, the temperature control module and heating device of the detection device perform the PCR reaction on the reaction chamber 7, and read the fluorescence data to complete the nucleic acid extraction and detection process.

[0047] In summary, the integrated nucleic acid detection chip of the present invention has a simple structure, convenient operation, good sealing effect, and small volume.

[0048] Finally, it should be emphasized that the above are only the preferred embodiments of the present invention and are not used to limit the present invention. For those skilled in the art, the present invention can have various changes and modifications. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. Integrated nucleic acid detection chip, characterized in that: It includes a lysis chamber, a first cavity, a first washing chamber, a second cavity, and a reaction chamber that are connected in sequence from top to bottom; The microfluidic chip further includes a sample addition port, an elution liquid addition port, and a first washing liquid addition port. The sample addition port is connected to the lysis chamber, the first washing liquid addition port is connected to the first washing chamber, and the elution liquid addition port is connected to the reaction chamber; The microfluidic chip further includes a storage chamber, a first filling port, a second filling port, a third filling port, a storage opening, and a storage cover. The storage opening is connected to the storage chamber, and the storage cover is closed at the storage opening; The first cavity is filled with a first isolation material; The second cavity is filled with a second isolation material; The storage chamber is connected to the reaction chamber. The storage chamber is located above the reaction chamber, and the storage chamber and the reaction chamber are separated by a third isolation material; The first isolation material, the second isolation material, and the third isolation material are all hydrophobic isolation materials; The first filling port is connected to the first cavity; The second filling port is connected to the second cavity; The third filling port is located between the storage chamber and the reaction chamber, and the third filling port is respectively connected to the storage chamber and the reaction chamber.

2. The integrated nucleic acid detection chip according to claim 1, characterized in that: The microfluidic chip is provided with a first positioning blind hole for assisting in the positioning of the ultrasonic device, and the first positioning blind hole is correspondingly arranged with the lysis chamber; And / or the microfluidic chip is further provided with a second positioning blind hole for assisting in the positioning of the ultrasonic device, and the second positioning blind hole is correspondingly arranged with the first washing chamber.

3. The integrated nucleic acid detection chip according to claim 2, characterized in that: The microfluidic chip further includes a second washing chamber, a second washing liquid addition port, a fourth filling port, and a third cavity. The third cavity is located between the first washing chamber and the second washing chamber, and the second washing chamber is located between the third cavity and the second cavity; The second washing liquid addition port is connected to the second washing chamber; The fourth filling port is connected to the third cavity; The third cavity is filled with a hydrophobic fourth isolation material.

4. The integrated nucleic acid detection chip according to claim 3, characterized in that: The microfluidic chip is further provided with a third positioning blind hole for assisting in the positioning of the ultrasonic device, and the third positioning blind hole is correspondingly arranged with the second washing chamber.

5. The integrated nucleic acid detection chip according to any one of claims 1 to 4, characterized in that: The second cavity includes a first cavity and a second cavity that are connected in sequence from top to bottom. Along the thickness direction of the microfluidic chip, the thickness of the second cavity is smaller than the thickness of the first cavity, and the second cavity is located between the first cavity and the reaction chamber; The second isolation material includes silicone oil, and both the first cavity and the second cavity are filled with silicone oil; Or, the second isolation material includes paraffin and silicone oil, the second cavity is filled with paraffin, and the first cavity is filled with silicone oil; The second filling port is connected to the first cavity.

6. The integrated nucleic acid detection chip according to claim 5, wherein: The first cavity includes a third cavity and a fourth cavity that are sequentially connected from top to bottom. Along the thickness direction of the microfluidic chip, the thickness of the third cavity is greater than that of the fourth cavity; The number of the first filling ports is two. One of the first filling ports is communicated with the third cavity, and the other first filling port is communicated with the fourth cavity; The first isolating material includes paraffin and silicone oil. The third cavity is filled with paraffin, and the fourth cavity is filled with silicone oil.

7. A method for adding reagents, which is applied to the integrated microfluidic chip according to claim 1 or 2. The method includes the following steps: S1: Add eluent to the elution liquid adding port until the reaction cavity is filled with the eluent; S2: Add the third isolating material to the third filling port to separate the reaction cavity from the storage cavity; S3: Add the second isolating material to the elution liquid adding port. The second isolating material fills the second cavity, and then add the second isolating material to the second filling port. The second isolating material fills the first cavity to separate the first cleaning cavity from the reaction cavity; S4: Add the first cleaning liquid to the first cleaning liquid adding port until the first cleaning cavity is filled with the first cleaning liquid; S5: Add the first isolating material to the first filling port to isolate the lysis cavity from the first cleaning cavity.

8. The method for adding reagents according to claim 7, wherein: The second cavity includes a first cavity and a second cavity that are communicated with each other. The second cavity is located between the first cavity and the reaction cavity; The second isolating material includes paraffin and silicone oil. The step S3 further includes: adding the paraffin to the elution liquid adding port until the second cavity is filled with the paraffin, and then adding the silicone oil to the second filling port until the first cavity is filled with the silicone oil to separate the first cleaning cavity from the reaction cavity.

9. The method for adding reagents according to claim 8, wherein: The first cavity includes a third cavity and a fourth cavity. The fourth cavity is located between the third cavity and the first cleaning cavity; The number of the first filling ports is two. One of the first filling ports is communicated with the third cavity, and the other first filling port is communicated with the fourth cavity; The first isolating material includes paraffin and silicone oil; The step S5 further includes: adding the silicone oil to one of the first filling ports until the fourth cavity is filled with the silicone oil, and then adding the paraffin to the other first filling port until the third cavity is filled with the paraffin.

10. The method for adding reagents according to any one of claims 7 to 9, wherein: The microfluidic chip further includes a second cleaning cavity, a fourth filling port, and a third cavity. The third cavity is located between the first cleaning cavity and the second cleaning cavity, and the second cleaning cavity is located between the third cavity and the second cavity; The fourth filling port is communicated with the third cavity; The third cavity is filled with a hydrophobic fourth insulator, and the fourth insulator includes silicone oil; Step S3 further includes: adding the second insulator into the elution liquid addition port, filling the second cavity with the second insulator, then adding the second insulator into the second filling port, filling the first cavity with the second insulator, separating the first cleaning cavity from the reaction cavity, then adding a second cleaning liquid into the second cleaning liquid addition port until the second cleaning cavity is filled with the second cleaning liquid, and finally adding the silicone oil into the fourth filling port until the third cavity is filled with the silicone oil.

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