Nucleic acid sequencing sample pretreatment device and method
Patent Information
- Application Number
- CN202280100884.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2022-12-14
- Publication Date
- 2025-06-17
AI Technical Summary
In the nanopore sequencing process, the utilization rate of long fragment nucleic acids is low, resulting in low sequencing accuracy. Existing technologies cannot effectively improve sample utilization rate and sequencing quality.
A nucleic acid sequencing sample pretreatment device is designed. By arranging a first electrode and a second electrode in the pretreatment chamber, and using a controller to apply an alternating current, the long fragment nucleic acid and the short fragment nucleic acid are separated, so that the long fragment nucleic acid flows to the lower layer of the fluid. Offset to obtain higher electrophoresis priority and improve sample utilization.
Preprocessing technology significantly improves the electrophoresis priority of long fragment nucleic acids, improves sample utilization, and thereby improves the accuracy of nucleic acid sequencing and the quality of sequencing results.
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Figure CN120167012A_ABST
Abstract
Description
Nucleic acid sequencing sample preprocessing device and method Technical Field
[0001] The present application relates to the field of gene sequencing technology, and for example, to a nucleic acid sequencing sample preprocessing device and method. Background Art
[0002] At present, nanopore sequencing technology has been widely used in gene sequencing. When using nanopore technology for nucleic acid sequencing, the nucleic acids to be tested, evenly distributed in the nucleic acid sample, will approach and pass through the nanopore according to the rules of electrophoresis under the action of the electric field, thereby generating an electrical signal that can be used to analyze the characteristics of the nucleic acid.
[0003] Electrophoresis refers to the phenomenon in which charged particles, under the influence of an electric field, migrate toward an electrode with opposite charge. In nucleic acid samples, short nucleic acid fragments have a smaller relative charge than long nucleic acid fragments and therefore have a higher electrophoretic velocity. When the endpoint position is fixed, the object that can reach the endpoint faster through electrophoresis has a higher electrophoretic priority. For the same object, the closer the initial position is to the endpoint, the higher the electrophoretic priority; when the initial position is fixed, the faster the electrophoretic speed, the higher the electrophoretic priority. Therefore, in a uniformly mixed nucleic acid sample, the nucleic acid sample is evenly distributed, and the initial electrophoretic positions can be considered the same, and short nucleic acid fragments have a higher electrophoretic priority.
[0004] When using nanopores to detect and analyze the characteristics of the nucleic acid to be tested to achieve nucleic acid sequencing, the sequencing data that can be provided by long-fragment nucleic acids is more valuable than that of short-fragment nucleic acids. The sequencing data based on long-fragment nucleic acids can be analyzed to obtain sequencing results with higher accuracy. Therefore, when the sequencing time is fixed, the proportion of long-fragment nucleic acids that complete electrophoresis through the nanopore and generate valid data represents the utilization rate of the sample to a certain extent. In the related art, the method of using nanopore sequencing samples is to use a pipette to directly push the mixed sample directly into the sample injection port. Since the mixed nucleic acid sample is not processed in any way, the short-fragment nucleic acid in the sample completes electrophoresis through the nanopore in a shorter time. Short-fragment nucleic acids have higher electrophoresis priority and sequencing utilization rate than long-fragment nucleic acids, which leads to insufficient sample utilization, low accuracy of sequencing results, and reduced sequencing quality.
[0005] Summary of the Invention
[0006] The present application provides a nucleic acid sequencing sample preprocessing device and method to avoid the low utilization rate of long-fragment nucleic acids and the resulting low sequencing accuracy during nanopore nucleic acid sequencing in the related art.
[0007] The present invention provides a nucleic acid sequencing sample preprocessing device, comprising:
[0008] A supporting seat, wherein a pre-treatment chamber is provided in the supporting seat, and a fluid inlet and a fluid outlet communicating with the pre-treatment chamber are opened on the supporting seat;
[0009] a first electrode, disposed on the top wall of the pretreatment chamber;
[0010] a second electrode, disposed on the bottom wall of the pretreatment chamber;
[0011] A controller, wherein the first electrode and the second electrode are electrically connected to the controller respectively.
[0012] The present invention provides a nucleic acid sequencing sample preprocessing method, which uses the nucleic acid sequencing sample preprocessing device described above, including:
[0013] Before nucleic acid sequencing is performed on the sample, the sample is guided from the fluid inlet into the pretreatment chamber;
[0014] The controller applies an alternating current to the first electrode and the second electrode to separate the first nucleic acid fragment from the second nucleic acid fragment. The second nucleic acid fragment deviates to the upper layer of the fluid, and the first nucleic acid fragment deviates to the lower layer of the fluid. BRIEF DESCRIPTION OF THE DRAWINGS
[0015] FIG1 is a schematic structural diagram of a nucleic acid sequencing sample preprocessing device provided in this embodiment;
[0016] FIG2 is a cross-sectional view of a nucleic acid sequencing sample pretreatment device provided in this embodiment;
[0017] Figure 3 is an enlarged view of point A in Figure 2;
[0018] FIG4 is a first schematic diagram of the decomposed structure of the nucleic acid sequencing sample preprocessing device provided in this embodiment;
[0019] FIG5 is a second schematic diagram of the decomposed structure of the nucleic acid sequencing sample preprocessing device provided in this embodiment;
[0020] FIG6 is a schematic diagram of the nucleic acid sequencing sample preprocessing device provided in this embodiment in an initial state;
[0021] FIG7 is a schematic diagram of a nucleic acid sequencing sample pretreatment device provided in this embodiment having a pre-filled liquid therein;
[0022] FIG8 is a first schematic diagram of adding a primer solution into the nucleic acid sequencing sample pretreatment device provided in this embodiment;
[0023] FIG9 is a second schematic diagram of adding a primer solution into the nucleic acid sequencing sample pretreatment device provided in this embodiment;
[0024] FIG10 is a third schematic diagram of adding a primer solution into the nucleic acid sequencing sample pretreatment device provided in this embodiment;
[0025] FIG11 is a schematic diagram of the replacement of the base liquid in the nucleic acid sequencing sample pretreatment device provided in this embodiment;
[0026] FIG12 is a schematic diagram of adding a sample into the nucleic acid sequencing sample pretreatment device provided in this embodiment;
[0027] FIG13 is a schematic diagram of sample sinking in the nucleic acid sequencing sample pretreatment device provided in this embodiment.
[0028] In the picture:
[0029] 100, sample; 200, pre-setting solution; 300, base solution;
[0030] 10. Carrier; 101. Pretreatment chamber; 1011. Guide ramp; 102. Fluid inlet; 103. Fluid outlet; 104. Sequencing chamber; 105. Well array; 106. Waste liquid reservoir; 107. Sample exchange channel; 1071. Liquid exchange port; 1072. Straight channel; 1073. Waste liquid port; 108. Sample loading channel; 109. Gas guide channel;
[0031] 11. Upper cover; 111. First protrusion; 112. First groove; 12. Middle plate; 121. First assembly groove; 122. First connecting hole; 123. Second assembly groove; 13. Bottom plate; 131. Second connecting hole; 132. Third connecting hole;
[0032] 20. a first electrode;
[0033] 30. a second electrode;
[0034] 40. Sealing gasket. DETAILED DESCRIPTION
[0035] In the description of this application, unless otherwise expressly specified or limited, the terms "connected," "connected," and "fixed" should be understood in a broad sense. For example, they can refer to fixed connections, detachable connections, or integration; mechanical connections or electrical connections; direct connections or indirect connections through an intermediate medium; and can refer to internal communication between two components or interaction between two components. Those skilled in the art will understand the specific meanings of the above terms in this application based on the specific circumstances.
[0036] In this application, unless otherwise expressly specified or limited, a first feature being "above" or "below" a second feature may include the first and second features being in direct contact, or may include the first and second features being in contact not directly but through another feature between them. Moreover, a first feature being "above," "above," and "above" a second feature may include the first feature being directly above or obliquely above the second feature, or may simply mean that the first feature is higher in level than the second feature. A first feature being "below," "below," and "below" a second feature may include the first feature being directly below or obliquely below the second feature, or may simply mean that the first feature is lower in level than the second feature.
[0037] The technical solution of this application is explained below with reference to the accompanying drawings and through specific implementation methods.
[0038] 1 to 5 , the present embodiment provides a nucleic acid sequencing sample pretreatment device for pretreating a sample 100 before nucleic acid sequencing, so that the first nucleic acid fragment and the second nucleic acid fragment in the sample 100 are separated. The sample 100 mentioned in the present embodiment is a nucleic acid sample to be tested. The first nucleic acid fragment can be a long nucleic acid fragment, and the second nucleic acid fragment can be a short nucleic acid fragment. For example, the dividing line between short nucleic acid fragments and long nucleic acid fragments can be 1-4 kb (kilobase pairs). Generally, nucleic acid fragments with a length of less than 1 kb are regarded as short nucleic acid fragments, and nucleic acid fragments with a length of greater than 1 kb are regarded as long nucleic acid fragments; nucleic acid fragments with a length of less than 4 kb can also be regarded as short nucleic acid fragments, and nucleic acid fragments with a length of greater than 4 kb can be regarded as long nucleic acid fragments.
[0039] For example, a nucleic acid sequencing sample preprocessing device includes a support base 10, a first electrode 20, a second electrode 30, and a controller. A preprocessing chamber 101 is disposed within the support base 10, and a fluid inlet 102 and a fluid outlet 103 are provided on the support base 10, communicating with the preprocessing chamber 101. The first electrode 20 is disposed on the top wall of the preprocessing chamber 101; the second electrode 30 is disposed on the bottom wall of the preprocessing chamber 101; and both the first electrode 20 and the second electrode 30 are electrically connected to the controller. The controller is capable of adjusting the frequency and potential difference of the alternating current between the first electrode 20 and the second electrode 30. The structures and operating principles of the controller, the first electrode 20, and the second electrode 30 are related art and will not be further described here.
[0040] Before nucleic acid sequencing of sample 100, sample 100 is guided into the pretreatment chamber 101 through the fluid inlet 102, and an alternating current is applied to the first electrode 20 and the second electrode 30 by the controller to complete the separation of long-fragment nucleic acid and short-fragment nucleic acid, so that the short-fragment nucleic acid is offset to the upper layer of the fluid and the long-fragment nucleic acid is offset to the lower layer of the fluid. During the formal sequencing process, the long-fragment nucleic acid located in the lower layer is closer to the nanopore array, thereby obtaining a higher electrophoresis priority and being able to pass through the nanopore array faster. The utilization rate of sample 100 is improved, thereby improving the accuracy of the sequencing results.
[0041] The first electrode 20 and the second electrode 30 cooperate to form a separation electrode pair. A stable dielectrophoretic field is formed between the separation electrode pair, gradually pre-separating nucleic acid fragments with different relative charges, causing short nucleic acid fragments to be deflected toward the upper layer of the fluid and long nucleic acid fragments to be deflected toward the lower layer. Based on the downward flow of fluid, the nanopore array is positioned below the pretreatment chamber 101. This brings long nucleic acid fragments closer to the nanopore array, giving them higher electrophoresis priority and improving sample 100 utilization.
[0042] For example, the carrier 10 also includes a sequencing chamber 104, which is located below the pretreatment chamber 101 and connected to the pretreatment chamber 101 via a hole array 105. When the sample 100 passes through the pretreatment chamber 101 and sinks through the hole array 105 into the sequencing chamber 104, the long nucleic acids located in the lower layer of the fluid are closer to the hole array 105. Therefore, the long nucleic acids receive higher electrophoresis priority, thereby improving the utilization rate of the sample 100. The hole array 105 includes multiple nanopores arranged in an array.
[0043] It is understandable that during the nucleic acid sequencing process, the sample 100 needs to pass through the hole array 105. After passing through the hole array 105, the sample 100 enters the gene sequencing device, wherein the gene sequencing device can adopt a conventional sequencing device. In this embodiment, the sequencing chamber 104 should be connected to the sequencing chip of the gene sequencing device. The sample 100 entering the sequencing chamber 104 can contact the sequencing chip to complete the sequencing.
[0044] A waste liquid pool 106 is also provided in the carrier 10, and the waste liquid pool 106 is connected to the sequencing chamber 104, and the fluid outlet 103 is connected to the waste liquid pool 106. In the process of guiding the sample 100 from the fluid inlet 102 into the pretreatment chamber 101, fluid replacement is required. For example, in the initial state, a pre-liquid 200 is provided in the pretreatment chamber 101 and the sequencing chamber 104. The pre-liquid 200 is first replaced with a base liquid 300, and then the sample 100 is added to the pretreatment chamber 101. The sample 100 can float on the base liquid 300. After the pre-separation is completed, the base liquid 300 is sucked out so that the pre-separated sample 100 can sink into the sequencing chamber 104. It can be understood that the sample 100 and the base liquid 300 are not miscible, so the sample 100 and the base liquid 300 will be stratified. The sample 100 is light in weight, the sample 100 is located in the upper layer, and the base liquid 300 is located in the lower layer.
[0045] Replacing the pre-liquid 200 with the base liquid 300 includes: using a pipette to add the base liquid 300 to the fluid inlet 102, with the base liquid 300 floating on the pre-liquid 200; then aspirating waste liquid from the waste liquid pool 106 through the fluid outlet 103, so that the pre-liquid 200 in the pre-treatment chamber 101 gradually decreases until the base liquid 300 fills the sequencing chamber 104 and a portion of the base liquid 300 is located in the pre-treatment chamber 101 or fills the pre-treatment chamber 101. Adding the sample 100 to the pre-treatment chamber 101 includes: using a pipette to add the sample 100 to the fluid inlet 102, with the sample 100 floating on the base liquid 300, with a portion of the base liquid 300 being squeezed into the waste liquid pool 106; then closing the fluid inlet 102 and the fluid outlet 103. After the pre-separation is completed, the base liquid 300 is aspirated, including: opening the fluid inlet 102 and the fluid outlet 103, and aspirating waste liquid from the waste liquid pool 106 through the fluid outlet 103, so that the base liquid 300 in the pre-treatment chamber 101 is reduced, and the pre-separated sample 100 sinks through the hole array 105 and enters the sequencing chamber 104.
[0046] Among them, the pre-setting liquid 200 and the base liquid 300 are buffer solutions in the related art, which play a protective role. The base liquid 300 is used to replace the pre-setting liquid 200 before the sample 100 is pretreated, and ensures the floating position of the sample 100 during the pretreatment process.
[0047] The waste liquid reservoir 106 is positioned directly opposite the fluid outlet 103, facilitating a pipette to draw waste liquid from the waste liquid reservoir 106 through the fluid outlet 103. The waste liquid reservoir 106 is connected to the sequencing chamber 104 via a sample exchange channel 107, facilitating smooth flow of fluid within the sequencing chamber 104 into the waste liquid reservoir 106. In this embodiment, the fluid inlet 102 and the fluid outlet 103 are located on opposite sides of the pretreatment chamber 101. Consequently, the waste liquid reservoir 106 and the fluid inlet 102 are located on opposite sides of the pretreatment chamber 101.
[0048] The support base 10 is also provided with a sample loading channel 108, through which the fluid inlet 102 communicates with the pretreatment chamber 101. The sample loading channel 108 guides the fluid. In this embodiment, a guide slope 1011 is provided at the edge of the pretreatment chamber 101. The end of the sample loading channel 108 extends to the guide slope 1011, providing a certain degree of guidance for the fluid, allowing it to enter smoothly. The inclination angle of the guide slope 1011 can be 60 degrees.
[0049] An air guide channel 109 is provided in the supporting base 10, one end of the air guide channel 109 is connected to the pretreatment chamber 101, and the other end of the air guide channel 109 is connected to the fluid outlet 103. In the process of the fluid entering the pretreatment chamber 101, the gas in the pretreatment chamber 101 enters the fluid outlet 103 from the air guide channel 109 and is discharged, thereby ensuring the pressure balance in the pretreatment chamber 101. The gas and fluid share the fluid outlet 103, which simplifies the structure. It is understandable that the air guide channel 109 is located above the sample exchange channel 107 to prevent liquid from entering the air guide channel 109. The fluid inlet 102 is used for adding samples, and the fluid outlet 103 is used to absorb waste liquid and is also used to balance the pressure.
[0050] In this embodiment, the support base 10 is composed of several detachable parts to facilitate processing, production, and assembly of electrodes. For example, the support base 10 includes an upper cover plate 11, an intermediate plate 12, and a bottom plate 13, which are sequentially connected from top to bottom. The intermediate plate 12 is provided with a pretreatment chamber 101, and the upper cover plate 11 is provided with a fluid inlet 102 and a fluid outlet 103.
[0051] The sample loading channel 108, the gas guiding channel 109, and the first electrode 20 are all disposed on the upper cover 11 and are located on a side of the upper cover 11 close to the middle plate 12. For example, a first protrusion 111 is disposed on the upper cover 11, and a first groove 112 is disposed around the first protrusion 111. The first electrode 20 is disposed on the first protrusion 111, and the sample loading channel 108 and the gas guiding channel 109 are both connected to the first groove 112.
[0052] The waste liquid pool 106 is disposed on the middle plate 12. Both the pretreatment chamber 101 and the waste liquid pool 106 extend through the middle plate 12 along its thickness. The air guide channel 109 can communicate directly or indirectly with the fluid outlet 103. In this embodiment, the air guide channel 109 communicates with the waste liquid pool 106, which in turn communicates with the fluid outlet 103.
[0053] The middle plate 12 is provided with a first mounting groove 121, into which the pin of the first electrode 20 is located. For example, a first connection hole 122 is defined within the first mounting groove 121, through which the pin of the first electrode 20 extends. The middle plate 12 is also provided with a second mounting groove 123, into which the pin of the second electrode 30 is located. The first mounting groove 121 and the second mounting groove 123 are located on opposite sides of the middle plate 12.
[0054] The bottom plate 13 is provided with a hole array 105, which faces the pretreatment chamber 101. Therefore, the top surface of the bottom plate 13 serves as the bottom wall of the pretreatment chamber 101. It is understood that the arrangement of the second electrode 30 does not affect the flow of fluid in the hole array 105.
[0055] For example, a second connection hole 131 is provided on the bottom plate 13. The second connection hole 131 communicates with the second assembly groove 123, and the pin of the second electrode 30 extends through the second connection hole 131. A third connection hole 132 is provided on the bottom plate 13 opposite the first connection hole 122 to facilitate the pin of the first electrode 20 to extend through it.
[0056] Sequencing chamber 104 is formed in bottom plate 13, on the side of bottom plate 13 away from middle plate 12, and faces well array 105. Sequencing chamber 104 is edged with a sealing gasket 40, which seals chamber 104 when connected to a gene sequencing device.
[0057] Among them, the sample exchange channel 107 includes a liquid exchange port 1071, a straight channel 1072 and a waste liquid port 1073 which are connected in sequence. The straight channel 1072 is arranged on the top surface of the bottom plate 13. The liquid exchange port 1071 passes through the bottom plate 13 to connect the sequencing chamber 104 with the straight channel 1072. The waste liquid port 1073 is located in the waste liquid pool 106. The fluid in the sequencing chamber 104 can flow along the liquid exchange port 1071, the straight channel 1072 and the waste liquid port 1073 to the waste liquid pool 106.
[0058] The first electrode 20 and the second electrode 30 can be prepared by common techniques such as vapor deposition, electroplating, or screen printing. Electrical connection can be achieved by contacting the first electrode 20 with a pin, and copper is deposited in the second connection hole 131 to serve as an electrical connection point for the second electrode 30. The upper cover plate 11, the middle plate 12, and the bottom plate 13 can all be assembled by sealing processes such as hot pressing, laser welding, or ultrasonic welding. The sealing gasket 40 is made of a flexible material such as silicone or rubber.
[0059] In this embodiment, the upper cover plate 11 is made of transparent HIPS, the first electrode 20 and the second electrode 30 are both made of platinum, the middle plate 12 is made of black HIPS, and the bottom plate 13 is made of transparent HIPS. The fluid inlet 102 and fluid outlet 103 are both circular, the sample loading channel 108 and the gas guide channel 109 have the same width and depth, and the first protrusion 111 is a 45° inclined platform. The waste liquid reservoir 106 is a regular hexagon. The dimensions of the various components can be adjusted according to actual needs.
[0060] 6 to 13 , this embodiment further provides a nucleic acid sequencing sample preprocessing method, which uses the above-mentioned nucleic acid sequencing sample preprocessing device, including:
[0061] Before nucleic acid sequencing is performed on the sample 100 , the sample 100 is guided from the fluid inlet 102 into the pre-treatment chamber 101 ;
[0062] The controller applies an alternating current to the first electrode 20 and the second electrode 30 to separate the long nucleic acid from the short nucleic acid. The short nucleic acid is displaced toward the upper layer of the fluid, and the long nucleic acid is displaced toward the lower layer of the fluid.
[0063] It is understandable that before using the above-mentioned nucleic acid sequencing sample pretreatment device, it is necessary to first complete the assembly of the above-mentioned nucleic acid sequencing sample pretreatment device and connect the above-mentioned nucleic acid sequencing sample pretreatment device to the matching gene sequencing equipment so that the sequencing chamber 104 is selectively connected to the gene sequencing equipment.
[0064] The sample 100 is guided from the fluid inlet 102 into the pre-treatment chamber 101, comprising:
[0065] Replacement of the pre-liquid 200: replacing the pre-liquid 200 in the pre-treatment chamber 101 with the base liquid 300;
[0066] Adding the sample 100: Add the sample 100 to the fluid inlet 102 , and the sample 100 floats on the bottom liquid 300 , so that part of the bottom liquid 300 is squeezed into the waste liquid pool 106 , and then close the fluid inlet 102 and the fluid outlet 103 .
[0067] As shown in FIG7 , in the initial state, the pretreatment chamber 101 and the sequencing chamber 104 are filled with a pre-filled liquid 200. It is understood that since the waste liquid pool 106 is connected to the sequencing chamber 104, part of the pre-filled liquid 200 will also flow into the waste liquid pool 106.
[0068] As shown in FIG8 to FIG11 , the pre-liquid 200 is replaced with the base liquid 300, including:
[0069] A pipette is used to add base liquid 300 to the fluid inlet 102. The base liquid 300 floats on the pre-filled liquid 200, causing the pre-filled liquid 200 to flow into the waste liquid reservoir 106. A pipette is then used to draw waste liquid from the waste liquid reservoir 106 through the fluid outlet 103, thereby reducing the amount of pre-filled liquid 200 in the pre-treatment chamber 101 and the sequencing chamber 104. This decreases until the base liquid 300 fills the sequencing chamber 104 and a portion of the base liquid 300 remains in the pre-treatment chamber 101 or fills the pre-treatment chamber 101. It will be appreciated that because the waste liquid reservoir 106 is connected to the sequencing chamber 104, a portion of the base liquid 300 will also enter the waste liquid reservoir 106, completing the replacement of the pre-filled liquid 200.
[0070] As shown in FIG12 , during the process of adding the sample 100, a pipette is used to add the sample 100 to the fluid inlet 102. The sample 100 floats on the bottom liquid 300, causing the bottom liquid 300 to flow into the waste liquid pool 106. The pipette is used to draw waste liquid from the waste liquid pool 106 through the fluid outlet 103, thereby reducing the bottom liquid 300 in the pretreatment chamber 101 until the sample 100 is no lower than the bottom wall of the pretreatment chamber 101.
[0071] Next, a pre-separation is performed by applying an alternating current to the first electrode 20 and the second electrode 30 via a controller to separate the long nucleic acid fragments from the short nucleic acid fragments. The short nucleic acid fragments are deflected to the upper layer of the fluid, while the long nucleic acid fragments are deflected to the lower layer of the fluid. The alternating current can have a first set frequency and a first set potential difference, and last for a first set duration.
[0072] After pre-separation is complete, the fluid inlet 102 and fluid outlet 103 are opened, and waste liquid is drawn from the waste liquid reservoir 106 through the fluid outlet 103, thereby reducing the amount of base liquid 300 in the sequencing chamber 104. The pre-separated sample 100 then sinks through the pore array 105 and enters the sequencing chamber 104. When the sequencing chamber 104 is open, the pre-separated sample 100 can enter the gene sequencing device for gene sequencing.
[0073] In this embodiment, in the initial state, 100 μL of protective pre-liquid 200 is added to the nucleic acid sequencing sample pretreatment device, and the fluid inlet 102 and fluid outlet 103 are sealed with rubber plugs. After the base plate 13 and sequencing chip are assembled, the gene sequencing device is loaded and the instrument is ready. The rubber plugs on the fluid inlet 102 and fluid outlet 103 are removed to allow the fluid to vent to the atmosphere.
[0074] When replacing the pre-liquid 200, a total of 80 μl of base liquid 300 is added to the fluid inlet 102 using a pipette, and then 80 μl of waste liquid is drawn from the bottom of the waste liquid pool 106 through the fluid outlet 103. This is repeated once to replace the pre-liquid 200 with the base liquid 300.
[0075] When adding the sample 100 , a total of 80 μL of the sample 100 is added to the fluid inlet 102 using a pipette, and then the fluid inlet 102 and the fluid outlet 103 are closed.
[0076] During pre-separation, an alternating current with a frequency of 10 MHz and a potential difference of 10 V is applied to the first electrode 20 and the second electrode 30 by the controller for 3 minutes to complete the separation of long-fragment nucleic acid and short-fragment nucleic acid.
[0077] Next, the fluid inlet 102 and the fluid outlet 103 are opened, and 80 μL of waste liquid is aspirated from the bottom of the waste liquid pool 106 through the fluid outlet 103 using a pipette. The pre-separated sample 100 sinks, and most of the remaining bottom liquid 300 flows into the waste liquid pool 106 through the sample exchange channel 107.
[0078] In summary, the nucleic acid sequencing sample pretreatment device and method provided in this embodiment utilizes AC dielectrophoresis technology and an electrode pair to achieve pre-separation of sample 100, and utilizes laminar flow technology and a connecting vessel structure to achieve replacement of pre-liquid 200 and base solution 300. Automated liquid inlet and / or wash circuits and control systems can also be used to replace manual pipetting.
Claims
1. A nucleic acid sequencing sample preprocessing device, comprising: A supporting seat (10), wherein a pretreatment chamber (101) is provided in the supporting seat (10), and a fluid inlet (102) and a fluid outlet (103) communicating with the pretreatment chamber (101) are provided on the supporting seat (10); a first electrode (20) disposed on the top wall of the pretreatment chamber (101); a second electrode (30) disposed on the bottom wall of the pretreatment chamber (101); A controller, wherein the first electrode (20) and the second electrode (30) are electrically connected to the controller respectively.
2. The device according to claim 1, wherein A sequencing chamber (104) is further provided in the supporting seat (10), and the sequencing chamber (104) is located below the pre-processing chamber (101) and is connected to the pre-processing chamber (101) via a hole array (105).
3. The device according to claim 2, wherein A waste liquid pool (106) is also provided in the supporting seat (10), the waste liquid pool (106) is communicated with the sequencing chamber (104), and the fluid outlet (103) is communicated with the waste liquid pool (106).
4. The device according to claim 3, wherein The waste liquid pool (106) is arranged opposite to the fluid outlet (103).
5. The device according to claim 3, wherein The waste liquid pool (106) is connected to the sequencing chamber (104) through a sample exchange channel (107).
6. The device according to claim 1, wherein A sample loading channel (108) is further provided in the supporting seat (10), and the fluid inlet (102) is connected to the pre-processing chamber (101) through the sample loading channel (108).
7. The device according to claim 6, wherein A guiding slope (1011) is provided at the edge of the pre-treatment chamber (101), and the end of the sample loading channel (108) extends to the guiding slope (1011).
8. The device according to claim 1, wherein An air guide channel (109) is provided in the support seat (10), a first end of the air guide channel (109) is communicated with the pretreatment chamber (101), and a second end of the air guide channel (109) is communicated with the fluid outlet (103).
9. The device according to any one of claims 1 to 8, wherein: The supporting base (10) comprises an upper cover plate (11), an intermediate plate (12) and a bottom plate (13) connected in sequence from top to bottom; the pretreatment chamber (101) is provided on the intermediate plate (12); and the fluid inlet (102) and the fluid outlet (103) are provided on the upper cover plate (11).
10. A nucleic acid sequencing sample pretreatment method, using the nucleic acid sequencing sample pretreatment device according to any one of claims 1 to 9, comprising: Before nucleic acid sequencing is performed on the sample, the sample is guided from the fluid inlet into the pretreatment chamber; The controller applies an alternating current to the first electrode and the second electrode to separate the first nucleic acid fragment from the second nucleic acid fragment. The second nucleic acid fragment deviates to the upper layer of the fluid, and the first nucleic acid fragment deviates to the lower layer of the fluid.