Automated nucleic acid extraction and purification device

Abstract

The present invention provides an automated nucleic acid extraction and purification device, which includes: a nucleic acid extraction and purification turntable chip and an electromagnetic unit. The device integrates a nucleic acid extraction and purification chamber and an amplification reagent storage chamber, and combines nucleic acid extraction and purification with mixing and distribution with nucleic acid amplification reagents through the electromagnetic field provided by the electromagnetic unit and the centrifugal force provided by the turntable, so as to realize the automation and integration of all steps from sample to test on the chip, effectively improve the automation degree of nucleic acid detection, thereby improving the efficiency and accuracy of nucleic acid detection, reducing costs and reducing artificial pollution; in addition, by setting a micro-slit structure, the magnetic bead group is driven by the magnetic field and the mother liquid droplet is constricted here, so that the residual reagent carried by the magnetic bead group is greatly reduced, thereby improving the extraction and purification effect of nucleic acid; furthermore, by setting multiple reaction units on the turntable, it can be suitable for high-throughput detection of multiple infectious disease pathogen samples.

Description

Technical Field

[0001] The invention belongs to the technical field of biomolecule detection, and in particular relates to an automatic nucleic acid extraction and purification device. Background Art

[0002] Nucleic acid detection technology is widely used in molecular biology experiments and medical pathogen detection. Compared with immunological testing, nucleic acid testing has low sample content requirements, so it can more accurately detect patients in the early stages of infection, thereby preventing and controlling the epidemic in all aspects during the golden time. Traditional nucleic acid testing steps include: sample inactivation, nucleic acid extraction and purification, nucleic acid amplification and detection, etc. The entire detection process is cumbersome and takes several hours, and it is easy to cause cross contamination due to operation, so it has high requirements for the experimental ability and detection conditions of medical personnel.

[0003] The current nucleic acid extraction and purification kits often provide more than 5 reagents, and the operation steps provided are more than 10 steps. The whole process takes 20 to 40 minutes. This step is cumbersome and consumes a lot of time and energy. Based on this, some devices for auxiliary detection have emerged, which can integrate nucleic acid extraction, purification and amplification into chips or cartridges, and realize nucleic acid extraction, purification and amplification steps based on magnetic bead transfer and driving liquid. However, the current magnetic bead control often requires a handheld permanent magnet to drag the magnetic beads at the bottom, so that the magnetic beads shuttle through various reaction reagents to complete the reaction. When faced with multiple samples, it obviously requires a lot of manual labor. In addition, liquid-driven devices often cannot complete each operation independently, and require a pressure pump to provide positive or negative pressure to drive the liquid flow.

[0004] Chi-Han Chiou, Biosensors and Bioelectronics, 2013. Using electromagnetism to replace fixed magnetic fields, the process of magnetic beads clustering and dispersing in the reagent and moving in a directional manner is realized, but it is difficult to integrate into an automated all-in-one machine. Therefore, it is necessary to provide an automated all-in-one device for nucleic acid extraction and purification to help scientific research and prevention. Summary of the invention

[0005] In view of the shortcomings of the prior art described above, the object of the present invention is to provide an automated nucleic acid extraction and purification device to solve the problems in the prior art of nucleic acid detection, such as low automation of nucleic acid extraction and purification devices resulting in low detection efficiency and detection accuracy, and complicated steps, time-consuming and labor-intensive operations, and increased costs.

[0006] To achieve the above-mentioned and other related purposes, the present invention provides an automated nucleic acid extraction and purification device, which comprises, from top to bottom, a nucleic acid extraction and purification turntable chip and an electromagnetic unit;

[0007] The electromagnetic unit includes an external power interface, internal and external power connection points, a coil control circuit, an upper coil array and a lower coil array; wherein the coil control circuit is electrically connected to the external power interface through the internal and external power connection points, and the coil control circuit is electrically connected to the upper coil array and the lower coil array respectively, so as to realize electromagnetic control of the upper coil array and the lower coil array by the external power supply;

[0008] The upper coil array is composed of three upper coils, and the lower coil array is composed of two lower coils; the upper coil array is located above the lower coil array, and a projection overlap area is generated between each lower coil and two adjacent upper coils;

[0009] The nucleic acid extraction and purification turntable chip comprises a turntable and at least one reaction unit arranged on the turntable, wherein the reaction unit comprises an injection port, a connecting pipe, a nucleic acid extraction chamber, a micro-slit pipe, a washing chamber, an elution chamber, an amplification reagent storage chamber and an amplification area; wherein the injection port is connected to the inlet of the nucleic acid extraction chamber through the connecting pipe, the outlet of the nucleic acid extraction chamber is connected to the inlet of the washing chamber through the micro-slit pipe, the outlet of the washing chamber is connected to the first inlet of the elution chamber through the micro-slit pipe, the second inlet of the elution chamber is connected to the amplification reagent storage chamber through the connecting pipe, and the outlet of the elution chamber is connected to the amplification area through the connecting pipe;

[0010] The three upper coils are respectively arranged below the nucleic acid extraction chamber, the washing chamber and the elution chamber, and the projection overlap area covers the micro-slit pipeline at the same time.

[0011] Optionally, the reaction units disposed on the turntable are distributed on the turntable in a centrally symmetrical manner.

[0012] Optionally, the number of the reaction units arranged on the turntable is between 2 and 96.

[0013] Optionally, the electromagnetic unit is configured as a PCB coil array, the upper coil array is configured on the front side of the PCB board, and the lower coil array is configured on the back side of the PCB board.

[0014] Optionally, the upper coil and the lower coil are the same coils.

[0015] Optionally, the lower coil is arranged along the center line of the upper coil.

[0016] Optionally, the coil includes 7 to 9 windings, the height of the coil is between 20 μm and 60 μm, the width of the coil is between 90 μm and 180 μm, and the spacing between two adjacent windings is between 100 μm and 300 μm.

[0017] Optionally, the width of the connecting pipe is 0.75 mm to 1 mm, and the width of the micro-slit pipe is 0.75 mm to 1 mm.

[0018] Optionally, the amplification region is a digital amplification region.

[0019] As described above, the automated nucleic acid extraction and purification device of the present invention integrates the nucleic acid extraction and purification chamber and the nucleic acid amplification reagent storage chamber, and cleverly combines the nucleic acid extraction and purification with the mixing and distribution of the nucleic acid amplification reagent through the electromagnetic field provided by the electromagnetic unit and the centrifugal force provided by the turntable. The periodic change of the direction and size of the electromagnetic field causes the magnetic beads to repeatedly form a cluster and regularly distribute on the magnetic flux lines to achieve stirring so that the reaction can proceed fully. The superposition effect of the upper and lower electromagnetic fields is used to cause the magnetic bead cluster to be broken from the mother liquid droplet through the micro-slit pipe and then merge with the subsequent adjacent droplets to form a new mother liquid droplet, which is then driven by centrifugal force to separate the nucleic acid amplification reagent and the elution. The nucleic acids are mixed and distributed to the amplification area. Finally, driven by the electromagnetic field and centrifugal force, the automation and integration of all steps from sample to test are realized on the chip, which effectively improves the automation level of nucleic acid detection, thereby improving the efficiency and accuracy of nucleic acid detection and reducing the cost of detection. In addition, by setting up a micro-slit structure, the magnetic bead cluster is constricted with the mother liquid droplet here under the drive of the magnetic field, so that the residual reagent carried by the magnetic bead cluster is greatly reduced, thereby improving the extraction and purification effect of the nucleic acid. Furthermore, the nucleic acid extraction and purification turntable chip is suitable for high-throughput detection of multiple infectious disease pathogen samples by setting multiple reaction units on the turntable. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 It is a schematic diagram showing the structure of the nucleic acid extraction and purification turntable chip in the automated nucleic acid extraction and purification device of the present invention.

[0021] Figure 2 Shown is a schematic diagram of the structure of the reaction unit in the nucleic acid extraction and purification turntable chip of the present invention.

[0022] Figure 3 Shown is a schematic diagram of the structure of the electromagnetic unit in the automated nucleic acid extraction and purification device of the present invention.

[0023] Figure 4 It is a schematic diagram showing the process of the magnetic beads entering the washing chamber from the nucleic acid extraction chamber through the micro-slit pipe under the action of the electromagnetic unit during the operation of the automated nucleic acid extraction and purification device of the present invention.

[0024] Figure 5 Shown is a schematic diagram of the multi-layer structure of the automated nucleic acid extraction and purification device of the present invention.

[0025] Component number description

[0026] 1 Nucleic acid extraction and purification turntable chip

[0027] 10. Turntable

[0028] 11 Reaction Unit

[0029] 12 Inlet

[0030] 13 Connecting pipes

[0031] 14 Nucleic Acid Extraction Room

[0032] 15 Micro-slit pipe

[0033] 16 Washing Room

[0034] 17 Elution Chamber

[0035] 18 Amplification reagent storage room

[0036] 19 Amplification region

[0037] 2 Electromagnetic unit

[0038] 20 External power supply interface

[0039] 21 Internal and external power connection points

[0040] 22 Coil control circuit

[0041] 23 Upper coil

[0042] 24 Lower coil

[0043] 25 mother drop

[0044] 26 Magnetic beads

[0045] 27 Reaction layer

[0046] 28 Magnetron Layer

[0047] A Projection overlap area DETAILED DESCRIPTION

[0048] The following describes the embodiments of the present invention through specific examples, and those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and the details in this specification can also be modified or changed in various ways based on different viewpoints and applications without departing from the spirit of the present invention.

[0049] See also Figures 1 to 5 It should be noted that the illustrations provided in this embodiment are only used to illustrate the basic concept of the present invention in a schematic manner, and therefore the illustrations only show components related to the present invention rather than being drawn according to the number, shape and size of components in actual implementation. In actual implementation, the type, quantity and proportion of each component may be changed according to actual needs, and the component layout type may also be more complicated.

[0050] like Figures 1 to 3 As shown, this embodiment provides an automated nucleic acid extraction and purification device, which includes, from top to bottom, a nucleic acid extraction and purification turntable chip 1 (such as Figure 1 As shown) and electromagnetic unit 2 (as Figure 3 shown);

[0051] like Figure 3 As shown, the electromagnetic unit 2 includes an external power interface 20, an internal and external power connection point 21, a coil control circuit 22, an upper coil array and a lower coil array; wherein the coil control circuit 22 is electrically connected to the external power interface 20 through the internal and external power connection point 21, and the coil control circuit 22 is electrically connected to the upper coil array and the lower coil array respectively, so as to realize electromagnetic control of the upper coil array and the lower coil array by the external power supply;

[0052] The upper coil array is composed of three upper coils 23, and the lower coil array is composed of two lower coils 24; the upper coil array is located above the lower coil array, and a projection overlap area A is generated between each lower coil 24 and two adjacent upper coils 23;

[0053] like Figure 1 As shown, the nucleic acid extraction and purification turntable chip 1 includes a turntable 10 and at least one reaction unit 11 disposed on the turntable 10. Figure 2 As shown, the reaction unit 11 includes an injection port 12, a connecting pipe 13, a nucleic acid extraction chamber 14, a micro-slit pipe 15, a washing chamber 16, an elution chamber 17, an amplification reagent storage chamber 18 and an amplification area 19; wherein the injection port 12 is connected to the inlet of the nucleic acid extraction chamber 14 through the connecting pipe 13, the outlet of the nucleic acid extraction chamber 14 is connected to the inlet of the washing chamber 16 through the micro-slit pipe 15, the outlet of the washing chamber 16 is connected to the first inlet of the elution chamber 17 through the micro-slit pipe 15, the second inlet of the elution chamber 17 is connected to the amplification reagent storage chamber 18 through the connecting pipe 13, and the outlet of the elution chamber 17 is connected to the amplification area 19 through the connecting pipe 13;

[0054] like Figure 3 and Figure 5As shown, the three upper coils 23 are respectively disposed below the nucleic acid extraction chamber 14 , the washing chamber 16 and the elution chamber 17 , and the projection overlap area A covers the micro-slit pipe 15 at the same time.

[0055] Based on the above-mentioned automated nucleic acid extraction and purification device, its working process includes the following steps:

[0056] 1) Detection preparation: first, fill the area outside the amplification zone 19 of the reaction unit 11 with mineral oil, fill the nucleic acid extraction chamber 14 with nucleic acid extraction reagent and magnetic bead solution, fill the washing chamber 16 with washing solution, fill the elution chamber 17 with nucleic acid elution solution, and fill the amplification reagent storage chamber 18 with nucleic acid amplification reagent, wherein all reagents used are aqueous reagents, and the electromagnetic unit is controlled by an external programmable current output instrument.

[0057] 2) Nucleic acid extraction step: drive the sample through the injection port 12 by centrifugal force and introduce it into the nucleic acid extraction chamber 14 and merge it into a mother liquid droplet of oil-in-water structure; at the same time, turn on the electromagnetic unit 2, and under the action of the electromagnetic field of the electromagnetic unit 2, the nucleic acid extraction reagent and the magnetic bead solution fully lyse the sample; specifically, the turntable 10 provides centrifugal force and drives the sample from the injection port 12 through the connecting pipe 13 into the nucleic acid extraction chamber 14, and by opening the upper coil 23 below the nucleic acid extraction chamber in the electromagnetic unit 2, the upper coil 23 can disperse the magnetic bead group on the magnetic flux line while changing the direction of the magnetic field, so that the nucleic acid extraction reagent and the sample are fully and evenly stirred, so that the lysis reaction of the sample is more complete and more nucleic acids are enriched on the magnetic beads in each mother liquid droplet. It should be noted here that the centrifugal force of the turntable 10 can be achieved by existing conventional devices, which will not be repeated here.

[0058] 3) The first transfer step: Figure 4 As shown, the mother liquid droplet 25 is constricted at the micro-slit pipe 15 under the action of the electromagnetic field of the electromagnetic unit 11, so that the magnetic bead group 26 in the mother liquid droplet 25 passes through the micro-slit pipe 15 into the washing chamber 16 and forms a new mother liquid droplet 25; specifically, as Figure 4 As shown, after the nucleic acid extraction in step 2) is completed (as shown Figure 4-1 ), and then open the lower coil 24 below the nucleic acid extraction chamber 14 (as shown Figure 4-2), since the magnetic field strength of the overlapping area of ​​the projections of the upper coil 23 and the lower coil 24 is higher than the surrounding magnetic field strength, the mother liquid droplet 25 will be driven by the magnetic bead group 26 inside it to move toward the micro-slit pipe 15 between the nucleic acid extraction chamber 14 and the washing chamber 16; when moving to the micro-slit pipe 15, the upper coil 23 below the nucleic acid extraction chamber is closed, so that the magnetic bead group 26 and the mother liquid droplet 25 are constricted here, and the magnetic bead group 26 is constricted from the mother liquid droplet 25 and moves along the micro-slit pipe 15 toward the washing chamber 16 (such as Figure 4-3 Then open the upper coil 23 below the washing chamber 16 (as shown); Figure 4-4 As shown), the magnetic bead group 26 enters the washing chamber 16 through the micro-slit pipe 15 and merges with the droplets in the washing chamber 16 to form a new mother droplet 25 (as shown Figure 4-5 4) Washing step: Wash the magnetic beads in the washing chamber 16 to remove impurities; the impurities include proteins, fragments and other impurities other than nucleic acids enriched on the magnetic beads after sample lysis, which will affect the nucleic acid detection results and need to be removed.

[0059] 5) The second transfer step: Similar to the first transfer step, the specific process of the first transfer step can be referred to and will not be repeated here. The new mother liquid droplet 25 in the washing chamber 16 is constricted at the micro-slit pipe 15 under the action of the electromagnetic field of the electromagnetic unit 11, so that the magnetic bead group 26 in the mother liquid droplet 25 passes through the micro-slit pipe 15 and enters the elution chamber 17.

[0060] 6) Nucleic acid elution step: the elution reagent acts on the magnetic beads in the elution chamber 17 to elute the nucleic acid. Similar to the first transfer step: the magnetic beads are recovered to the washing chamber 16.

[0061] 7) Nucleic acid amplification step: The nucleic acid amplification reagent in the amplification reagent storage chamber 18 is introduced into the elution chamber 17 by centrifugal force, mixed with the nucleic acid elution solution in the elution chamber 17, and then distributed to the amplification area 19 for nucleic acid amplification.

[0062] The automated nucleic acid extraction and purification device of this embodiment integrates the nucleic acid extraction and purification chamber and the nucleic acid amplification reagent storage chamber, and cleverly combines the nucleic acid extraction and purification with the mixing and distribution of the nucleic acid amplification reagent through the electromagnetic field provided by the electromagnetic unit and the centrifugal force provided by the turntable. The periodic change of the direction and size of the electromagnetic field causes the magnetic beads to repeatedly form a cluster and regularly distribute on the magnetic flux lines to achieve stirring so that the reaction can proceed fully. The superposition effect of the upper and lower electromagnetic fields is used to cause the magnetic bead cluster to break from the mother liquid droplet through the micro-slit pipe and then merge with the subsequent adjacent droplets to form a new mother liquid droplet, which is then driven by centrifugal force to separate the nucleic acid amplification reagent and the eluted The nucleic acids are mixed and distributed to the amplification area. Finally, driven by the electromagnetic field and centrifugal force, the automation and integration of all steps from the sample to the test are realized on the chip, which effectively improves the automation level of nucleic acid detection, thereby improving the efficiency and accuracy of nucleic acid detection and reducing the detection cost. In addition, by setting a micro-slit structure, the magnetic bead cluster is constricted with the mother liquid droplet here under the drive of the magnetic field, so that the residual reagent carried by the magnetic bead cluster is greatly reduced, thereby improving the extraction and purification effect of the nucleic acid. Furthermore, the nucleic acid extraction and purification turntable chip is suitable for high-throughput detection of multiple infectious disease pathogens by setting multiple reaction units on the turntable.

[0063] like Figure 1 As shown, as an example, the number of the reaction units 11 arranged on the turntable 10 can be set to 4. In this way, when the turntable is rotated, the centrifugal force on each reaction unit 11 is the same, and the magnetic beads and the liquid can be separated, so that the purified sample is set aside for enrichment. Considering the process difficulty and detection efficiency, the number of the reaction units 11 arranged on the turntable 10 is preferably between 2 and 96.

[0064] like Figure 3 As shown, as an example, the electromagnetic unit 1 is configured as a PCB coil array, the upper coil array is configured on the front side of the PCB board, and the lower coil array is configured on the back side of the PCB board.

[0065] like Figure 3 As shown, as an example, the upper coil 23 and the lower coil 24 are the same coils. Preferably, the lower coil 24 is arranged along the center line position of the upper coil 23. As another preferred example, the coil includes 7 to 9 windings, the height of the coil is between 20μm and 60μm, the width is between 90μm and 180μm, and the spacing between two adjacent windings is between 100μm and 300μm. In this embodiment, the coil is selected to be 8 windings, the height of the coil is 35μm, the width is 150μm, the spacing between two adjacent windings is 150μm, the coil is a copper coil, and the spacing between the two coils is 3mm.

[0066] As an example, the width of the connecting pipe 13 is between 0.75 mm and 1 mm, and the width of the micro-slit pipe 15 is between 0.75 mm and 1 mm. In this embodiment, the width of the connecting pipe 13 is selected to be 1 mm, and the width of the micro-slit pipe 15 is selected to be 1 mm.

[0067] As an example, the amplification region 19 is selected as a digital amplification region.

[0068] In summary, the present invention provides an automated nucleic acid extraction and purification device that integrates a nucleic acid extraction and purification chamber and a nucleic acid amplification reagent storage chamber, and cleverly combines nucleic acid extraction and purification with mixing and distribution with nucleic acid amplification reagents through the electromagnetic field provided by the electromagnetic unit and the centrifugal force provided by the turntable. The periodic change of the direction and size of the electromagnetic field causes the magnetic beads to repeatedly form a cluster and regularly distribute on the magnetic flux lines to achieve stirring to fully carry out the reaction. The superposition of the upper and lower electromagnetic fields is used to cause the magnetic bead cluster to be broken from the mother liquid droplet through the micro-slit pipe and then merge with the subsequent adjacent droplets to form a new mother liquid droplet (such as Figure 4 ), and then the centrifugal force drives the nucleic acid amplification reagent to mix with the eluted nucleic acid and distribute it to the amplification area. The entire multilayer structure is as follows Figure 5 As shown. Finally, driven by the electromagnetic field and centrifugal force, the automation and integration of all steps from the sample to the test are realized on the chip, which effectively improves the automation degree of nucleic acid detection, thereby improving the efficiency and accuracy of nucleic acid detection and reducing the detection cost; in addition, by setting a micro-slit structure, the magnetic bead group is driven by the magnetic field and the mother liquid droplet is constricted here, so that the residual reagent carried by the magnetic bead group is greatly reduced, thereby improving the extraction and purification effect of nucleic acid; furthermore, the nucleic acid extraction and purification turntable chip is suitable for high-throughput detection of multiple infectious disease pathogen samples by setting multiple reaction units on the turntable. Therefore, the present invention effectively overcomes the various shortcomings of the prior art and has a high industrial utilization value.

[0069] The above embodiments are merely illustrative of the principles and effects of the present invention, and are not intended to limit the present invention. Anyone familiar with the art may modify or alter the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or alterations made by a person of ordinary skill in the art without departing from the spirit and technical ideas disclosed by the present invention shall still be covered by the claims of the present invention.

Claims

1. An automated nucleic acid extraction and purification device, characterized in that: The device comprises, from top to bottom, a nucleic acid extraction and purification turntable chip and an electromagnetic unit; The electromagnetic unit includes an external power interface, internal and external power connection points, a coil control circuit, an upper coil array and a lower coil array; wherein the coil control circuit is electrically connected to the external power interface through the internal and external power connection points, and the coil control circuit is electrically connected to the upper coil array and the lower coil array respectively, so as to realize electromagnetic control of the upper coil array and the lower coil array by the external power supply; The upper coil array is composed of three upper coils, and the lower coil array is composed of two lower coils; the upper coil array is located above the lower coil array, and a projection overlap area is generated between each lower coil and two adjacent upper coils; The nucleic acid extraction and purification turntable chip comprises a turntable and at least one reaction unit arranged on the turntable, wherein the reaction unit comprises an injection port, a connecting pipe, a nucleic acid extraction chamber, a micro-slit pipe, a washing chamber, an elution chamber, an amplification reagent storage chamber and an amplification area; wherein the injection port is connected to the inlet of the nucleic acid extraction chamber through the connecting pipe, the outlet of the nucleic acid extraction chamber is connected to the inlet of the washing chamber through the micro-slit pipe, the outlet of the washing chamber is connected to the first inlet of the elution chamber through the micro-slit pipe, the second inlet of the elution chamber is connected to the amplification reagent storage chamber through the connecting pipe, and the outlet of the elution chamber is connected to the amplification area through the connecting pipe; The reaction units disposed on the turntable are distributed on the turntable in a centrally symmetrical manner; the width of the connecting pipe is between 0.75 mm and 1 mm, and the width of the micro-slit pipe is between 0.75 mm and 1 mm; The three upper coils are respectively arranged below the nucleic acid extraction chamber, the washing chamber and the elution chamber, and the projection overlap area covers the micro-slit pipeline at the same time.

2. The automated nucleic acid extraction and purification device according to claim 1, characterized in that: The number of the reaction units arranged on the turntable is between 2 and 96.

3. The automated nucleic acid extraction and purification device according to claim 1, characterized in that: The electromagnetic unit is configured as a PCB coil array, the upper coil array is configured on the front side of the PCB board, and the lower coil array is configured on the back side of the PCB board.

4. The automated nucleic acid extraction and purification device according to claim 1, characterized in that: The upper coil and the lower coil are the same coils.

5. The automated nucleic acid extraction and purification device according to claim 4, characterized in that: The lower coil is arranged along the center line of the upper coil.

6. The automated nucleic acid extraction and purification device according to claim 4, characterized in that: The coil comprises 7 to 9 windings, the height of the coil is between 20 μm and 60 μm, the width is between 90 μm and 180 μm, and the spacing between two adjacent windings is between 100 μm and 300 μm.

7. The automated nucleic acid extraction and purification device according to claim 1, characterized in that: The amplification region is a digital amplification region.

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