CN112871230A - Vertical micro-fluidic chip for nucleic acid amplification - Google Patents

Vertical micro-fluidic chip for nucleic acid amplification Download PDF

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Publication number
CN112871230A
CN112871230A CN202110244578.1A CN202110244578A CN112871230A CN 112871230 A CN112871230 A CN 112871230A CN 202110244578 A CN202110244578 A CN 202110244578A CN 112871230 A CN112871230 A CN 112871230A
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amplification
chamber
rotary piston
cavity
communicated
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颜菁
章志伟
邹长华
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Jiangsu Huixian Pharmaceutical Technology Co ltd
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Jiangsu Huixian Pharmaceutical Technology Co ltd
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Priority to CN202110244578.1A priority Critical patent/CN112871230A/en
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    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6848—Nucleic acid amplification reactions characterised by the means for preventing contamination or increasing the specificity or sensitivity of an amplification reaction
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    • B01L2200/026—Fluid interfacing between devices or objects, e.g. connectors, inlet details
    • B01L2200/027—Fluid interfacing between devices or objects, e.g. connectors, inlet details for microfluidic devices
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    • C12Q2531/00—Reactions of nucleic acids characterised by
    • C12Q2531/10—Reactions of nucleic acids characterised by the purpose being amplify/increase the copy number of target nucleic acid
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    • C12Q2549/00—Reactions characterised by the features used to influence the efficiency or specificity
    • C12Q2549/10—Reactions characterised by the features used to influence the efficiency or specificity the purpose being that of reducing false positive or false negative signals
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Abstract

The invention discloses a vertical micro-fluidic chip for nucleic acid amplification. This vertical micro-fluidic chip is including the body of seting up the cavity, the cavity includes: a first amplification chamber for performing a first round of amplification on a nucleic acid extract; and a second amplification chamber for performing a second round of amplification on the products of the first round of amplification; the body is provided with a first liquid outlet channel communicated with the first amplification cavity and a liquid flow channel communicated with the second amplification cavity; the vertical micro-fluidic chip further comprises: the amplification rotary piston is rotatably arranged in the body around a horizontally extending rotating shaft, the first amplification cavity is positioned above the amplification rotary piston, the second amplification cavity is positioned below the amplification rotary piston, and the amplification rotary piston is provided with an amplification communicating groove which can communicate the liquid flow channels of the first liquid outlet channel and the second amplification cavity. The invention can amplify nucleic acid, and has simple structure and accurate reagent quantification.

Description

Vertical micro-fluidic chip for nucleic acid amplification
Technical Field
The invention belongs to the technical field of nucleic acid detection, and relates to a vertical micro-fluidic chip for nucleic acid amplification.
Background
Microfluidics is a technology for precisely controlling and controlling microscale fluids, and particularly relates to a technology for automatically completing the whole analysis process by integrating basic operation units of sample preparation, reaction, separation, detection and the like in the processes of biological, chemical and medical analysis on a microfluidic chip with the square centimeter. At present, a microfluidic chip has been applied to the field of PCR nucleic acid amplification, for example, a microfluidic chip of an integrated liquid path switching valve disclosed in chinese patent CN111760601A, which includes a microfluidic chip body, a reagent channel, a liquid path switching valve, a waste liquid storage chamber, a sample storage chamber, a first cleaning solution storage chamber, a second cleaning solution storage chamber, an amplification solution storage chamber, a nucleic acid extraction and amplification detection chamber, and an integrated nucleic acid detection microfluidic chip is formed integrally; the connection between different storage cavities and the nucleic acid extraction and amplification detection cavity is switched by using a liquid path switching valve; during nucleic acid extraction and amplification detection, the liquid path switching valve needs to be switched to different positions, different reagent storage cavities and nucleic acid extraction and amplification detection cavities are conducted, and an external power source is in butt joint with pressure caps of the different storage cavities. Most of the existing microfluidic chips for nucleic acid detection are horizontal chips, more parts connected and matched with an external power source are required to be arranged, the structure is complex, and the detection precision needs to be further improved.
Disclosure of Invention
The invention aims to provide a vertical micro-fluidic chip for nucleic acid amplification, which has a simple structure and relatively accurate reagent quantification.
In order to achieve the purpose, the invention adopts the following technical scheme:
a vertical micro-fluidic chip for nucleic acid amplification comprises a body provided with a chamber, wherein the chamber comprises:
a first amplification chamber for performing a first round of amplification on the extracted nucleic acids; and
a second amplification chamber for performing a second round of amplification on the products of the first round of amplification;
the body is provided with a first liquid outlet channel communicated with the first amplification cavity and a liquid flow channel communicated with the second amplification cavity;
the vertical micro-fluidic chip further comprises:
the amplification rotary piston is rotatably arranged in the body around a horizontally extending rotating shaft, the first amplification cavity is positioned above the amplification rotary piston, the second amplification cavity is positioned below the amplification rotary piston, and the amplification rotary piston is provided with an amplification communicating groove which can communicate the liquid flow channels of the first liquid outlet channel and the second amplification cavity.
Preferably, the amplification communicating groove is provided on an outer circumferential surface of the amplification rotary piston.
More preferably, the amplification communication channel has a starting end and a terminating end, and a central angle between the starting end and the terminating end is greater than zero.
Further, the central angle is 180 degrees.
More preferably, the amplification rotary piston includes a body and a sealing layer covering the body, and the amplification communicating groove is provided on the sealing layer or on the body and penetrates through the sealing layer.
More preferably, the number of the second amplification chambers is plural, each of the second amplification chambers is provided with one liquid flow channel, each of the second amplification chambers corresponds to one of the amplification communicating grooves, and the plural amplification communicating grooves are arranged in parallel in the circumferential direction of the amplification rotary piston.
More preferably, the starting ends of the amplification communicating grooves are arranged at the same circumferential position of the amplification rotary piston at intervals.
Further, the lengths of the amplification communicating grooves are different from each other, and the terminating ends of any two adjacent amplification communicating grooves are spaced at a distance in the axial direction and the circumferential direction of the amplification rotary piston.
Preferably, vertical micro-fluidic chip is still including locating the extraction rotary piston of first amplification chamber top, it can locate with rotating around the axis of rotation that a level extends to extract rotary piston in the body, set up on the body with the first inlet channel of first amplification chamber intercommunication, it can with the nucleic acid transfer groove of first inlet channel intercommunication to extract last being equipped with of rotary piston.
More preferably, the chamber further comprises a buffer chamber for receiving the amplification product of the first amplification chamber, the buffer chamber is located between the extraction rotary piston and the amplification rotary piston, and the first liquid outlet channel can be communicated with the amplification communication groove through the buffer chamber.
More preferably, the body is provided with a second liquid inlet channel and a second liquid outlet channel which are communicated with the buffer cavity, the extraction rotary piston is further provided with a liquid transfer communication groove which can be communicated with the first liquid outlet channel and the second liquid inlet channel, and the amplification communication groove can be communicated with the second liquid outlet channel and a liquid flow channel of the second amplification cavity.
Further, when the pipetting communication tank communicates the first effluent channel with the second effluent channel, the nucleic acid transfer tank communicates with the first effluent channel.
Preferably, the chamber further comprises a buffer chamber for receiving the amplification product of the first amplification chamber, the buffer chamber is located above the amplification rotary piston, the first liquid outlet channel can be communicated with the amplification communicating groove through the buffer chamber, the buffer chamber is communicated with a quantitative chamber arranged in the body through an air flow channel, and the vertical microfluidic chip further comprises a quantitative piston slidably arranged in the quantitative chamber.
Preferably, the first amplification chamber is provided with amplification reagents therein.
Compared with the prior art, the invention has the following advantages by adopting the scheme:
the micro-fluidic chip for nucleic acid amplification can perform PCR nested amplification, adopts a vertical structure, effectively eliminates the influence of bubbles, ensures accurate liquid inflow and can realize accurate quantification of liquid; the liquid channel of the microfluidic chip is switched by rotating the piston, so that the operation is convenient and controllable, and the automation is easy; the structure is compact and simple, and the batch production is easy; no aerosol pollution.
Drawings
In order to more clearly illustrate the technical solution of the present invention, the drawings needed to be used in the description of the embodiments will be briefly introduced below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings based on the drawings without creative efforts.
Fig. 1 is a schematic perspective view of a vertical microfluidic chip according to an embodiment of the present invention;
fig. 2 is a side view of a vertical microfluidic chip according to an embodiment of the present invention;
fig. 3 is an internal perspective view of a vertical microfluidic chip according to an embodiment of the present invention;
FIGS. 4a, 4b and 4c are schematic views of an extraction rotary piston according to an embodiment of the present invention from different viewing angles;
FIG. 5 is a cross-sectional view of an extraction rotary piston along its length in accordance with an embodiment of the present invention;
FIGS. 6a and 6b are schematic diagrams of an amplification rotary piston according to an embodiment of the present invention at different viewing angles;
FIG. 7 is a cross-sectional view of an amplification rotary piston along its length in accordance with an embodiment of the present invention.
Wherein the content of the first and second substances,
1. a body; 11. uniformly mixing the pistons; 12. a magnetic body; 13. an interface; 14. a dosing piston; 15. sealing the cover;
100. an aperture; 101. a sample chamber; 102. a reagent chamber; 103. a waste fluid chamber; 104. a first amplification chamber; 105. a buffer chamber; 106. a dosing chamber; 107. a second amplification chamber; 108. a flow channel; 109. a waste liquid channel, 110, a first liquid inlet channel; 111. a first liquid outlet channel; 112. a second liquid inlet channel; 113. a second liquid outlet channel;
2. extracting a rotary piston; 21. a reagent communicating groove; 22. a waste liquid communicating tank; 23. a nucleic acid transfer tank; 24. a liquid transfer communicating tank; 200. a curve segment; 201. a first body; 202. a first sealing layer; 203. connecting holes;
3. an amplification rotary piston; 31. amplifying the communication groove; 300. a corner section; 301. a second body; 302. a second sealing layer; 303. and connecting the holes.
Detailed Description
Preferred embodiments of the present invention will be described in detail below with reference to the accompanying drawings so that the advantages and features of the invention may be more readily understood by those skilled in the art. It should be noted that the description of the embodiments is provided to help understanding of the present invention, but the present invention is not limited thereto.
As used in this specification and the appended claims, the terms "comprises" and "comprising" are intended to only encompass the explicitly identified steps and elements, which do not constitute an exclusive list, and that a method or apparatus may include other steps or elements. As used herein, the term "and/or" includes any combination of one or more of the associated listed items.
It should be noted that, unless otherwise specified, when a feature is referred to as being "fixed" or "connected" to another feature, it may be directly fixed or connected to the other feature or indirectly fixed or connected to the other feature. Further, the description of the upper, lower, left, right, etc. used in the present invention is only with respect to the positional relationship of the respective components of the present invention with respect to each other in the drawings.
It is further understood that the use of "a plurality" in this disclosure means two or more, as other terms are analogous. "and/or" describes the association relationship of the associated objects, meaning that there may be three relationships, e.g., a and/or B, which may mean: a exists alone, A and B exist simultaneously, and B exists alone.
It will be further understood that the terms "first," "second," and the like are used to describe various information and that such information should not be limited by these terms. These terms are only used to distinguish one type of information from another and do not denote a particular order or importance. Indeed, the terms "first," "second," and the like are fully interchangeable. For example, first information may also be referred to as second information, and similarly, second information may also be referred to as first information, without departing from the scope of the present disclosure.
Referring to fig. 1 to 7, according to an embodiment of the present invention, a vertical microfluidic chip for nucleic acid amplification includes a body 1 having a cavity, and the body 1 is vertically disposed in a plate shape. The vertical micro-fluidic chip also comprises an extraction rotary piston 2 and an amplification rotary piston 3 which are integrally and respectively in a cylindrical shape; the extracting rotary piston 2 is rotatably inserted into the body 1, and the amplifying rotary piston 3 is positioned right below the extracting rotary piston 2 and rotatably inserted into the body 1. The extraction rotary piston 2 and the amplification rotary piston 3 are capable of rotating around a horizontally extending rotation axis line, respectively, the rotation axis lines of which are parallel to each other and are located in the same vertical plane, that is, the rotation axis lines of which extend horizontally in the left-right direction in fig. 3, respectively. The length to diameter ratio of the two rotary pistons is greater than 1, preferably greater than 5. The vertical micro-fluidic chip is divided into three functional areas by an extraction rotary piston 2 and an amplification rotary piston 3, the extraction rotary piston 2 and the part above the extraction rotary piston are nucleic acid extraction and purification areas, and nucleic acid samples finish nucleic acid extraction and purification in the areas; extracting a part between the rotary piston 2 and the amplification rotary piston 3 to be a first amplification area, and performing first round PCR amplification on the extracted and purified piston in the first amplification area; the part below the amplification rotary piston 3 is a second amplification area, the amplification product after the first round of PCR amplification is subjected to second round PCR amplification in the second amplification area, and the fluorescence signal of the amplification product is measured in the second amplification area through a fluorescence detection device of a PCR instrument.
As shown in FIG. 3, the chambers of the nucleic acid extraction and purification section (i.e., the chambers located above the extraction rotary piston 2) include a sample chamber 101, a reagent chamber 102, and a waste liquid chamber 103, which are arranged in parallel in the upper portion of the body 1, wherein the number of the reagent chambers 102 is plural, and the reagent chambers 102 are arranged in parallel between the sample chamber 101 on the left side and the waste liquid chamber 103 on the right side, i.e., the sample chamber 101, the plural reagent chambers 102, and the waste liquid chamber 103 are arranged at intervals in the axial direction of the extraction rotary piston 2. The sample chamber 101 and the reagent chamber 102 each have a hole 100 provided at an upper end portion of the body 1 (preferably, an upper end surface of the body 1), and a sample or a filling reagent can be introduced into these chambers through these holes 100, and the outside atmosphere can be communicated through these holes 100 to facilitate pipetting. When the vertical microfluidic chip is not in use, the holes 100 at the upper end of the body 1 are closed by a sealing film; in use, the sealing film is removed; after the nucleic acid sample is added to the sample chamber 101, the hole 100 in the sample chamber 101 is closed by a cover 15, and the cover 15 is removable. The sample chamber 101 is used for containing a nucleic acid sample; the reagent cavity 102 is used for storing reagents required for nucleic acid extraction, such as lysis solution, rinsing solution, eluent and the like; the waste liquid cavity 103 is used for storing waste liquid generated in the process of extracting and purifying nucleic acid. Specifically, in this embodiment, the number of the reagent chambers 102 is five, and lysis buffer, protease, rinsing buffer and eluent are sequentially stored from left to right. Correspondingly, the plurality of reagent chambers 102 includes a first reagent chamber for storing a lysis solution, a second reagent chamber for storing a protease, third and fourth reagent chambers for storing a rinsing solution, and a fifth reagent chamber for storing an elution solution.
The chambers are connected or disconnected by microchannels provided in the extraction rotary piston 2. As shown in FIG. 3, the main body 1 is further provided with a liquid flow channel 108 communicating with the sample chamber 101 and each reagent chamber 102, and the main body 1 is further provided with a waste liquid channel 109 communicating with the waste liquid chamber 103. The extraction rotary piston 2 can selectively connect two of the liquid flow channel 108 and the waste liquid channel 109 and disconnect the other channels; as the extraction rotary piston 2 rotates by a certain angle, the extraction rotary piston 2 communicates the other two of the liquid flow channel 108 and the waste liquid channel 109, while disconnecting the other channels. Two chambers of the extraction rotary piston 2 are communicated by a communication groove arranged on the outer surface of the extraction rotary piston.
The layout of the communication grooves on the extraction rotary piston 2 is shown in fig. 4a to 4 c. As shown in fig. 4a to 4c, the extraction rotary piston 2 is provided with a reagent communicating groove 21 capable of communicating the flow channel 108 of the sample chamber 101 with the flow channel 108 of the reagent chamber 102, and the extraction rotary piston 2 is further provided with a waste liquid communicating groove 22 capable of communicating the flow channel 108 of the sample chamber 101 with the waste liquid channel 109. Each reagent chamber 102 is provided with one liquid flow channel 108, and thus, the extraction rotary piston 2 is provided with a plurality of reagent communicating grooves 21. The reagent communication grooves 21 and the waste liquid communication groove 22 extend entirely in the axial direction of the extraction rotary piston 2, and the plurality of reagent communication grooves 21 and the plurality of waste liquid communication grooves 22 are arranged in parallel in the circumferential direction of the extraction rotary piston 2. The starting ends of each of the reagent communication grooves 21 and the above-mentioned waste liquid communication grooves 22 are alignably abutted with the liquid flow channel 108 of the sample chamber 101 to achieve communication with the sample chamber 101, so that the starting ends of each of the reagent communication grooves 21 and the above-mentioned waste liquid communication grooves 22 are located at the same circumferential position of the extracting rotary piston 2, that is, the axial distance thereof is zero. The terminating end of each reagent communicating groove 21 can be aligned with the flow channel 108 of the corresponding reagent chamber 102 to communicate with the reagent chamber 102, and the terminating end of the waste communicating groove 22 can be aligned with the waste channel 109 to communicate with the waste chamber 103, so that the terminating ends of each reagent communicating groove 21 and the waste communicating groove 22 are located at different circumferential positions of the extraction rotary piston 2, respectively, i.e., the axial distance of any two terminating ends is greater than zero. At least a portion of the reagent communication channel 21 and the waste communication channel 22 each have one or more curved segments 200, the curved segments 200 corresponding to the flow channels 108 of the non-target reagent chambers 102 to be passed and being located below the flow channels 108 so as to avoid the flow channels 108 and thereby avoid contact with the flow channels 108. The reagent communication groove 21 and the waste liquid communication groove 22 described above extend in a straight line in portions other than the curved line segment 200, and are parallel to each other. The "non-target agent communication channel" herein is explained as follows: for example, for the reagent communication groove 21 communicating the fifth reagent chamber storing the eluent with the sample chamber 101, the first to fourth reagent chambers located therebetween are non-target reagent chambers; for the waste communication channel 22, all reagent chambers are non-target reagent chambers; and so on.
As shown in fig. 1 and 2, the vertical microfluidic chip further includes positive and negative pressure pumps for providing positive and negative pressures to the sample chamber 101, and the sample chamber 101 and the positive and negative pressure pumps are communicated with each other. The nucleic acid extraction and purification area is only connected with the sample cavity 101 and the positive and negative pressure pumps, and the power for liquid circulation in the process of nucleic acid extraction, purification and nucleic acid extracting solution transferring to the first amplification area can be provided through the positive and negative pressure pumps. The upper end of the body 1 (preferably the upper end of the body 1) is provided with a port 13 for connecting a positive pressure sample and a negative pressure sample, and the port 13 is communicated with the sample cavity 101 through a micro-channel arranged in the body 1. When the positive and negative pressure pumps provide positive pressure to the sample cavity 101, the liquid in the sample cavity 101 can be pushed into the waste liquid cavity 103 or the cavity of the first amplification area; the positive and negative pressure pumps provide negative pressure to the sample chamber 101, which draws reagent from the reagent chamber 102 into the sample chamber 101.
As shown in fig. 1 to 3, the vertical microfluidic chip further includes a mixing piston 11 capable of moving or rotating to mix the material in the sample chamber 101. The mixing piston 11 is movably or rotatably disposed in the sample chamber 101, or the mixing piston 11 is movably or rotatably disposed in the body 1 and communicates with the sample chamber 101. By moving the mixing piston 11 back and forth, the reagent and the like in the sample chamber 101 can be disturbed, and the materials can be mixed uniformly.
Magnetic beads (not shown) for adsorbing nucleic acids are provided in the sample chamber 101. As shown in fig. 2 and 3, a magnetic body 12 is provided at a portion of the body 1 near the sample chamber 101, and the magnetic body 12 is specifically embedded in a magnet hole of the body 1. The magnetic body 12 is preferably an electromagnet, and when the electromagnet is energized, the magnetic beads are capable of adsorbing nucleic acids; when the electromagnet is powered off, the magnetic beads release the nucleic acids. In another embodiment, the magnetic body 12 may be a magnet, which is removable.
As shown in fig. 3, the chamber of the first amplification zone (i.e. the chamber located between the extraction rotary piston 2 and the amplification rotary piston 3) comprises a first amplification chamber 104, and the first amplification chamber 104 is used for performing a first round of PCR amplification on the extracted nucleic acid. The first amplification chamber 104 is pre-loaded with amplification reagents. The body 1 is provided with a first inlet channel 110 and a first outlet channel 111 which are communicated with the first amplification chamber 104. The first amplification chamber 104 is located below the extraction rotary piston 2, and the extraction rotary piston 2 is further provided with a nucleic acid transfer groove 23 capable of communicating the liquid flow channel 108 of the sample chamber 101 with the first liquid inlet channel 110. The nucleic acid transfer groove 23 is provided on the outer circumferential surface of the extraction rotary piston 2, and the central angle between the starting end and the terminating end of the nucleic acid transfer groove 23 is larger than zero, preferably larger than 90 degrees. When the extraction rotary piston 2 is rotated to a certain position, the starting end of the nucleic acid transfer groove 23 is aligned with the liquid flow channel 108 of the sample chamber 101 above, the terminating end is aligned with the first liquid inlet channel 110 below, and the positive and negative pressure pumps supply positive pressure to the sample chamber 101 to transfer the nucleic acid extract from the sample chamber 101 to the first amplification chamber 104.
The chamber of the first amplification zone further comprises a buffer chamber 105 for receiving the amplification product of the first amplification chamber 104, and the first amplification chamber 104 may be in communication with a second amplification chamber within the second amplification zone via the buffer chamber 105. The buffer chamber 105 is located between the extraction rotary piston 2 and the amplification rotary piston 3. The body 1 is provided with a second liquid inlet channel 112 and a second liquid outlet channel 113 which are communicated with the buffer cavity 105. The extraction rotary piston 2 is further provided with a pipetting communicating groove 24 capable of communicating the first liquid outlet channel 111 and the second liquid inlet channel 112, and the pipetting communicating groove 24 extends linearly in the axial direction of the extraction rotary piston 2 and is located entirely on the right side of the termination end of the nucleic acid transfer groove 23. When the pipetting communication groove 24 communicates the first outlet channel 111 and the second inlet channel 112, the nucleic acid transfer groove 23 communicates the flow channel 108 of the sample chamber 101 and the first inlet channel 110.
As shown in fig. 4a to 5, the extraction rotary piston 2 includes a first body 201 and a first sealing layer 202 covering the first body 201. The reagent communication groove 21, the waste liquid communication groove 22, the nucleic acid transfer groove 23, and the pipette communication groove 24 are provided on the first sealing layer 202; alternatively, the reagent communication groove 21, the waste liquid communication groove 22, the nucleic acid transfer groove 23, and the pipette communication groove 24 are provided in the first main body 201 so as to penetrate the first sealing layer 202, and the reagent communication groove 21, the waste liquid communication groove 22, the nucleic acid transfer groove 23, and the pipette communication groove 24 are surrounded on the periphery by the first sealing layer 202. First seal layer 202 is made of a flexible material, such as rubber.
The buffer chamber 105 is communicated with a quantitative chamber 106 arranged in the body 1 through an air flow channel, and the vertical microfluidic chip further comprises a quantitative piston 14 which is slidably arranged in the quantitative chamber 106. By moving the quantitative piston 14, the amplification product in the first amplification chamber 104 can be sucked into the buffer chamber 105, and the amplification product in the buffer chamber 105 can be quantitatively dispensed into the second amplification region.
As shown in fig. 3, the chamber of the second amplification zone (i.e. the chamber located below the amplification rotary piston 3) comprises a plurality of second amplification chambers 107, and the second amplification chambers 107 are used for performing the second round PCR amplification on the products of the first round amplification. The plurality of second amplification chambers 107 are arranged side by side from left to right in the axial direction of the amplification rotary piston 3. The body 1 is provided with liquid flow channels 108 respectively communicating with the second amplification chambers 107.
The connection and disconnection between the second outlet channel 113 of the buffer chamber 105 and the liquid flow channel 108 of each second amplification chamber 107 are realized by a connection groove provided in the amplification rotary piston 3. Referring to FIGS. 6a and 6b, the amplification rotary piston 3 is provided with an amplification communicating groove 31 capable of communicating the first liquid outlet channel 111 with the liquid flow channel 108 of the second amplification chamber 107. Specifically, the first outlet channel 111 is communicated with the amplification communicating groove 31 via the second outlet channel 113 of the buffer chamber 105. The amplification communicating groove 31 can communicate the second liquid outlet channel 113 and the liquid flow channel 108 of the second amplification chamber 107. The amplification rotary piston 3 can selectively connect the second liquid outlet channel 113 with the liquid flow channel 108 of one of the second amplification cavities 107 and disconnect the other liquid flow channels 108; as the amplification rotary piston 3 rotates a certain angle, the amplification rotary piston 3 connects the second liquid outlet channel 113 with the liquid flow channel 108 of another second amplification chamber 107, and disconnects the other liquid flow channels 108.
The layout of the amplification communication grooves 31 in the amplification rotary piston 3 is shown in fig. 6a and 6 b. As shown in fig. 6a and 6b, amplification-communicating grooves 31 are provided on the outer circumferential surface of the amplification rotary piston 3, one amplification-communicating groove 31 for each second amplification chamber 107. The amplification communication groove is provided with an initial end and a termination end, and the central angle between the initial end and the termination end is larger than zero; specifically, the central angle is 180 degrees. The amplification communicating grooves 31 are arranged in parallel in the circumferential direction of the amplification rotary piston 3. Each amplification communication groove 31 comprises three straight line segments connected in sequence, the adjacent straight line segments are perpendicular to each other, and the two straight line segments are in circular arc transition to form a corner segment 300. The starting ends of the amplification communicating grooves 31 are arranged at intervals in the circumferential direction of the amplification rotary piston 3 and are located at the same circumferential position of the amplification rotary piston 3. The amplification communicating grooves 31 are different in length from each other, and the terminating ends of any two adjacent amplification communicating grooves 31 are spaced apart in both the axial direction and the circumferential direction of the amplification rotary piston 3.
Referring to fig. 6a to 7, the amplification rotary piston 3 includes a second body 301 and a second sealing layer 302 covering the second body 301. The amplification communication groove 31 is opened in the second sealing layer 302; alternatively, the amplification communicating grooves 31 are formed in the second body 301 so as to penetrate the second sealing layer 302, and the amplification communicating grooves 31 are surrounded on the periphery by the second sealing layer 302. The second sealant layer 302 is made of a flexible material, such as rubber.
The extraction rotary piston 2 and the amplification rotary piston 3 can be driven to rotate by a power source respectively, and the power source can be a motor. Referring to fig. 2, the extraction rotary piston 2 has a driving end portion for engagement with a power source, the driving end portion having a connection hole 203, which is a polygonal hole or a special-shaped hole, into which an output shaft of a motor can be inserted and connected. The amplification rotary piston 3 has a driving end portion for engagement with a power source, the driving end portion having a connection hole 303 of a polygonal hole or a shaped hole into which an output shaft of a motor can be inserted and connected.
The present embodiment also provides a method for nucleic acid amplification, which employs the above vertical microfluidic chip. The method comprises the following steps:
s1, adding the nucleic acid sample into the sample cavity 101;
s2, rotating the extraction rotary piston 2 to enable the reagent communicating groove 21 to communicate the reagent cavity 102 with the sample cavity 101, and enabling the reagent to be transferred into the sample cavity 101 and uniformly mixed for reaction;
s3, rotating the extraction rotary piston 2 to enable the waste liquid communicating groove 22 on the extraction rotary piston to communicate the sample cavity 101 with the waste liquid cavity 103 arranged on the body 1, and transferring the waste liquid after reaction into the waste liquid cavity 103;
repeating the steps S2 and S3, adding the lysis solution, the protease, the rinsing solution and the eluent into the sample cavity 101 in sequence for reaction, and discharging the waste liquid into the waste liquid cavity 103 after each reaction;
s4, rotating the extraction rotary piston 2 to enable the nucleic acid communicating groove to communicate the sample cavity 101 with the first amplification cavity 104, and transferring the nucleic acid extracting solution in the sample cavity 101 to the first amplification cavity 104;
s5, rotating the extraction rotary piston 2, closing the micro-channel between the sample cavity 101 and the first amplification cavity 104, and carrying out a first round of amplification reaction; specifically, the extraction rotary piston 2 closes the microchannel between the first amplification chamber 104 and the buffer chamber 105;
s6, rotating the rotary extraction piston 2 to make the liquid transfer connecting groove 24 on the rotary extraction piston communicate the first amplification chamber 104 with a buffer chamber 105 arranged on the main body 1, and transferring the amplification product into the buffer chamber 105;
s7, the amplification rotary piston 3 is rotated to connect the amplification communicating groove 31 and the second amplification chamber 107, and the first round of amplification products are transferred to the second amplification chamber 107. Specifically, the amplification communication groove 31 communicates the buffer chamber 105 with the second amplification chamber 107.
Step S7 is specifically implemented as follows:
step S7-1, rotating the amplification rotary piston 3 to make the first amplification communicating groove 31 communicate with the corresponding first second amplification chamber 107, moving the quantitative piston 14 in the quantitative chamber 106 communicating with the buffer chamber 105, and quantitatively pushing the amplification product in the buffer chamber 105 into the first second amplification chamber 107;
step S7-2, continuing to rotate the amplification rotary piston 3 to make the second amplification communicating groove 31 communicate with the corresponding second amplification cavity 107, moving the quantitative piston 14 in the quantitative cavity 106 communicated with the buffer cavity 105, and quantitatively pushing the amplification product in the buffer cavity 105 to the second amplification cavity 107;
the above steps S7-1 and S7-2 are repeated to sequentially push the amplification products into the respective second amplification chambers 107.
The procedure of using the microfluidic chip for PCR nucleic acid extraction and amplification of the present example is described below.
Firstly, the operation steps of extracting and purifying nucleic acid
1. The cover 15 is opened, the sample is added to the sample chamber 101 through the hole above the sample chamber 101, and then the cover 15 is closed.
2. The extraction rotary piston 2 is rotated by a certain angle, the first reagent cavity 102 is communicated with the sample cavity 101, then negative pressure is generated by a positive and negative pressure pump to transfer lysate in the first reagent cavity 102 to the sample cavity 101, and then the blending piston 11 is driven to blend the lysate and the nucleic acid sample and perform reaction.
3. The magnetic body 12 starts to generate a magnetic field to adsorb magnetic beads, the extraction rotary piston 2 continues to rotate for a certain angle, the sample cavity 101 is communicated with the waste liquid cavity 103, and positive pressure is generated by the positive and negative pressure pumps to discharge waste liquid which is not adsorbed on the magnetic beads in the sample cavity 101 to the waste liquid cavity 103.
4. The extraction rotary piston 2 is rotated by a certain angle, the second reagent chamber 102 is communicated with the sample chamber 101, negative pressure is generated by the positive and negative pressure pumps to transfer the protease in the second reagent chamber 102 to the sample chamber 101, meanwhile, the magnetic body 12 stops generating a magnetic field to stop the adsorption of the magnetic beads, and then the mixing piston 11 is driven to perform mixing reaction.
5. The magnetic body 12 starts to generate a magnetic field to adsorb magnetic beads, the extraction rotary piston 2 continues to rotate for a certain angle, the sample cavity 101 is communicated with the waste liquid cavity 103, and positive pressure is generated by the positive and negative pressure pumps to discharge waste liquid which is not adsorbed on the magnetic beads in the sample cavity 101 to the waste liquid cavity 103.
6. The extraction rotary piston 2 is rotated by a certain angle, the third reagent chamber 102 is communicated with the sample chamber 101, negative pressure is generated by a positive and negative pressure pump to transfer the rinsing liquid in the third reagent chamber 102 to the sample chamber 101, meanwhile, the magnetic body 12 stops generating a magnetic field to stop the adsorption of magnetic beads, and then the mixing piston 11 is driven to carry out mixing reaction to carry out first rinsing on nucleic acid.
7. The magnetic body 12 starts to generate a magnetic field to adsorb magnetic beads, the extraction rotary piston 2 continues to rotate for a certain angle, the sample cavity 101 is communicated with the waste liquid cavity 103, and positive pressure is generated by the positive and negative pressure pumps to discharge waste liquid which is not adsorbed on the magnetic beads in the sample cavity 101 to the waste liquid cavity 103.
8. The extraction rotary piston 2 is rotated by a certain angle, the fourth reagent chamber 102 is communicated with the sample chamber 101, negative pressure is generated by a positive and negative pressure pump to transfer the rinsing liquid in the fourth reagent chamber 102 to the sample chamber 101, meanwhile, the magnetic body 12 stops generating a magnetic field to stop the adsorption of magnetic beads, and then the mixing piston 11 is driven to carry out mixing reaction to carry out secondary rinsing on nucleic acid.
9. The magnetic body 12 starts to generate a magnetic field to adsorb magnetic beads, the extraction rotary piston 2 continues to rotate for a certain angle, the sample cavity 101 is communicated with the waste liquid cavity 103, and positive pressure is generated by the positive and negative pressure pumps to discharge waste liquid which is not adsorbed on the magnetic beads in the sample cavity 101 to the waste liquid cavity 103.
10. The extraction rotary piston 2 is rotated by a certain angle, the fifth reagent chamber 102 is communicated with the sample chamber 101, then the negative pressure generated by the positive and negative pressure pump transfers the eluent in the fifth reagent chamber 102 to the sample chamber 101, meanwhile, the magnetic body 12 stops generating the magnetic field to stop the adsorption of the magnetic beads, then the mixing piston 11 is driven to carry out mixing reaction, and the extracted nucleic acid is eluted to form the nucleic acid extract.
Second, first round PCR amplification operation step
1. The extraction rotary piston 2 is rotated to connect the first amplification chamber 104 with the sample chamber 101, and then the nucleic acid extract in the sample chamber 101 is discharged into the first amplification chamber 104 by positive pressure generated by the positive and negative pressure pumps.
2. The micro-channel is closed by rotating the extraction rotary piston 2, and the first round of PCR amplification reaction is carried out.
3. The extraction rotary piston 2 is rotated to connect the first amplification chamber 104 with the buffer chamber 105, and then the quantitative piston 14 transfers the liquid into the buffer chamber 105 to mix the liquid.
Third and second round PCR amplification operation steps
1. The amplification rotary piston 3 was rotated to connect the buffer chamber 105 to the first second amplification chamber 107 (chamber L1), and then the liquid was transferred into the chamber L1 by the fixed displacement piston 14.
2. The amplification rotary piston 3 was rotated to transfer the liquid into the chambers L2-L12 in turn.
3. A second round of PCR amplification was performed.
The embodiment can realize PCR nested amplification, and integrates nucleic acid extraction, amplification and detection; can simultaneously realize the PCR real-time fluorescence detection of 1-48 gene loci; the vertical structure is adopted, so that the influence of bubbles is effectively eliminated, the liquid inlet amount is accurate, and the accurate quantification of the liquid can be realized; compact and simple structure and easy batch production. The liquid channel of the micro-fluidic chip is switched by rotating the piston, so that the operation is convenient and controllable, and the automation is easy. The full-sealing and aerosol-free pollution is realized, and an additional gas circuit is not needed. The power of the flowing of the liquid in the whole operation process can be provided only by controlling one positive and negative pressure pump and one quantitative piston, the operation is convenient, and the structure is simple.
The above embodiments are merely illustrative of the technical ideas and features of the present invention, and are preferred embodiments, which are intended to enable those skilled in the art to understand the contents of the present invention and implement the present invention, and not to limit the scope of the present invention. All equivalent changes or modifications made according to the principles of the present invention should be covered within the protection scope of the present invention.

Claims (10)

1. The vertical micro-fluidic chip for nucleic acid amplification comprises a body provided with a cavity, and is characterized in that the cavity comprises:
a first amplification chamber for performing a first round of amplification on a nucleic acid extract; and
a second amplification chamber for performing a second round of amplification on the products of the first round of amplification;
the body is provided with a first liquid outlet channel communicated with the first amplification cavity and a liquid flow channel communicated with the second amplification cavity;
the vertical micro-fluidic chip further comprises:
the amplification rotary piston is rotatably arranged in the body around a horizontally extending rotating shaft, the first amplification cavity is positioned above the amplification rotary piston, the second amplification cavity is positioned below the amplification rotary piston, and the amplification rotary piston is provided with an amplification communicating groove which can communicate the liquid flow channels of the first liquid outlet channel and the second amplification cavity.
2. The vertical microfluidic chip according to claim 1, wherein the amplification communication groove is formed on the outer circumferential surface of the amplification rotary piston.
3. The vertical microfluidic chip according to claim 2, wherein the amplification communication groove has a start end and a stop end, and a central angle between the start end and the stop end is greater than zero.
4. The vertical microfluidic chip according to claim 1, wherein the amplification rotary piston comprises a body and a sealing layer covering the body, and the amplification communicating groove is provided on the sealing layer or on the body and penetrates through the sealing layer.
5. The vertical microfluidic chip according to claim 1, wherein the number of the second amplification chambers is plural, each of the second amplification chambers has a liquid flow channel, each of the second amplification chambers corresponds to one of the amplification communication grooves, and the amplification communication grooves are arranged in parallel along a circumferential direction of the amplification rotary piston.
6. The vertical microfluidic chip according to claim 5, wherein the starting ends of the amplification communicating grooves are arranged at intervals at the same circumferential position of the amplification rotary piston.
7. The vertical microfluidic chip according to claim 6, wherein the amplification communication grooves have different lengths, and the terminating ends of any two adjacent amplification communication grooves are spaced apart in the axial direction and the circumferential direction of the amplification rotary piston.
8. The vertical microfluidic chip according to claim 1, further comprising an extraction rotary piston disposed above the first amplification chamber, wherein the extraction rotary piston is rotatably disposed in the body around a horizontally extending rotation axis, the body is provided with a first liquid inlet channel communicated with the first amplification chamber, and the extraction rotary piston is provided with a nucleic acid transfer groove capable of being communicated with the first liquid inlet channel.
9. The vertical microfluidic chip according to claim 8, wherein the first amplification chamber is filled with amplification reagents, the chamber further comprises a buffer chamber for receiving amplification products of the first amplification chamber, the buffer chamber is located between the extraction rotary piston and the amplification rotary piston, the first liquid outlet channel can be communicated with the amplification communicating groove through the buffer chamber, the body is provided with a second liquid inlet channel and a second liquid outlet channel which are communicated with the buffer chamber, the extraction rotary piston is further provided with a liquid transfer communicating groove which can be communicated with the first liquid outlet channel and the second liquid inlet channel, and the amplification communicating groove can be communicated with liquid flow channels of the second liquid outlet channel and the second amplification chamber.
10. The vertical microfluidic chip according to claim 1, wherein the chamber further comprises a buffer chamber for receiving the amplification product of the first amplification chamber, the buffer chamber is located above the amplification rotary piston, the first liquid outlet channel can be communicated with the amplification communicating groove through the buffer chamber, the buffer chamber is communicated with a quantitative chamber provided in the body through an air flow channel, and the vertical microfluidic chip further comprises a quantitative piston slidably provided in the quantitative chamber.
CN202110244578.1A 2021-03-05 2021-03-05 Vertical micro-fluidic chip for nucleic acid amplification Pending CN112871230A (en)

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113667598A (en) * 2021-08-17 2021-11-19 圣湘生物科技股份有限公司 Nucleic acid detection device and nucleic acid detection method
CN114177963A (en) * 2022-01-13 2022-03-15 深圳市刚竹医疗科技有限公司 Nucleic acid analysis device
WO2023274301A1 (en) * 2021-06-30 2023-01-05 恒泰医疗有限公司 Chip device and instrument for nucleic acid detection, and application thereof
WO2023040477A1 (en) * 2021-09-17 2023-03-23 上海微创惟微诊断技术有限公司 In-vitro diagnostic analysis apparatus and reagent kit
WO2023071049A1 (en) * 2021-11-01 2023-05-04 江苏汇先医药技术有限公司 Vertical micro-fluidic chip and method for pcr test

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2023274301A1 (en) * 2021-06-30 2023-01-05 恒泰医疗有限公司 Chip device and instrument for nucleic acid detection, and application thereof
CN113667598A (en) * 2021-08-17 2021-11-19 圣湘生物科技股份有限公司 Nucleic acid detection device and nucleic acid detection method
WO2023040477A1 (en) * 2021-09-17 2023-03-23 上海微创惟微诊断技术有限公司 In-vitro diagnostic analysis apparatus and reagent kit
WO2023071049A1 (en) * 2021-11-01 2023-05-04 江苏汇先医药技术有限公司 Vertical micro-fluidic chip and method for pcr test
CN114177963A (en) * 2022-01-13 2022-03-15 深圳市刚竹医疗科技有限公司 Nucleic acid analysis device

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