CN112980650A - Vertical micro-fluidic chip and method for nucleic acid extraction - Google Patents
Vertical micro-fluidic chip and method for nucleic acid extraction Download PDFInfo
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- 108020004707 nucleic acids Proteins 0.000 title claims abstract description 85
- 150000007523 nucleic acids Chemical class 0.000 title claims abstract description 85
- 102000039446 nucleic acids Human genes 0.000 title claims abstract description 85
- 238000000605 extraction Methods 0.000 title claims abstract description 26
- 238000000034 method Methods 0.000 title claims abstract description 22
- 239000007788 liquid Substances 0.000 claims abstract description 148
- 239000003153 chemical reaction reagent Substances 0.000 claims abstract description 116
- 230000003321 amplification Effects 0.000 claims description 120
- 238000003199 nucleic acid amplification method Methods 0.000 claims description 120
- 239000002699 waste material Substances 0.000 claims description 63
- 238000004891 communication Methods 0.000 claims description 51
- 239000000872 buffer Substances 0.000 claims description 36
- 238000012546 transfer Methods 0.000 claims description 33
- 238000007789 sealing Methods 0.000 claims description 20
- 238000002156 mixing Methods 0.000 claims description 19
- 239000011324 bead Substances 0.000 claims description 17
- 238000006243 chemical reaction Methods 0.000 claims description 17
- 239000000463 material Substances 0.000 claims description 6
- 238000011002 quantification Methods 0.000 abstract description 5
- 239000000243 solution Substances 0.000 description 11
- 238000012408 PCR amplification Methods 0.000 description 9
- 238000001514 detection method Methods 0.000 description 9
- 238000003860 storage Methods 0.000 description 8
- 239000003480 eluent Substances 0.000 description 6
- 108091005804 Peptidases Proteins 0.000 description 5
- 239000004365 Protease Substances 0.000 description 5
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 description 5
- 238000000746 purification Methods 0.000 description 5
- 238000001179 sorption measurement Methods 0.000 description 4
- 230000009089 cytolysis Effects 0.000 description 3
- 239000006166 lysate Substances 0.000 description 3
- 239000012487 rinsing solution Substances 0.000 description 3
- 238000004458 analytical method Methods 0.000 description 2
- 238000010923 batch production Methods 0.000 description 2
- 238000004140 cleaning Methods 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 239000012530 fluid Substances 0.000 description 2
- 238000001917 fluorescence detection Methods 0.000 description 2
- 239000000443 aerosol Substances 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 238000007599 discharging Methods 0.000 description 1
- 238000006073 displacement reaction Methods 0.000 description 1
- 238000010828 elution Methods 0.000 description 1
- 238000011049 filling Methods 0.000 description 1
- 210000001503 joint Anatomy 0.000 description 1
- 239000012139 lysis buffer Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 239000000565 sealant Substances 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 230000007704 transition Effects 0.000 description 1
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- C12N15/09—Recombinant DNA-technology
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
- C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
- C12N15/1006—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers
- C12N15/1013—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers by using magnetic beads
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Abstract
The invention discloses a vertical micro-fluidic chip for nucleic acid extraction and a method thereof. This vertical micro-fluidic chip is including the body of seting up the cavity, the cavity includes: a sample chamber for holding a nucleic acid sample; and a reagent chamber for storing a reagent required for nucleic acid extraction; the body is also provided with a liquid flow channel which is respectively communicated with the sample cavity and the reagent cavity; the vertical micro-fluidic chip further comprises: the reagent chamber and the reagent chamber are positioned above the first rotary piston, and the first rotary piston is provided with a reagent communicating groove which can communicate the liquid flow channel of the sample chamber with the liquid flow channel of the reagent chamber. The invention can extract nucleic acid, and has simple structure and accurate reagent quantification.
Description
Technical Field
The invention belongs to the technical field of nucleic acid extraction, and relates to a vertical micro-fluidic chip and a method for extracting nucleic acid.
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 microfluidic chip for nucleic acid extraction, which has a simple structure and relatively accurate reagent quantification.
Another object of the present invention is to provide a method for nucleic acid extraction, which is simple and convenient to operate and has accurate reagent quantification.
In order to achieve the purpose, the invention adopts the following technical scheme:
a vertical micro-fluidic chip for nucleic acid extraction comprises a body provided with a cavity, wherein the cavity comprises:
a sample chamber for holding a nucleic acid sample; and
a reagent chamber for storing a reagent required for nucleic acid extraction;
the body is also provided with a liquid flow channel which is respectively communicated with the sample cavity and the reagent cavity;
the vertical micro-fluidic chip further comprises:
the reagent chamber and the reagent chamber are positioned above the first rotary piston, and the first rotary piston is provided with a reagent communicating groove which can communicate the liquid flow channel of the sample chamber with the liquid flow channel of the reagent chamber.
Preferably, magnetic beads for adsorbing nucleic acid are arranged in the sample cavity.
More preferably, a magnetic body is disposed at a portion of the body near the sample chamber.
Preferably, the chamber further comprises a waste liquid cavity located above the first rotary piston, the body is provided with a waste liquid channel communicated with the waste liquid cavity, and the first rotary piston is further provided with a waste liquid communicating groove capable of communicating the liquid flow channel of the sample cavity with the waste liquid channel.
More preferably, the sample chamber, the reagent chamber and the waste chamber are arranged at intervals in the axial direction of the first rotary piston.
Preferably, the number of the reagent chambers is multiple, the reagent chambers are arranged at intervals along the axial direction of the first rotary piston, each reagent chamber is correspondingly provided with one liquid flow channel, and the first rotary piston is correspondingly provided with a plurality of reagent communicating grooves.
More preferably, the plurality of reagent chambers include a first reagent chamber for storing a lysate, a second reagent chamber for storing a protease, a third reagent chamber and a fourth reagent chamber for storing a rinsing liquid, and a fifth reagent chamber for storing an eluent, five reagent communicating grooves are correspondingly arranged on the first rotary piston, and the five reagent communicating grooves are arranged on the outer circumferential surface of the first rotary piston at intervals along the circumferential direction of the first rotary piston.
More preferably, at least a portion of the reagent communication channel passes under at least one non-target reagent chamber with a curved segment located under the flow channel of the non-target reagent chamber to avoid communication with the flow channel.
Further, the plurality of reagent communication grooves are provided in parallel in the circumferential direction of the first rotary piston.
Preferably, the chamber further comprises a first amplification cavity for amplifying the extracted nucleic acid, the body is provided with a first liquid inlet channel communicated with the first amplification cavity, the first amplification cavity is located below the first rotary piston, and the first rotary piston is further provided with a nucleic acid transfer groove capable of communicating the liquid flow channel of the sample cavity with the first liquid inlet channel.
More preferably, the nucleic acid transfer groove is provided on the outer circumferential surface of the first rotary piston, and a central angle between a start end and a termination end of the nucleic acid transfer groove is greater than zero.
Preferably, the first rotary piston includes a first body and a first sealing layer covering the first body, and the reagent communication groove and the nucleic acid transfer groove are provided in the first sealing layer or in the first body and penetrate the first sealing layer.
More preferably, the first rotary piston has a drive end for engagement with a power source.
Preferably, the chamber further comprises a buffer chamber for receiving amplification products of the first amplification chamber, the buffer chamber being located below the first rotary piston.
More preferably, the body is further provided with a first liquid outlet channel communicated with the first amplification cavity, a second liquid inlet channel communicated with the buffer cavity, and a second liquid outlet channel, and the first rotary piston is further provided with a liquid transfer communicating groove capable of communicating the first liquid outlet channel with the second liquid inlet channel.
More preferably, the first amplification chamber is provided with amplification reagents therein.
More preferably, when the pipetting communication groove communicates the first liquid outlet channel and the second liquid inlet channel, the nucleic acid transfer groove communicates the liquid flow channel of the sample chamber and the first liquid inlet channel.
More preferably, 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 vertical microfluidic chip further comprises a positive and negative pressure pump for providing positive and negative pressure to the sample chamber, and the sample chamber and the positive and negative pressure pump are communicated with each other.
More preferably, the upper part of the body is provided with a port for connecting the positive pressure sample and the negative pressure sample, and the port is communicated with the sample cavity.
Preferably, the vertical microfluidic chip further comprises a mixing piston capable of moving or rotating to mix the materials in the sample cavity.
More preferably, the mixing piston is movably or rotatably disposed in the sample chamber, or the mixing piston is movably or rotatably disposed in the body and is in communication with the sample chamber.
Preferably, the sample chamber and the reagent chamber each have a hole provided at an upper end of the body.
The invention also adopts the following technical scheme:
a method for extracting nucleic acid, which adopts the vertical microfluidic chip, comprises the following steps:
A. adding a nucleic acid sample to the sample chamber;
B. and rotating the first rotary piston to enable the reagent communicating groove to communicate the reagent cavity with the sample cavity, so that the reagent is transferred into the sample cavity and is uniformly mixed for reaction.
Preferably, the method further comprises the steps of:
C. rotating the first rotary piston to enable the nucleic acid communication groove to communicate the sample cavity with the first amplification cavity, and transferring the nucleic acid extracting solution in the sample cavity into the first amplification cavity;
D. and rotating the first rotary piston to close the micro-channel between the sample cavity and the first amplification cavity, and performing a first round of amplification reaction.
Preferably, the method further comprises the following steps after step B: and rotating the first rotary piston to enable a waste liquid communicating groove on the first rotary piston to communicate the sample cavity with a waste liquid cavity arranged on the body, and transferring the waste liquid after reaction to the waste liquid cavity.
Preferably, the method further comprises the following steps after step D: rotating the first rotary piston to enable a liquid transfer communication groove on the first rotary piston to communicate the first amplification cavity with a buffer cavity arranged on the body, and transferring an amplification product into the buffer cavity;
in the step D, the first rotary piston closes the micro-channel between the first amplification chamber and the buffer chamber.
Compared with the prior art, the invention has the following advantages by adopting the scheme:
the micro-fluidic chip and the method for extracting nucleic acid can realize the extraction and purification of nucleic acid, adopt the vertical structure, effectively eliminate the influence of bubbles, ensure the accurate liquid inflow and realize the 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 microfluidic chip according to an embodiment of the present invention;
FIGS. 4a, 4b and 4c are schematic views of a first rotary piston according to an embodiment of the invention, respectively, from different perspectives;
FIG. 5 is a cross-sectional view of a first rotary piston along its length in accordance with an embodiment of the present invention;
FIGS. 6a and 6b are schematic views of a second rotary piston according to an embodiment of the present invention, respectively, from different perspectives;
fig. 7 is a cross-sectional view of a second rotary piston along its length in accordance with an embodiment of the present invention.
Wherein,
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. a first 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. a second 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 extraction includes a body 1 with a cavity, and the body 1 is vertically disposed in a plate shape. The vertical micro-fluidic chip also comprises a first rotary piston 2 and a second rotary piston 3 which are integrally and respectively in a cylindrical shape; the first rotary piston 2 is rotatably inserted into the body 1, and the second rotary piston 3 is located right below the first rotary piston 2 and rotatably inserted into the body 1. The first rotary piston 2 and the second rotary piston 3 are each rotatable about a horizontally extending axis of rotation, the axes of rotation of which are parallel to each other and lie in the same vertical plane, i.e. the axes of rotation 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 a first rotary piston 2 and a second rotary piston 3, the first rotary piston 2 and the part above the first rotary piston are nucleic acid extraction and purification areas, and nucleic acid samples are extracted and purified in the areas; the part between the first rotary piston 2 and the second rotary piston 3 is a first amplification area, and the extracted and purified pistons are subjected to first round of PCR amplification in the first amplification area; the part below the second 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 first 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 arranged in parallel between the left sample chamber 101 and the right waste liquid chamber 103, 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 first 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 first 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 first rotary piston 2 is able to selectively connect two of the flow channel 108 and the waste channel 109, while disconnecting the other channels; as the first rotary piston 2 rotates by a certain angle, the first rotary piston 2 communicates the other two of the liquid flow channel 108 and the waste liquid channel 109, while disconnecting the other channels. In particular, two of the chambers are connected by a connecting channel provided on the outer surface of the first rotary piston 2.
The layout of the communication grooves on the first rotary piston 2 is shown in fig. 4a to 4 c. As shown in fig. 4a to 4c, the first rotary piston 2 is provided with a reagent communicating groove 21 capable of communicating the liquid flow channel 108 of the sample chamber 101 with the liquid flow channel 108 of the reagent chamber 102, and the first rotary piston 2 is further provided with a waste liquid communicating groove 22 capable of communicating the liquid flow channel 108 of the sample chamber 101 with the waste liquid channel 109. One liquid flow channel 108 is provided for each reagent chamber 102, and thus a plurality of reagent communication grooves 21 are provided for each first rotary piston 2. The reagent communication grooves 21 and the waste liquid communication grooves 22 extend entirely in the axial direction of the first 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 first 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 connected with the liquid flow path 108 of the sample chamber 101 to communicate 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 first rotary piston 2, i.e., 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 end of each reagent communicating groove 21 and the terminating end of the waste communicating groove 22 are located at different circumferential positions of the first 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 reciprocating or rotating the mixing piston 11, 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 first rotary piston 2 and the second 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 first rotary piston 2, and the first 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 first 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 first rotary piston 2 rotates to a certain position, the starting end of the nucleic acid transfer groove 23 is aligned with the liquid flow channel 108 of the upper sample chamber 101, the terminating end is aligned with the first liquid inlet channel 110, 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 first rotary piston 2 and the second 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 first 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 first rotary piston 2 and is located entirely on the right side of the terminating 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 first 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 second rotary piston 3) comprises a plurality of second amplification chambers 107, and the second amplification chambers 107 are used for performing a second round of PCR amplification on the products of the first round of amplification. The plurality of second amplification chambers 107 are arranged side by side from left to right in the axial direction of the second 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 of the second liquid outlet channel 113 of the buffer chamber 105 and the liquid flow channel 108 of each second amplification chamber 107 are realized by a communication groove provided in the second rotary piston 3. Referring to FIGS. 6a and 6b, the second rotary piston 3 is provided with an amplification communication groove 31 capable of communicating the first liquid outlet channel 111 with the liquid 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 second rotary piston 3 can selectively connect the second liquid outlet channel 113 with the liquid flow channel 108 of one of the second amplification chambers 107 and disconnect the other liquid flow channels 108; as the second rotary piston 3 rotates a certain angle, the second 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 channel 108.
The layout of the amplification communication grooves 31 on the second 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 second 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 communication grooves 31 are arranged in parallel in the circumferential direction of the second 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 communication grooves 31 are arranged at intervals in the circumferential direction of the second rotary piston 3 and are located at the same circumferential position of the second rotary piston 3. The amplification communication grooves 31 are different in length from each other, and the terminating ends of any two adjacent amplification communication grooves 31 are spaced apart by a distance in both the axial direction and the circumferential direction of the second rotary piston 3.
Referring to fig. 6a to 7, the second 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 first rotary piston 2 and the second rotary piston 3 can be driven to rotate by a power source, which can be a motor. Referring to fig. 2, the first 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 second rotary piston 3 has a driving end portion for engagement with a power source, the driving end portion having a connection hole 303 which is a polygonal hole or a profiled hole into which an output shaft of the motor can be inserted and connected.
The present embodiment also provides a method for nucleic acid extraction, which uses 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 first 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 first rotary piston 2 to enable the waste liquid communicating groove 22 on the first 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 first 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 first rotary piston 2 to close the micro-channel between the sample cavity 101 and the first amplification cavity 104, and performing a first round of amplification reaction; specifically, the first rotary piston 2 closes the microchannel between the first amplification chamber 104 and the buffer chamber 105;
s6, rotating the first rotary piston 2 to make the liquid transfer connecting groove 24 on the first rotary piston communicate the first amplification chamber 104 with a buffer chamber 105 arranged on the body 1, and transferring the amplification product into the buffer chamber 105;
s7, the second rotary piston 3 is rotated to connect the amplification communication 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 second 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 second rotary piston 3, so that the second amplification communicating groove 31 communicates with the corresponding 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 second amplification chamber 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 first 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 the positive and negative pressure pumps to transfer the 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 first rotary piston 2 continues to rotate by 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 pump 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 first 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 first rotary piston 2 continues to rotate by 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 pump 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 first 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 the positive and negative pressure pumps 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 rinse nucleic acid for the first time.
7. The magnetic body 12 starts to generate a magnetic field to adsorb magnetic beads, the first rotary piston 2 continues to rotate by 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 pump 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 first 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 the positive and negative pressure pumps 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 first rotary piston 2 continues to rotate by 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 pump 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 first 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 uniform mixing piston 11 is driven to carry out uniform mixing reaction, and the extracted nucleic acid is eluted to form the nucleic acid extract.
Second, first round PCR amplification operation step
1. The first 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 first rotary piston 2 is rotated to close the micro-channel, and a first round of PCR amplification reaction is carried out.
3. The first 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 for mixing.
Third and second round PCR amplification operation steps
1. The second rotary piston 3 is rotated to connect the buffer chamber 105 to the first second amplification chamber 107 (chamber L1), and then the liquid is transferred into the chamber L1 by the fixed displacement piston 14.
2. The second rotary piston 3 is 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 14, 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 (12)
1. The utility model provides a vertical micro-fluidic chip of nucleic acid extraction usefulness, is including the body of seting up the cavity, its characterized in that, the cavity includes:
a sample chamber for holding a nucleic acid sample; and
a reagent chamber for storing a reagent required for nucleic acid extraction;
the body is also provided with a liquid flow channel which is respectively communicated with the sample cavity and the reagent cavity;
the vertical micro-fluidic chip further comprises:
the reagent chamber and the reagent chamber are positioned above the first rotary piston, and the first rotary piston is provided with a reagent communicating groove which can communicate the liquid flow channel of the sample chamber with the liquid flow channel of the reagent chamber.
2. The vertical microfluidic chip according to claim 1, wherein the sample chamber is provided with magnetic beads for adsorbing nucleic acid, and a magnetic body is disposed at a position of the body close to the sample chamber; and/or the vertical micro-fluidic chip further comprises a positive and negative pressure pump for providing positive pressure and negative pressure for the sample cavity, and the sample cavity and the positive and negative pressure pump are communicated with each other; and/or the vertical micro-fluidic chip also comprises a mixing piston which can move or rotate to mix the materials in the sample cavity uniformly; and/or the sample cavity and the reagent cavity are respectively provided with holes arranged at the upper end part of the body.
3. The vertical microfluidic chip according to claim 1, wherein the chamber further comprises a waste liquid chamber located above the first rotary piston, the body is provided with a waste liquid channel communicated with the waste liquid chamber, and the first rotary piston is further provided with a waste liquid communicating groove capable of communicating the liquid flow channel of the sample chamber with the waste liquid channel.
4. The vertical microfluidic chip according to claim 1, wherein the number of the reagent chambers is plural and is arranged at intervals along the axial direction of the first rotary piston, each of the reagent chambers is provided with one of the liquid flow channels, the first rotary piston is correspondingly provided with a plurality of the reagent communicating grooves, and the plurality of the reagent communicating grooves are arranged in parallel along the circumferential direction of the first rotary piston.
5. The vertical microfluidic chip according to claim 1, wherein the chamber further comprises a first amplification chamber for amplifying the extracted nucleic acid, an amplification reagent is disposed in the first amplification chamber, the body is provided with a first liquid inlet channel communicated with the first amplification chamber, the first amplification chamber is located below the first rotary piston, the first rotary piston is further provided with a nucleic acid transfer groove capable of communicating the liquid flow channel of the sample chamber with the first liquid inlet channel, the nucleic acid transfer groove is disposed on an outer circumferential surface of the first rotary piston, and a central angle between a start end and a stop end of the nucleic acid transfer groove is greater than zero.
6. The vertical microfluidic chip according to claim 5, wherein said chamber further comprises a buffer chamber for receiving amplification products of said first amplification chamber, said buffer chamber being located below said first rotary piston.
7. The vertical microfluidic chip according to claim 6, wherein the body further comprises a first liquid outlet channel communicated with the first amplification chamber, a second liquid inlet channel communicated with the buffer chamber, and a second liquid outlet channel, and the first rotary piston further comprises a liquid transfer communicating groove capable of communicating the first liquid outlet channel with the second liquid inlet channel.
8. The vertical microfluidic chip according to claim 7, wherein when the pipetting communication groove communicates the first liquid outlet channel and the second liquid inlet channel, the nucleic acid transfer groove communicates the liquid flow channel of the sample cavity and the first liquid inlet channel.
9. The vertical microfluidic chip according to claim 6, wherein the buffer chamber is connected to a quantitative chamber disposed in the body via an air flow channel, and the vertical microfluidic chip further comprises a quantitative piston slidably disposed in the quantitative chamber.
10. The vertical microfluidic chip according to claim 1, wherein the first rotary piston comprises a first body and a first sealing layer covering the first body, and the reagent communicating groove is disposed on the first sealing layer or on the first body and penetrates through the first sealing layer.
11. A method for nucleic acid extraction, using the vertical microfluidic chip according to any one of claims 1 to 10, comprising the steps of:
A. adding a nucleic acid sample to the sample chamber;
B. and rotating the first rotary piston to enable the reagent communicating groove to communicate the reagent cavity with the sample cavity, so that the reagent is transferred into the sample cavity and is uniformly mixed for reaction.
12. The method of claim 11, further comprising the steps of:
C. rotating the first rotary piston to enable the nucleic acid communicating groove to communicate the sample cavity with a first amplification cavity, and transferring the nucleic acid extracting solution in the sample cavity into the first amplification cavity;
D. rotating the first rotary piston to close the micro-channel between the sample cavity and the first amplification cavity, and performing a first round of amplification reaction;
the method further comprises the following steps after step D: rotating the first rotary piston to enable a liquid transfer communication groove on the first rotary piston to communicate the first amplification cavity with a buffer cavity arranged on the body, and transferring an amplification product into the buffer cavity;
in the step D, the first rotary piston closes the micro-channel between the first amplification chamber and the buffer chamber.
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