CN111019804B - Nucleic acid amplification device, system and method - Google Patents
Nucleic acid amplification device, system and method Download PDFInfo
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- CN111019804B CN111019804B CN201911303885.1A CN201911303885A CN111019804B CN 111019804 B CN111019804 B CN 111019804B CN 201911303885 A CN201911303885 A CN 201911303885A CN 111019804 B CN111019804 B CN 111019804B
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- 108020004707 nucleic acids Proteins 0.000 title claims abstract description 71
- 150000007523 nucleic acids Chemical class 0.000 title claims abstract description 71
- 102000039446 nucleic acids Human genes 0.000 title claims abstract description 71
- 238000003199 nucleic acid amplification method Methods 0.000 title claims abstract description 36
- 230000003321 amplification Effects 0.000 title claims abstract description 32
- 238000000034 method Methods 0.000 title claims abstract description 23
- 239000012295 chemical reaction liquid Substances 0.000 claims abstract description 37
- 238000004720 dielectrophoresis Methods 0.000 claims abstract description 29
- 238000003940 alternating current dielectrophoresis Methods 0.000 claims description 6
- 238000012546 transfer Methods 0.000 claims description 2
- 238000006243 chemical reaction Methods 0.000 abstract description 20
- 239000012634 fragment Substances 0.000 abstract description 12
- 230000008569 process Effects 0.000 abstract description 9
- 230000000630 rising effect Effects 0.000 abstract description 4
- 238000010438 heat treatment Methods 0.000 description 23
- 239000005662 Paraffin oil Substances 0.000 description 19
- 108020004414 DNA Proteins 0.000 description 14
- 239000012530 fluid Substances 0.000 description 8
- 238000007599 discharging Methods 0.000 description 6
- 239000007788 liquid Substances 0.000 description 6
- 238000003752 polymerase chain reaction Methods 0.000 description 6
- 239000000047 product Substances 0.000 description 6
- 239000000376 reactant Substances 0.000 description 6
- 238000001816 cooling Methods 0.000 description 4
- 230000000694 effects Effects 0.000 description 4
- 238000002474 experimental method Methods 0.000 description 4
- 102100031181 Glyceraldehyde-3-phosphate dehydrogenase Human genes 0.000 description 3
- 108091028043 Nucleic acid sequence Proteins 0.000 description 3
- 108010006785 Taq Polymerase Proteins 0.000 description 3
- 239000008367 deionised water Substances 0.000 description 3
- 229910021641 deionized water Inorganic materials 0.000 description 3
- 108020004445 glyceraldehyde-3-phosphate dehydrogenase Proteins 0.000 description 3
- 238000011027 product recovery Methods 0.000 description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- 230000004544 DNA amplification Effects 0.000 description 2
- 239000006227 byproduct Substances 0.000 description 2
- 238000004891 communication Methods 0.000 description 2
- 238000006073 displacement reaction Methods 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 description 1
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 description 1
- 108700039887 Essential Genes Proteins 0.000 description 1
- 101100013786 Mus musculus Gapdh gene Proteins 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 230000005684 electric field Effects 0.000 description 1
- 230000017525 heat dissipation Effects 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 230000002277 temperature effect Effects 0.000 description 1
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
- 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
- B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
- B01L3/502707—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by the manufacture of the container or its components
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
- B01L2200/12—Specific details about manufacturing devices
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- Dispersion Chemistry (AREA)
- Analytical Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Hematology (AREA)
- Clinical Laboratory Science (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Apparatus Associated With Microorganisms And Enzymes (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention provides a nucleic acid amplification device, a system and a method, which relate to the technical field of nucleic acid amplification and comprise the following steps: the chip body is provided with at least two temperature areas; at least one main channel is formed in the chip body, the main channel comprises at least two channel sections, at least one channel section corresponds to each temperature zone, and the channel sections positioned in adjacent temperature zones are positioned on different layers of the chip body; dielectrophoresis electrodes, and the dielectrophoresis electrodes are correspondingly arranged at the juncture positions of the adjacent channel sections. In the technical scheme, in the process of shifting nucleic acid from one temperature zone to another temperature zone, the flow mode of the reaction liquid in each temperature zone is kept unchanged, only the nucleic acid shifts, and the reaction liquid containing the nucleic acid is not operated to shift in different temperature zones, so that the temperature rising and falling rate of the nucleic acid can be obviously improved, and the overall time consumption can be obviously shortened. In the process, the temperature control is directly aimed at the target DNA fragment, so that the temperature control efficiency can be greatly improved, and the reaction rate of nucleic acid amplification is improved.
Description
Technical Field
The invention relates to the technical field of nucleic acid amplification, in particular to a nucleic acid amplification device, a system and a method.
Background
For nucleic acid amplification, the Polymerase Chain Reaction (PCR) is the most commonly used nucleic acid amplification method, which takes a target DNA sequence as a template, and uses a specific primer designed for the target DNA sequence, and the number of target DNA sequences is exponentially increased after a plurality of cycles of high-temperature denaturation-low Wen Fuxing-temperature rising extension under the action of heat-resistant DNA polymerase, so that the purpose of rapidly amplifying target genes is realized. However, the reaction rate of nucleic acid amplification in the prior art is low, and thus, by-product generation is easily caused and the amplification specificity is lowered or failed.
Disclosure of Invention
The invention aims to provide a nucleic acid amplification device, a system and a method, which are used for solving the technical problem of low nucleic acid amplification reaction rate in the prior art.
The invention provides a microfluidic chip, comprising:
the chip body is provided with at least two temperature areas;
at least one main channel is formed in the chip body, the main channel comprises at least two channel sections, at least one channel section corresponds to each temperature zone, and the channel sections positioned in adjacent temperature zones are positioned on different layers of the chip body;
and the dielectrophoresis electrode is arranged corresponding to the juncture position of the adjacent channel section.
Further, the microfluidic chip further includes:
at least one temperature control channel, at least one said temperature control channel communicating with at least one said main channel.
Further, at least one buffer cavity is arranged in the temperature control channel.
Further, the microfluidic chip further includes:
at least one heater and at least one temperature sensor, the temperature sensor in data connection with the heater;
the heater is arranged corresponding to the buffer cavity, and the temperature sensor is arranged corresponding to the channel section.
Further, part of channel ends of adjacent channel sections positioned at different layers are overlapped and communicated along the layer direction to form the junction position.
Further, the microfluidic chip further includes:
at least one micro valve disposed at an inlet and/or an outlet of the main channel.
Further, the number of the main channels is a plurality, and the main channels are parallel to each other.
Further, the temperature zone comprises a first temperature zone, a second temperature zone and a third temperature zone, and the main channel comprises a first channel section, a second channel section and a third channel section;
the first channel section is located in the first temperature zone, the second channel section is located in the second temperature zone, and the third channel section is located in the third temperature zone.
The invention also provides a nucleic acid amplification device, which comprises at least one reciprocating power source and the microfluidic chip;
the reciprocating power source is arranged at the inlet and/or the outlet of the main channel.
The invention also provides a nucleic acid amplification method based on the microfluidic chip or the nucleic acid amplification device, which comprises the following steps:
and driving the nucleic acid contained in the reaction liquid to transfer to the adjacent channel section at the junction position by using dielectrophoresis force generated by the dielectrophoresis electrode, so that the nucleic acid in the reaction liquid is transferred in the adjacent temperature region.
In the technical scheme, in the process of shifting nucleic acid from one temperature zone to another temperature zone, the flow mode of the reaction liquid in each temperature zone is kept unchanged, only the nucleic acid shifts, and the reaction liquid containing the nucleic acid is not operated to shift in different temperature zones, so that the temperature rising and falling rate of the nucleic acid can be obviously improved, and the overall time consumption can be obviously shortened. Because the temperature control in the process is directly aimed at the target DNA fragment, the temperature control efficiency can be greatly improved, and the reaction rate of nucleic acid amplification is improved. Moreover, the simple and easy reciprocating flow control is used for replacing a complicated circulating temperature control program, and the cost is reduced.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are needed in the description of the embodiments or the prior art will be briefly described, and it is obvious that the drawings in the description below are some embodiments of the present invention, and other drawings can be obtained according to the drawings without inventive effort for a person skilled in the art.
Fig. 1 is a schematic diagram of a channel structure of a chip body according to an embodiment of the present invention;
FIG. 2 is an exploded view of a channel segment of a main channel according to one embodiment of the present invention;
FIG. 3 is a perspective view of a main channel according to one embodiment of the present invention;
FIG. 4 is a plan view of a primary channel provided by one embodiment of the present invention;
FIG. 5 is a perspective view of an interface location according to one embodiment of the present invention;
FIG. 6 is a plan view of an interface location provided by one embodiment of the present invention;
fig. 7 is an assembly view of a dielectrophoresis electrode according to an embodiment of the present invention.
Reference numerals:
1. a chip body; 2. a warm zone;
3. a main channel; 4. dielectrophoresis electrodes;
5. a temperature control channel; 6. a buffer chamber;
21. a first temperature zone; 22. a second temperature zone;
23. a third temperature zone;
31. a channel section; 32. a junction position;
33. a first channel segment; 34. a second channel segment;
35. and a third channel segment.
Detailed Description
The following description of the embodiments of the present invention will be made apparent and fully in view of the accompanying drawings, in which some, but not all embodiments of the invention are shown. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
In the description of the present invention, it should be noted that the directions or positional relationships indicated by the terms "center", "upper", "lower", "left", "right", "vertical", "horizontal", "inner", "outer", etc. are based on the directions or positional relationships shown in the drawings, are merely for convenience of describing the present invention and simplifying the description, and do not indicate or imply that the devices or elements referred to must have a specific orientation, be configured and operated in a specific orientation, and thus should not be construed as limiting the present invention. Furthermore, the terms "first," "second," and "third" are used for descriptive purposes only and are not to be construed as indicating or implying relative importance.
In the description of the present invention, it should be noted that, unless explicitly specified and limited otherwise, the terms "mounted," "connected," and "connected" are to be construed broadly, and may be either fixedly connected, detachably connected, or integrally connected, for example; can be mechanically or electrically connected; can be directly connected or indirectly connected through an intermediate medium, and can be communication between two elements. The specific meaning of the above terms in the present invention will be understood in specific cases by those of ordinary skill in the art.
Analysis and research on the principle of the PCR technology show that the core of the PCR technology is temperature control, namely, continuous, rapid and accurate control on the temperature of liquid in a reaction system is realized. Theoretically, the temperature control of the PCR process is directed to the DNA fragment of interest, i.e. the temperature at which the nucleic acid is manipulated. However, in the conventional PCR method, the temperature of the reaction solution in which the target DNA fragment is located is controlled by using a heating/heat dissipation module, so that the temperature of the target DNA fragment is indirectly changed. The volume of the reaction solution is much larger than that of the target DNA fragment (compared with more than 10 5 Multiple times) such a manner of indirectly controlling the temperature of the DNA fragment by controlling the overall temperature of the reaction solution is inefficient and time-consuming.
For example, in the current method for controlling the overall temperature of the reaction solution, the heating rate is generally 3-5 ℃/s, the cooling rate is 2-3 ℃/s, and the time wasted in the heating (55-72 ℃,72-94 ℃) and cooling (94-55 ℃) processes is equivalent to 1/3 of the single cycle time, so that the reaction rate is seriously affected, the generation of byproducts is easily caused, and the amplification specificity is reduced or failed.
Therefore, the application provides a temperature control method capable of directly controlling the temperature of nucleic acid, thereby fundamentally improving the reaction rate of nucleic acid amplification.
As shown in fig. 1 to 7, a microfluidic chip provided in this embodiment includes:
a chip body 1, wherein the chip body 1 is provided with at least two temperature areas 2;
at least one main channel 3 is formed in the chip body 1, the main channel 3 comprises at least two channel sections 31, at least one channel section 31 corresponds to each temperature zone 2, and the channel sections 31 positioned in adjacent temperature zones 2 are positioned on different layers of the chip body 1;
dielectrophoresis electrodes 4, and the dielectrophoresis electrodes 4 are arranged corresponding to the boundary positions 32 of the adjacent channel segments 31.
The laminar flow is a fluid flow state corresponding to turbulent flow, and refers to a laminar flow of fluid. The fluid streamlines in laminar flow can be parallel to the tube wall. Laminar flow conditions occur when the reynolds number (which is proportional to the flow rate, density, and tube diameter of the fluid, and inversely proportional to the viscosity) is less than 3000. In laminar flow, when several different colored fluids enter the same microchannel from different inlets, even if they are mutually dissolved, a distinct multiphase parallel flow is formed. While the micro-channels generally exhibit laminar flow conditions due to the limitations in channel size and fluid flow rate.
The micro-fluidic chip controls the temperature by utilizing the laminar flow effect of fluid in the micro-channel, and referring to fig. 1 and 2, the channel of the micro-fluidic chip adopts a layered and segmented structure, namely, a main channel 3 is arranged in the micro-fluidic chip, the main channel 3 is formed by a plurality of channel sections 31, and the main channel 3 is arranged to enable each temperature zone 2 to correspond to at least one channel section 31.
Therefore, the reaction liquid containing nucleic acid is poured into the main channel 3, and the reaction liquid can be driven by a reciprocating power source, so that the reaction liquid respectively flows back and forth in a laminar flow manner in each channel section 31 of the main channel 3, and the reaction liquid temperatures in different temperature areas 2 can be ensured not to have serious interference. The dielectrophoresis electrodes 4 for nucleic acid manipulation are arranged at the junctions of the adjacent temperature regions 2, namely at the junction positions 32 of the adjacent channel sections 31, and the dielectrophoresis electric field formed by the dielectrophoresis electrodes 4 can enable the nucleic acid subjected to dielectrophoresis effect at the junctions to be displaced from one temperature region 2 to the other temperature region 2, so that rapid and efficient temperature switching of the target DNA fragments is realized, and only the temperature of the target DNA fragments themselves is changed.
It should be noted that, in the process of shifting the nucleic acid from one temperature zone 2 to another temperature zone 2, the flow pattern of the reactant flow in each temperature zone 2 is kept unchanged, only the nucleic acid shifts, and the reactant flow containing the nucleic acid is not operated to shift in different temperature zones 2, so that the temperature rising and falling rate of the nucleic acid can be remarkably improved, and the overall time consumption can be remarkably shortened. In the process, the reaction liquid can be controlled to flow reciprocally and continuously by utilizing the external reciprocating power source, so that the rapid and efficient temperature control of the target DNA fragment is realized, and the amplification of the nucleic acid is finished. Because the temperature control in the process is directly aimed at the target DNA fragment, the temperature control efficiency can be greatly improved, and the reaction rate of nucleic acid amplification is improved. Moreover, the simple and easy reciprocating flow control is used for replacing a complicated circulating temperature control program, and the cost is reduced.
Wherein dielectrophoresis formed by the dielectrophoresis electrodes 4 is double-frequency alternating-current dielectrophoresis. When the nucleic acid is controlled, as the volume of the nucleic acid (especially the DNA fragment) is very small and the dielectric constant difference of the nucleic acid is small, the two factors have very high requirements on the dielectrophoresis mode, so that the dielectrophoresis electrode 4 is adopted to form double-frequency alternating current dielectrophoresis, the sensitivity of the nucleic acid control can be improved, and the displacement of the nucleic acid is ensured to be more accurate. In addition, the reciprocating power source refers to a mechanism for providing reciprocating power for liquid in the chip, such as a reciprocating electromagnetic pump, a reciprocating plunger pump, a reciprocating piezoelectric pump, a reciprocating diaphragm pump and the like. After the liquid enters the channel, the reciprocating power source can provide pressure (pushing force) and suction (pulling force) which are periodically changed according to the requirement, so that the reciprocating power source periodically advances and retreats in the channel, and the reciprocating motion is realized. While the number of the warm regions 2 and the number of the main channels 3 may be set according to the need, for example, the warm regions 2 may be set to three, and the channel sections 31 of the main channels 3 are set to three, that is, one channel section 31 corresponds to one warm region 2.
In one embodiment, the warm zone 2 comprises a first warm zone 21, a second warm zone 22 and a third warm zone 23, and the main channel 3 comprises a first channel section 33, a second channel section 34 and a third channel section 35; the first channel section 33 is located in the first temperature zone 21, the second channel section 34 is located in the second temperature zone 22, and the third channel section 35 is located in the third temperature zone 23. The temperatures in the first temperature zone 21, the second temperature zone 22 and the third temperature zone 23 are respectively set as a low temperature zone 2, a middle temperature zone 2 and a high temperature zone 2, and the specific temperature ranges of the low temperature zone 2, the middle temperature zone 2 and the high temperature zone 2 can be set according to requirements, so that the reciprocating displacement of the nucleic acid in the reaction liquid in the low temperature zone 2, the middle temperature zone 2 and the high temperature zone 2 can be controlled, and the temperature of the nucleic acid can be accurately and rapidly controlled.
In the case of controlling the temperature of each of the temperature zones 2, a heating element, for example, a heating electrode, may be embedded in the position corresponding to the temperature zone 2, and the temperature of the reaction solution and nucleic acid in the main channel 3 may be controlled by heating the periphery of the main channel 3 with the heating element. The specific type and number of heating elements can be set by those skilled in the art according to the need and will not be described in detail herein.
In one embodiment, the microfluidic chip further comprises: at least one temperature control channel 5, at least one of said temperature control channels 5 being in communication with at least one of said main channels 3. With continued reference to fig. 1, the channel sections 31 in the different temperature areas 2 are all connected with a temperature control channel 5 for controlling the reaction liquid in the main channel 3 to keep constant temperature. In cooperation with this, at least one buffer chamber 6 can also be provided in the temperature-controlled channel 5. In order to construct temperature zones 2 in the microfluidic chip independent of each other, at least one heater and at least one temperature sensor may also be provided within the microfluidic chip.
The heater (e.g., heating electrode) may be disposed corresponding to the buffer chamber 6, or may be disposed corresponding to other parts of the temperature control channel, so as to heat the reaction liquid in the buffer chamber 6 or directly heat the reaction liquid at other parts of the temperature control channel. The temperature sensors may be provided corresponding to the respective channel segments 31 to monitor the temperature of each channel segment 31 in real time. The temperature sensor and the heater can be installed in the micro-fluidic chip in a pre-buried mode, or an external heat source (such as a heating plate) can be used for carrying out contact heating on a temperature zone, and the temperature sensor is in data connection with the heater or the matched external heat source. Any heating means can be adopted by those skilled in the art according to the requirements, and the method is not limited herein.
For example, the heating electrode is pre-buried at the bottom of the buffer cavity 6 to heat the reaction liquid flowing through the buffer cavity 6, and at this time, the buffer cavity 6 is communicated with the main channel 3 through the temperature control channel. The temperature sensor monitors the temperature in the main channel 3 in real time, when the temperature in the main channel 3 does not meet the requirement, the temperature of the reaction liquid in the buffer cavity 6 can be changed by adjusting the heating power of the heating electrode, the temperature in the temperature control channel is indirectly changed, and finally the temperature in the main channel 3 is adjusted.
The purpose of setting up buffer chamber 6 is in order to increase the stability of reaction solution temperature in the control by temperature change passageway, because this amplification method has used a plurality of independent constant temperature district 2, and the circulation cooling down in independent cavity, reduce each temperature district 2 temperature fluctuation and be the key of accuse temperature, the buffer chamber 6 of big volume, big inertia can play obvious accuse temperature effect this moment. Of course, those skilled in the art may choose to directly heat other portions of the temperature control channel 5, or heat the temperature control channel 5 in other manners, which are not limited herein.
As shown in fig. 3 to 6, the partial channel ends of the adjacent channel segments 31 at different levels overlap and communicate in the layer direction, constituting the junction 32. In this structure, the junction 32 of the adjacent channel segments 31 is formed by stacking the ends of the adjacent channel segments 31, and at this time, the channel structures of the junction 32 are mutually consecutive with the channel interiors of the adjacent channel segments 31, and an obvious stacked structure is formed in the layer direction, and the obvious stacked structure can enable the fluid to maintain a stable laminar flow effect in the micro-channel, so that the mutual interference between the reaction liquid temperatures of different temperature areas 2 is avoided.
Wherein, the microfluidic chip further comprises: at least one micro valve arranged at the inlet and/or outlet of the main channel 3. The micro valve can be used for controlling the opening or closing of the inlet or outlet of the main channel 3, thereby being matched with the smooth progress of nucleic acid amplification and being convenient for the subsequent recovery of products.
With continued reference to fig. 1, the number of the main channels 3 is plural, and plural main channels 3 are parallel to each other. At this time, since the inlet and the outlet of each main channel 3 can be independent from each other, multiplexing of different samples can be achieved. The number of the specific main channels 3 can be set according to the needs, and will not be described herein. Preferably, the temperature control channel 5 and the main channel 3 form an included angle of 120 degrees.
When the microfluidic chip is used for nucleic acid amplification, sample loading and preheating can be performed firstly, namely, a reciprocating power source is started firstly, 5 microliter of paraffin oil is added into each main channel 3 or the designated main channel 3, 5N microliter of paraffin oil (N is the number of parallel main channels 3) is added into the temperature control channel 5, and redundant air is discharged. Then 20 microliter of reaction solution is added to each main channel 3, 20N (N is the number of parallel main channels 3) microliter of reaction solution is added to the temperature control channel 5, and redundant air is discharged. Then 5 microliter of paraffin oil was added to each channel, 5N (N is the number of parallel main channels 3) microliter of paraffin oil was added to the temperature-controlled channel 5, and the excess air was removed. And closing the inlet and the outlet of the microfluidic chip, and controlling the reaction liquid at the inlet position. At this time, heating is started until the temperature of each temperature zone 2 is stable, and then the nucleic acid is controlled to stay in the high temperature zone 2 for preheating for a required time.
Paraffin oil is added to the main channel 3 and placed at both ends of the reaction liquid, and its main function is to prevent a large amount of bubbles from occurring in the reaction liquid, volatilizing and causing liquid loss. At the same time, when a plurality of different samples are simultaneously amplified in the main channel 3, different reaction liquids can be separated to avoid mutual interference between adjacent reaction liquids.
Then amplifying nucleic acid, namely connecting dielectrophoresis electrode 4, controlling nucleic acid to stay in high temperature region 2, low temperature region 2 and middle temperature region 2 for corresponding time, repeating operation until reaching the cycle number required by amplification.
And finally, recovering the product, namely controlling the nucleic acid to a low-temperature region 2, stopping cooling for required time, closing a heating electrode, and opening an outlet of the microfluidic chip after the reactant is cooled to room temperature, and recovering the product or entering a rear-end integrated channel for subsequent experiments.
The invention also provides a nucleic acid amplification device, which comprises at least one reciprocating power source and the microfluidic chip; the reciprocating power source is arranged at the inlet and/or outlet of the main channel 3. Since the specific structure, functional principle and technical effect of the microfluidic chip are described in detail above, the detailed description is omitted here. Any technical content related to the microfluidic chip can be referred to in the foregoing description.
The invention also provides a nucleic acid amplification method based on the microfluidic chip or the nucleic acid amplification device, which comprises the following steps: the dielectrophoresis force generated by the dielectrophoresis electrode 4 is used for driving the nucleic acid contained in the reaction solution to be transferred to the adjacent channel section 31 at the boundary position 32, so that the nucleic acid in the reaction solution is transferred in the adjacent temperature region 2. In order to clearly describe the technical scheme of the nucleic acid amplification method, the following specific examples will be described.
Example 1 Single target Single drop mouse housekeeping Gene GAPDH amplification
The chip configuration parameters used in this embodiment are as follows: the inlet, main channel 3 and outlet are of total sub-total structure, the inlet and outlet dimensions are 500 μm x 2mm (width x height x length), the main channel 3 dimensions are 100 μm x 500 μm x 6mm (width x height x length), the number of parallel main channels 3 is 4; the dimensions of the boundary position 32 are 100 μm×500 μm×200 μm (width×height×length); the 20. Mu.l reaction liquid system used included: 0.1. Mu. l Taq DNA Polymerase (5U/. Mu.l), 1.6. Mu.l GAPDH primer mix (10. Mu.M each), 2. Mu.l template DNA (0.2. Mu.g), 1.6. Mu.l dNTP (2.5 mM each), 2. Mu.l 10 XPCR Buffer (with Mg2+), deionized water; dielectrophoresis uses dual-frequency alternating-current dielectrophoresis with voltage amplitudes of 18V and 24V, respectively.
The method for extracting and purifying nucleic acid based on the system comprises the following steps:
sample addition and preheating: firstly, starting a reciprocating power source, adding 5 microliters of paraffin oil into each main channel 3, adding 20 microliters of paraffin oil into a temperature control channel 5, and discharging redundant air; then 20 microliter of reaction solution is added into each main channel 3, 80 microliter of reaction solution is added into the temperature control channel 5, and redundant air is discharged; then 5 microliter of paraffin oil is added into each channel, 20 microliter of paraffin oil is added into the temperature-controlled channel 5, and redundant air is discharged; closing the inlet and the outlet of the chip, and controlling the reaction liquid at the inlet position; heating is started until the temperature of each temperature zone 2 is stable, and then the nucleic acid is controlled to stay in the high temperature zone 2 for 5min.
Amplification: and the dielectrophoresis electrode 4 is communicated, the nucleic acid is controlled to stay in the high temperature region 2, the low temperature region 2 and the middle temperature region 2 for corresponding time, and the operation is repeated for 30 times.
And (3) product recovery: controlling the nucleic acid to a low temperature region 2, and closing the heating electrode after staying for 2 min; and opening the chip outlet after the reactant is cooled to room temperature, and recovering the product or entering a back-end integrated channel for subsequent experiments.
Example 2 Single target Multi-drop mouse GAPDH Gene amplification
The chip configuration parameters used in this embodiment are as follows: the inlet, the main channel 3 and the outlet are of a total sub-total structure, the size of the inlet and the outlet is 500 mu m multiplied by 2mm (width multiplied by height multiplied by length), the size of the main channel 3 is 100 mu m multiplied by 500 mu m multiplied by 6mm (width multiplied by height multiplied by length), the number of the parallel main channels 3 is 3, and the number of liquid drops in a single channel is 3; the dimensions of the boundary position 32 are 100 μm×500 μm×200 μm (width×height×length); the 10. Mu.l reaction liquid system used included: 0.05. Mu. l Taq DNA Polymerase (5U/. Mu.l), 0.8. Mu.l GAPDH primer mix (10. Mu.M each), 1. Mu.l template DNA (0.2. Mu.g), 0.8. Mu.l dNTP (2.5 mM each), 1. Mu.l 10 XPCR Buffer (with Mg2+), deionized water; dielectrophoresis uses dual-frequency alternating-current dielectrophoresis with voltage amplitudes of 18V and 24V, respectively.
The method for extracting and purifying nucleic acid based on the system comprises the following steps:
sample addition and preheating: firstly, starting a reciprocating power source, adding 2.5 microliter of paraffin oil into each main channel 3, adding 7.5 microliter of paraffin oil into a temperature control channel 5, and discharging redundant air; adding 10 microliters of reaction liquid into each main channel 3, adding 30 microliters of reaction liquid into the temperature control channel 5, and discharging redundant air; then adding 2.5 microliter of paraffin oil into each channel, adding 7.5 microliter of paraffin oil into the temperature-controlled channel 5, and discharging redundant air; repeating the operation until 3 reaction liquid drops are clamped in paraffin oil, closing the inlet and the outlet of the chip, and controlling the reaction liquid at the inlet position; heating is started until the temperature of each temperature zone 2 is stable, and then the nucleic acid is controlled to stay in the high temperature zone 2 for 5min.
Amplification: and the dielectrophoresis electrode 4 is communicated, all the nucleic acids are controlled to stay in the high temperature region 2, the low temperature region 2 and the middle temperature region 2 for corresponding time, and the operation is repeated for 33 times.
And (3) product recovery: controlling all the nucleic acids to a low temperature area 2, and closing a heating electrode after staying for 2 min; and opening the chip outlet after the reactant is cooled to room temperature, and recovering the product or entering a back-end integrated channel for subsequent experiments.
Example 3 Multi-target Multi-drop mouse Gene amplification
Parameters of the present embodiment: the chip configuration parameters used in this embodiment are as follows: the inlet, the main channel 3 and the outlet are of a multi-channel parallel structure, the sizes of the inlet and the outlet are 90 mu m multiplied by 500 mu m multiplied by 2mm (width multiplied by height multiplied by length), the sizes of the main channel 3 are 100 mu m multiplied by 500 mu m multiplied by 6mm (width multiplied by height multiplied by length), the number of the parallel main channels 3 is 4, and the number of liquid drops in a single channel is 4; the dimensions of the boundary position 32 are 100 μm×500 μm×200 μm (width×height×length); the 5. Mu.l reaction liquid system used included: 0.025. Mu. l Taq DNA Polymerase (5U/. Mu.l), 0.4. Mu.l primer mix (10. Mu.M each), 0.5. Mu.l template DNA (0.2. Mu.g), 0.4. Mu.l dNTP (2.5 mM each), 0.5. Mu.l 10 XPCR Buffer (with Mg2+), deionized water; dielectrophoresis uses dual-frequency alternating-current dielectrophoresis with voltage amplitudes of 18V and 24V, respectively.
The method for extracting and purifying nucleic acid based on the system comprises the following steps:
sample addition and preheating: firstly, starting a reciprocating power source, adding 2.5 microliter of paraffin oil into each main channel 3, adding 10 microliter of paraffin oil into a temperature control channel 5, and discharging redundant air; then 5 microliter of reaction liquid is added into each main channel 3, 20 microliter of reaction liquid is added into the temperature control channel 5, and redundant air is discharged; then adding 2.5 microliter of paraffin oil into each channel, adding 10 microliter of paraffin oil into the temperature-controlled channel 5, and discharging redundant air; repeating the operation until 4 reaction liquid drops are clamped in paraffin oil, closing the inlet and the outlet of the chip, and controlling the reaction liquid at the inlet position; heating is started until the temperature of each temperature zone 2 is stable, and then the nucleic acid is controlled to stay in the high temperature zone 2 for 5min.
Amplification: and the dielectrophoresis electrode 4 is communicated, all the nucleic acids are controlled to stay in the high temperature region 2, the low temperature region 2 and the middle temperature region 2 for corresponding time, and the operation is repeated for 30 times.
And (3) product recovery: controlling all the nucleic acids to a low temperature area 2, and closing a heating electrode after staying for 2 min; and opening the chip outlet after the reactant is cooled to room temperature, and recovering the product or entering a back-end integrated channel for subsequent experiments.
Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention, and not for limiting the same; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some or all of the technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit of the invention.
Claims (7)
1. A microfluidic chip, comprising:
the chip body is provided with at least two temperature areas;
at least one main channel is formed in the chip body, the main channel comprises at least two channel sections, at least one channel section corresponds to each temperature zone, and the channel sections positioned in adjacent temperature zones are positioned on different layers of the chip body; the end parts of partial channels of the adjacent channel sections positioned at different layers are overlapped and communicated along the layer direction to form a junction position;
the dielectrophoresis electrode is double-frequency alternating current dielectrophoresis and is arranged corresponding to the junction position of the adjacent channel sections;
at least one temperature control channel, at least one of which communicates with at least one of the main channels; at least one buffer cavity is arranged in the temperature control channel;
and the at least one heater is arranged corresponding to the buffer cavity.
2. The microfluidic chip according to claim 1, wherein the microfluidic chip further comprises:
at least one temperature sensor in data connection with the heater; the temperature sensor is arranged corresponding to the channel section.
3. The microfluidic chip according to claim 1, wherein the microfluidic chip further comprises:
at least one micro valve disposed at an inlet and/or an outlet of the main channel.
4. A microfluidic chip according to any one of claims 1-3, wherein the number of said main channels is plural, and a plurality of said main channels are parallel to each other.
5. The microfluidic chip according to claim 4, wherein the temperature zone comprises a first temperature zone, a second temperature zone, and a third temperature zone, and the main channel comprises a first channel section, a second channel section, and a third channel section;
the first channel section is located in the first temperature zone, the second channel section is located in the second temperature zone, and the third channel section is located in the third temperature zone.
6. A nucleic acid amplification device comprising at least one reciprocating power source and a microfluidic chip according to any one of claims 1-5;
the reciprocating power source is arranged at the inlet and/or the outlet of the main channel.
7. A method of nucleic acid amplification based on the microfluidic chip of any one of claims 1-5 or based on the nucleic acid amplification device of claim 6, comprising the steps of:
and driving the nucleic acid contained in the reaction liquid to transfer to the adjacent channel section at the junction position by using dielectrophoresis force generated by the dielectrophoresis electrode, so that the nucleic acid in the reaction liquid is transferred in the adjacent temperature region.
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| AU2002307152A1 (en) * | 2001-04-06 | 2002-10-21 | California Institute Of Technology | Nucleic acid amplification utilizing microfluidic devices |
| KR101840530B1 (en) * | 2016-01-08 | 2018-05-04 | 고려대학교 산학협력단 | Surface measurement sensing-based realtime nucleic acid amplification measuring apparatus |
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| CN101169403A (en) * | 2006-10-25 | 2008-04-30 | 中国科学院大连化学物理研究所 | A microfluidic chip and its preparation and application |
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