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CN113244970A - Microfluidic chip for nucleic acid extraction and PCR detection and application thereof - Google Patents

Microfluidic chip for nucleic acid extraction and PCR detection and application thereof Download PDF

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CN113244970A
CN113244970A CN202110408434.5A CN202110408434A CN113244970A CN 113244970 A CN113244970 A CN 113244970A CN 202110408434 A CN202110408434 A CN 202110408434A CN 113244970 A CN113244970 A CN 113244970A
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acid extraction
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CN113244970B (en
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杨宇
赖天然
齐润姿
周永运
聂聪
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Chinese Academy of Inspection and Quarantine CAIQ
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Abstract

本发明涉及一种用于提取核酸和PCR检测的微流控芯片及其应用。微流控芯片核酸提取反应仓、纯化反应仓和PCR反应仓，核酸提取反应仓和纯化反应仓的前端均连接有带有两分支进液仓，后端连通一个取液仓，PCR反应仓前端连通一个三分支进液仓，后端连通一个取液仓。核酸提取反应仓、纯化反应仓可制成一个芯片，PCR反应仓在另一芯片上，PCR反应仓可设有一个或多个。本发明提供的用于提取核酸和PCR检测的微流控芯片，有效的将提取核酸的步骤及纯化过程与PCR扩增反应有效结合起来，实现全密封式操作，避免二次污染，保证实验结果的可靠、真实、有效。

Figure 202110408434

The invention relates to a microfluidic chip for nucleic acid extraction and PCR detection and its application. Microfluidic chip nucleic acid extraction reaction chamber, purification reaction chamber and PCR reaction chamber, the front end of the nucleic acid extraction reaction chamber and the purification reaction chamber are connected with two branch liquid inlet chambers, the back end is connected with a liquid extraction chamber, and the front end of the PCR reaction chamber is connected It is connected with a three-branch liquid inlet silo, and the rear end is connected with a liquid taking silo. The nucleic acid extraction reaction chamber and the purification reaction chamber can be made into one chip, the PCR reaction chamber is on another chip, and there can be one or more PCR reaction chambers. The microfluidic chip for nucleic acid extraction and PCR detection provided by the present invention effectively combines the nucleic acid extraction steps and the purification process with the PCR amplification reaction, realizes a fully sealed operation, avoids secondary pollution, and ensures the experimental results reliable, authentic and effective.

Figure 202110408434

Description

Microfluidic chip for nucleic acid extraction and PCR detection and application thereof
Technical Field
The invention relates to a micro-fluidic chip for nucleic acid extraction and PCR detection and application thereof, belonging to the technical field of micro-fluidic chip analysis.
Background
The microfluidic chip technology (Microfluidics) integrates basic operation units of sample preparation, reaction, separation, detection and the like in the processes of biological, chemical and medical analysis on a micron-scale chip by taking a micro-pipeline network as a structural characteristic, and automatically completes the whole analysis process. Due to its great potential in the fields of biology, chemistry, medicine and the like, the method has been developed into a new research field crossing the disciplines of biology, chemistry, medicine, fluid, electronics, materials, machinery and the like. The existing micro-fluidic chip is mostly composed of a plurality of layers or an upper layer and a lower layer, and the chip is made of materials such as monocrystalline silicon, glass, quartz, high molecular polymers and the like.
The automatic sample changing of the conventional microfluidic chip adopts an array type multi-liquid-groove sample disc, introduces a sample into a sample inlet hole of a chip channel through a probe, and controls a sample introduction probe to be inserted into a specified sample liquid groove for sample introduction by adopting an X-Y-Z three-dimensional robot. The capillary is coupled with the chip, the capillary which is vertical to the chip and is communicated with the channel absorbs liquid and samples, and is combined with the array type sample disk, and the control also adopts a three-dimensional manipulator. The common characteristic is that the sample changing operation can be realized only by the probe or the sample plate moving in three dimensions, and the defects are that the three-dimensional control mechanism is complicated, the volume is large, the cost is high, the sample changing speed is slow, the analysis flux is low, the PCR amplification detection can not be completed by the integration of nucleic acid extraction and purification and PCR amplification, and can be completed only by separation, and the operation is inconvenient.
Disclosure of Invention
Technical problem to be solved
In order to solve the above problems in the prior art, the present invention provides a microfluidic chip for nucleic acid extraction and PCR detection, which realizes that nucleic acid extraction and purification and PCR amplification detection can be completed in the same chip.
(II) technical scheme
In order to achieve the purpose, the invention adopts the main technical scheme that:
a micro-fluidic chip for extracting nucleic acid and detecting PCR comprises a nucleic acid extraction reaction bin, a purification reaction bin and a PCR reaction bin, wherein the front ends of the nucleic acid extraction reaction bin and the purification reaction bin are respectively connected with a liquid inlet bin with two branches, the rear ends of the nucleic acid extraction reaction bin and the purification reaction bin are communicated with a liquid taking bin, the front end of the PCR reaction bin is communicated with a liquid inlet bin with three branches, and the rear end of the PCR reaction bin is communicated with a liquid taking bin.
In the microfluidic chip, preferably, the nucleic acid extraction reaction chamber and the purification reaction chamber are made into one chip, the PCR reaction chamber is arranged on another chip, and one or more PCR reaction chambers are provided.
In the microfluidic chip as described above, preferably, the volumes of the chambers of the nucleic acid extraction reaction chamber, the purification reaction chamber and the PCR reaction chamber are 5-10 μ L.
In the microfluidic chip, preferably, the nucleic acid extraction reaction chamber, the purification reaction chamber and the PCR reaction chamber are cylindrical chambers or square chambers.
A method for using a microfluidic chip for extracting nucleic acid and detecting PCR (polymerase chain reaction) comprises the steps that cells are cracked outside the chip to obtain cracked sample liquid, the cracked sample liquid enters a liquid inlet bin through a capillary, the cracked sample liquid in the liquid inlet bin is added into a nucleic acid extraction reaction bin with pre-embedded magnetic beads for magnetic bead adsorption, and after adsorption, the sample liquid is washed by a buffer or pure water under the condition that the magnetic beads are fixed by an external magnetic field; if the elution of nucleic acid is needed, adding an eluent, moving a washing liquid containing magnetic beads and nucleic acid into a liquid inlet bin communicated with a purification reaction bin through a capillary, and adding the eluent into the purification reaction bin through a liquid inlet bin of the other branch for elution; if the magnetic beads adsorbing the nucleic acid are directly conveyed to a PCR reaction bin without elution, the nucleic acid is conveyed to a liquid inlet bin of the PCR reaction bin through a liquid taking bin after the nucleic acid is eluted, the nucleic acid enters the PCR reaction bin, PCR reaction liquid and enzyme are added into the liquid inlet bins of the other two branches communicated with the PCR reaction bin, PCR reaction is carried out in the PCR reaction bin, and a PCR amplification product is moved out through the liquid taking bin of the PCR reaction bin after the reaction.
In the above method, preferably, the nucleic acid extraction reaction chamber further contains a nucleic acid adsorption solution under a high-salt and low-pH solution, and the eluent contained in the purification reaction chamber is a low-salt and high-pH solution to elute the nucleic acid into the solution.
Wherein the high-salt low-pH solution is 4-8 mol of guanidine hydrochloride solution, and the pH value is 5.0-5.5; the low salt high pH solution was 0.1mM Tris, pH 8.0.
Using the method described above, preferably, the aspiration and aspiration of all solutions is controlled by a MFCS type pressure-driven microfluidic control system.
(III) advantageous effects
The invention has the beneficial effects that:
the microfluidic chip for extracting nucleic acid and detecting PCR provided by the invention effectively combines the steps of extracting nucleic acid, the purification process and the PCR amplification reaction, realizes full-sealed operation, avoids secondary pollution, and ensures the reliability, the reality and the effectiveness of the experimental result.
Drawings
FIG. 1 is a schematic view of a preferred microfluidic chip provided in the present invention;
fig. 2 is a schematic diagram of another preferred operation process of the microfluidic chip provided by the present invention.
[ description of reference ]
1: a nucleic acid extraction reaction bin;
2: a purification reaction bin;
3: and (3) a PCR reaction bin.
Detailed Description
For the purpose of better explaining the present invention and to facilitate understanding, the present invention will be described in detail by way of specific embodiments with reference to the accompanying drawings.
Example 1
The utility model provides a micro-fluidic chip for extracting nucleic acid and PCR detect, including nucleic acid extraction reaction storehouse, purification reaction storehouse and PCR reaction storehouse, these three reaction storehouse can design on same chip, as shown in figure 1, nucleic acid extraction reaction storehouse 1 and purification reaction storehouse 2 all connect the feed liquor storehouse that has two branches in its front end, it is buffer effect when being used for solution to get into to enter the liquid storehouse with capillary connection, the rear end communicates a liquid taking storehouse, it is connected with capillary and draws out the solution in the reaction storehouse through the imbibition pump, PCR reaction storehouse front end communicates a three branch and gets the liquid storehouse, three branch's feed liquor storehouses can get into three kinds of reaction solutions respectively, PCR reaction storehouse rear end communicates a liquid taking storehouse. The nucleic acid extraction reaction bin 1 is a reaction liquid for adding magnetic bead method to extract nucleic acid, namely a reaction chamber which is used for adsorbing nucleic acid under the environment of high salt and low pH value after the surface of magnetic bead is treated by silicon, the purification reaction bin 2 is a reaction chamber which is used for eluting nucleic acid under the environment of low salt and high pH value, and the PCR reaction bin is a reaction chamber for carrying out PCR amplification. The material of the chip is high temperature resistant material, because the PCR reaction has a thermal cycle process, 35-40 cycles are possible, and the temperature gradient of each cycle is between 50-95 ℃. The PCR reaction chip also has a requirement on light transmittance because a fluorescent signal of the PCR reaction is collected. PMMA or other high polymer materials (e.g., PDMS, epoxy or PU resins) may be used.
The volume of the chambers of the nucleic acid extraction reaction chamber, the purification reaction chamber and the PCR reaction chamber is set to be 5 muL or 10 muL, the shape of the reaction chamber and the field of view for collecting the fluorescence signal have certain requirements, the specific position of the reaction chamber is designed according to the corresponding light path during design, the shape of the reaction chamber can be set to be a square chamber, and the size of the reaction chamber enables the volume of the reaction chamber to be 10 muL.
Example 2
The microfluidic chip of the embodiment is arranged on two chips for extracting nucleic acid and PCR detection, wherein one chip is provided with a nucleic acid extraction reaction bin and a purification reaction bin, and the other chip is provided with one or more PCR reaction bins. Other settings are required by the embodiment.
The reaction flow of the microfluidic chip of this example is specifically as follows: firstly, cells are cracked outside a chip to obtain cracked sample liquid, a sample injection pump is used for controlling the cracked sample liquid to enter a liquid inlet bin communicated with a nucleic acid extraction reaction bin through a capillary under the control of an MFCS type pressure-driven microfluidic control system, pre-embedded magnetic beads are added into the nucleic acid extraction reaction bin in advance, the cracked sample liquid in the liquid inlet bin is added into the nucleic acid extraction reaction bin with the pre-embedded magnetic beads for magnetic bead adsorption, after adsorption, a buffer or pure water is added into the nucleic acid extraction reaction bin through another liquid inlet bin, and the magnetic beads are washed under the condition that the magnetic beads are fixed by an external magnetic field. If the elution of nucleic acid and the elution of the nucleic acid are needed, the washing liquid containing the magnetic beads and the nucleic acid is moved into a liquid inlet bin communicated with a purification reaction bin through a capillary, and the elution is added into the purification reaction bin through a liquid inlet bin of the other branch for elution. If elution is not needed, the magnetic beads adsorbing the nucleic acid are directly conveyed to a PCR reaction bin. After nucleic acid is eluted, the nucleic acid is conveyed to a liquid inlet bin of a PCR reaction bin through a liquid taking bin and enters the PCR reaction bin, PCR reaction liquid such as PCR Buffer solution and enzyme is respectively added into the liquid inlet bins of the other two branches communicated with the PCR reaction bin, PCR amplification reaction is carried out in the PCR reaction bin, and after the reaction, a PCR amplification product is moved out through the liquid taking bin of the PCR reaction bin.
Before adding the cracked sample liquid into the nucleic acid extraction reaction bin, adding a high-salt low-pH solution (4-8 mol guanidine hydrochloride solution) in advance, wherein the pH value is 5.0-5.5, the nucleic acid is adsorbed on magnetic beads, adding an eluent into the purification reaction bin in advance, wherein the eluent is a low-salt high-pH solution and can adopt 0.1mM Tris, the pH value is 8.0, the eluent is used for eluting the nucleic acid, and the nucleic acid can enter the solution.
The above description is only a preferred embodiment of the present invention, and is not intended to limit the present invention in other forms, and any person skilled in the art can change or modify the technical content disclosed above into an equivalent embodiment with equivalent changes. However, any simple modification, equivalent change and modification of the above embodiments according to the technical essence of the present invention are within the protection scope of the technical solution of the present invention.

Claims (7)

1.一种用于提取核酸和PCR检测的微流控芯片，其特征在于，其包括核酸提取反应仓、纯化反应仓和PCR反应仓，核酸提取反应仓和纯化反应仓的前端均连接有带有两分支进液仓，后端连通一个取液仓，PCR反应仓前端连通一个三分支进液仓，后端连通一个取液仓。1. a microfluidic chip for extracting nucleic acid and PCR detection, is characterized in that, it comprises nucleic acid extraction reaction chamber, purification reaction chamber and PCR reaction chamber, and the front ends of nucleic acid extraction reaction chamber and purification reaction chamber are all connected with belts. There are two branch liquid inlet chambers, the rear end is connected with a liquid taking chamber, the front end of the PCR reaction chamber is connected with a three-branch liquid inlet chamber, and the rear end is connected with a liquid taking chamber. 2.如权利要求1所述的微流控芯片，其特征在于，所述核酸提取反应仓、纯化反应仓制成一个芯片，PCR反应仓在另一芯片上，PCR反应仓设有一个或多个。2. The microfluidic chip according to claim 1, wherein the nucleic acid extraction reaction chamber and the purification reaction chamber are made into one chip, the PCR reaction chamber is on another chip, and the PCR reaction chamber is provided with one or more indivual. 3.如权利要求1所述的微流控芯片，其特征在于，所述核酸提取反应仓、纯化反应仓、PCR反应仓的仓室体积大小为5～10μL。3 . The microfluidic chip according to claim 1 , wherein the volume of the nucleic acid extraction reaction chamber, the purification reaction chamber and the PCR reaction chamber is 5-10 μL. 4 . 4.如权利要求1所述的微流控芯片，其特征在于，所述核酸提取反应仓、纯化反应仓、PCR反应仓的形状为圆柱形仓室或方形仓室。4 . The microfluidic chip according to claim 1 , wherein the nucleic acid extraction reaction chamber, the purification reaction chamber and the PCR reaction chamber are in the shape of a cylindrical chamber or a square chamber. 5 . 5.如权利要求1-4中任一项所述微流控芯片的使用方法，其特征在于，其包括细胞在芯片外裂解获得裂解后的样本液体，通过毛细管进入进液仓中，进液仓中裂解后的样本液体加入已经预埋好磁珠的核酸提取反应仓内，进行磁珠吸附，吸附后用buffer或者纯水在外加磁场固定磁珠情况下冲洗；如需要洗脱核酸加洗脱液，将含磁珠-核酸的冲洗液通过毛细管移入连通纯化反应仓的进液仓中，纯化反应仓通过另一分支的进液仓加入洗脱液进行洗脱；如果不需洗脱就直接将吸附核酸的磁珠运送到PCR反应仓，洗脱核酸后将核酸通过取液仓运送到PCR反应仓的进液仓，进入PCR反应仓，在连通PCR反应仓的另两个分支的进液仓中加入PCR反应液及酶，在PCR反应仓进行PCR反应，反应后通过PCR反应仓的取液仓移出PCR扩增产物。5. The use method of the microfluidic chip according to any one of claims 1-4, characterized in that, it comprises that cells are lysed outside the chip to obtain a lysed sample liquid, enter the liquid inlet chamber through a capillary, and enter the liquid The lysed sample liquid in the chamber is added to the nucleic acid extraction reaction chamber with pre-embedded magnetic beads for magnetic bead adsorption. After adsorption, use buffer or pure water to fix the magnetic beads with an external magnetic field; if necessary, elute nucleic acid and add washing Dehydration, move the rinse solution containing magnetic beads-nucleic acid into the inlet chamber connected to the purification reaction chamber through the capillary, and the purification reaction chamber is eluted by adding eluent to the inlet chamber of another branch; if no elution is required, The magnetic beads for adsorbing nucleic acid are directly transported to the PCR reaction chamber, and after the nucleic acid is eluted, the nucleic acid is transported to the liquid inlet chamber of the PCR reaction chamber through the liquid extraction chamber, and then enters the PCR reaction chamber. The PCR reaction solution and enzyme are added to the liquid chamber, and the PCR reaction is carried out in the PCR reaction chamber. After the reaction, the PCR amplification product is removed through the liquid extraction chamber of the PCR reaction chamber. 6.如权利要求5所述的使用方法，其特征在于，所述核酸提取反应仓内还含有高盐低pH溶液下核酸吸附液，纯化反应仓内含有的洗脱液为低盐高pH溶液，将核酸洗脱下来，进入溶液中。6. The method of use according to claim 5, wherein the nucleic acid extraction reaction chamber also contains nucleic acid adsorption solution under a high-salt low-pH solution, and the eluent contained in the purification reaction chamber is a low-salt high-pH solution , the nucleic acid is eluted into solution. 7.如权利要求5所述的使用方法，其特征在于，所有溶液的吸入与吸出由MFCS型压力驱动型微流体控制系统控制。7. The method of claim 5, wherein the suction and suction of all solutions are controlled by an MFCS-type pressure-driven microfluidic control system.
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Cited By (3)

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CN115445677A (en) * 2022-08-23 2022-12-09 深圳市第二人民医院(深圳市转化医学研究院) A microfluidic chip combined with MALDI-TOF-MS analysis
CN115873691A (en) * 2023-02-24 2023-03-31 北京凡知医学科技有限公司 Micro-fluidic chip and nucleic acid extraction and purification method and device
TWI797820B (en) * 2021-11-08 2023-04-01 財團法人工業技術研究院 Pcr rapid detection device and method thereof

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