CN110819698B - High-pressure liquid immersion type digital PCR method, digital PCR chip and preparation method thereof - Google Patents
High-pressure liquid immersion type digital PCR method, digital PCR chip and preparation method thereof Download PDFInfo
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- CN110819698B CN110819698B CN201911030850.5A CN201911030850A CN110819698B CN 110819698 B CN110819698 B CN 110819698B CN 201911030850 A CN201911030850 A CN 201911030850A CN 110819698 B CN110819698 B CN 110819698B
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- B01L7/52—Heating or cooling apparatus; Heat insulating devices with provision for submitting samples to a predetermined sequence of different temperatures, e.g. for treating nucleic acid samples
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Abstract
The invention discloses a high-pressure liquid immersion type digital PCR method, a digital PCR chip and a preparation method thereof, wherein the digital PCR method comprises the following steps: s1, immersing a digital PCR chip subjected to sample injection treatment into liquid in a high-pressure reaction chamber; s2, exhausting air in the high-pressure reaction chamber, and pressurizing the high-pressure reaction chamber after air is exhausted; s3, placing the pressurized high-pressure reaction chamber on a PCR instrument for PCR reaction; s4, cooling the high-pressure reaction chamber after the PCR reaction; s5, reducing the pressure of the cooled high-pressure reaction chamber; s6, taking out the digital PCR chip in the high-pressure reaction chamber, and carrying out fluorescence signal analysis on the digital PCR chip. The chip sample introduction does not depend on complex equipment such as a pump, a valve and the like, high-viscosity thermal polymerization separation oil is not required, the chip does not need to be sealed after the sample introduction is finished, and the operation is simple; the chip has small thickness, fast heat conduction and fast reaction; the chip has the advantages of simple structure, easy manufacture, low cost and high automation degree.
Description
Technical Field
The invention relates to the technical field of nucleic acid detection, in particular to a high-pressure liquid immersion type digital PCR method, a digital PCR chip and a preparation method thereof.
Background
Polymerase Chain Reaction (PCR) is a molecular biology technique for amplifying specific DNA fragments, which can be regarded as specific DNA replication in vitro. The method is widely applied to the molecular biology fields such as gene detection, gene amplification, gene engineering and the like, and plays an irreplaceable role in the aspects of clinical medicine, forensic medicine, paternity testing, environmental detection and the like. However, the PCR reaction is amplified by exponential order, and can amplify millions of times in tens of minutes, and it is difficult to determine the content of the original PCR template through the PCR product. In order to accurately quantitatively analyze the content of nucleic acid, a digital PCR (dPCR) technology is invented.
The basic principle of digital PCR (dPCR) is to distribute a PCR sample equally to a number of different micro-reaction units, each containing a different number of template molecules, in each of which an independent PCR amplification is performed. After the amplification is completed, the micro-reaction unit containing the template molecule is marked as positive because of the fluorescence signal, and the micro-reaction unit not containing the template molecule is marked as negative because of no fluorescence signal. The number of templates in different micro-reaction units conforms to the poisson distribution, so that the concentration of the PCR template in the initial sample can be accurately obtained according to a formula by counting the number of the positive and negative micro-reaction units. The number of reaction units, the volume accuracy, the uniformity of the micro reaction units and the reaction quality of the micro reaction units determine the quality of the entire dPCR. The existing dPCR system mainly comprises a valve type dPCR chip, a water-in-oil micro-drop type dPCR chip and an open type array dPCR chip. These chips are costly and complex to operate. Some of the chips also have the problems of slow thermal reaction, poor volume accuracy, poor uniformity and the like.
Polydimethylsiloxane (PDMS) has the characteristics of transparency, good biocompatibility, low value, easy manufacturing and the like, and is widely applied to dPCR. PDMS is a high molecular polymer that can permeate gas and store a certain amount of air, so it is easy to realize end filling of liquid micro-reaction units on a PDMS chip. However, the gas storage and permeability of PDMS poses a serious problem in that the PDMS chip is susceptible to bubbles when heated. In addition, as the temperature increases, the micro reaction units may be contaminated with each other during the PCR process, and the water in the PCR solution may be volatilized and lost, thereby affecting the PCR reaction. And, the smaller the liquid micro-reaction unit cavity is, the more obvious this phenomenon is. Currently, glass, parylene, oil-containing PDMS prepolymers, and the like are used to prevent bubble formation and reduce water evaporation. However, the adoption of the method causes great operation difficulty, high cost and long reaction time of the dPCR chip.
Disclosure of Invention
Through the mechanism research of heating the PDMS chip to generate bubbles, the invention provides a high-pressure liquid immersion type digital PCR method, a digital PCR chip and a preparation method thereof, and aims to solve the problems of high operation difficulty, high cost and long reaction time of the digital PCR method in the prior art.
In order to solve the technical problems, the invention adopts the following technical scheme:
the first aspect of the present invention provides a high pressure liquid immersion type digital PCR method, comprising the steps of:
s1, immersing a digital PCR chip subjected to sample injection treatment into liquid in a high-pressure reaction chamber;
s2, exhausting air in the high-pressure reaction chamber, and pressurizing the high-pressure reaction chamber after the air is exhausted;
s3, placing the pressurized high-pressure reaction chamber on a PCR instrument for PCR reaction;
s4, cooling the high-pressure reaction chamber after the PCR reaction;
s5, reducing the pressure of the cooled high-pressure reaction chamber;
and S6, taking out the digital PCR chip in the high-pressure reaction chamber, and carrying out fluorescence signal analysis on the digital PCR chip.
Preferably, the sample injection treatment in the step S1 includes the following steps:
s11, combining a sample injector with a digital PCR chip, so that a sample inlet and a sample outlet of a reaction layer of the digital PCR chip are aligned with a sample inlet pool and a sample outlet of the sample injector respectively and are tightly attached to the sample inlet pool and the sample outlet;
s12, adding a PCR reaction solution into a sample inlet pool of the sample injector, and pumping air outwards at a sample outlet of the sample injector by using an injector to enable the PCR reaction solution to flow into a main runner of a reaction layer of the digital PCR chip through a sample inlet of the reaction layer of the digital PCR chip until part of the PCR reaction solution flows out of the sample outlet of the sample injector through the sample outlet of the reaction layer of the digital PCR chip;
s13, using an injector to exhaust air from an air exhaust port of a negative pressure cavity of the sample injector outwards until the PCR reaction liquid is filled in each branch flow channel communicated with the main flow channel and each PCR micro-reaction unit communicated with each branch flow channel;
s14, sucking off redundant PCR reaction liquid at a sample injection pool of the sample injector, and dripping isolation oil;
and S15, exhausting air at a sample outlet of the sample injector by using an injector to enable the main runner of the reaction layer to be filled with the isolation oil, so as to complete isolation of each PCR micro-reaction unit.
Preferably, the high pressure reaction chamber is evacuated in step S2 by injecting a neutral solution.
Preferably, the pressure of the high-pressure reaction chamber is 210-500KPa.
A second aspect of the present invention provides a digital PCR chip for performing the above-described high-pressure liquid immersion digital PCR method.
Preferably, the digital PCR chip comprises a substrate layer, a blank layer and a reaction layer which are arranged in sequence,
wherein the reaction layer has a main flow channel, branch flow channels communicated with the main flow channel, and PCR micro-reaction units communicated with the branch flow channels; the reaction layer still has introduction port and appearance mouth, the introduction port on reaction layer is linked together with the introduction sample cell of injector, the appearance mouth of reaction layer is linked together with the appearance mouth of injector.
Preferably, the substrate layer is made of a support material for supporting the blank layer and the reaction layer.
Preferably, the blank layer and the reaction layer are made of polydimethylsiloxane.
The third aspect of the present invention provides a method for preparing the above digital PCR chip, which comprises the steps of:
1) Cleaning a silicon wafer, and manufacturing a reaction layer die on the silicon wafer;
2) Uniformly mixing polydimethylsiloxane and a curing agent, degassing, pouring onto the reaction layer mold, and performing thermocuring to form a polydimethylsiloxane membrane with a main flow channel, branch flow channels and PCR micro-reaction units; taking the polydimethylsiloxane membrane off the reaction layer die and punching to form a sample inlet and a sample outlet, so as to prepare a reaction layer with the sample inlet, the sample outlet, a main flow channel, branch flow channels and PCR micro-reaction units;
3) Uniformly mixing polydimethylsiloxane and a curing agent, and spin-coating the mixture on a silicon wafer, and curing to form a blank layer;
4) Carrying out plasma activation treatment on the reaction layer obtained in the step 2) and the blank layer obtained in the step 3) and then bonding the reaction layer and the blank layer together;
5) And (3) bonding the two polydimethylsiloxane bonding structures obtained in the step (4) and the substrate layer together after plasma activation treatment, thus obtaining the digital PCR chip.
Preferably, the bonding in step 4) comprises baking at 80 ℃ for 1-2h after bonding to achieve permanent bonding.
The invention has the following beneficial effects:
1. the sample introduction of the chip does not depend on complex equipment such as a pump, a valve and the like, high-viscosity thermal polymerization separation oil is not needed, the chip does not need to be sealed after the sample introduction is finished, and the operation is simple;
2. the sample injector is used for assisting in rapid sample injection, and the negative pressure sample injection mode is used for reducing gas residue in the chip, so that the sample injection is rapid;
3. the chip completes the reaction in water environment, the water loss in the PCR micro-reaction unit can be ignored, the volume of the micro-reaction unit can be made smaller, more micro-reaction units can be made on the chip in unit area, and the overall flux of the chip is higher;
4. each micro-reaction unit of the chip has accurate volume and uniform array, and is easy for imaging analysis;
5. the high-pressure reaction chamber is easily compatible with the existing PCR instrument and is also easily integrated on a metal temperature control module of the PCR instrument;
6. the chip has small thickness, fast heat conduction and fast reaction;
7. the chip has simple structure, easy manufacture, low cost and high automation degree;
8. reduce water evaporation and is compatible with other dPCR chips.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the embodiments or the prior art descriptions will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.
FIG. 1 is a schematic diagram of the bubble formation mechanism and water evaporation mechanism when PDMS chip is heated;
FIG. 2 is a schematic cross-sectional view of a digital PCR chip and an injector according to an embodiment of the present invention;
FIG. 3 is a schematic diagram of an exemplary injector;
FIG. 4 is a schematic diagram of a reaction layer of a digital PCR chip according to an embodiment of the present invention;
FIG. 5 is an enlarged view of A in FIG. 4;
FIG. 6 is a schematic diagram of a main channel of a PCR reaction solution entering a reaction layer of a digital PCR chip according to an embodiment of the present invention;
FIG. 7 is a schematic diagram of PCR reaction solutions entering each branch flow channel and each PCR micro-reaction unit of the reaction layer of the digital PCR chip according to the embodiment of the present invention;
FIG. 8 is a schematic diagram of the isolation of each PCR micro-reaction unit by the isolation oil in the embodiment of the present invention;
in the figure: the kit comprises 1-PDMS chip, 2-PCR solution, 3-air bubble, 4-micro-droplet, 5-substrate layer, 6-blank layer, 7-reaction layer, 71-sample inlet of reaction layer, 72-sample outlet of reaction layer, 73-main flow channel, 74-branch flow channel, 75-micro-reaction unit, 8-sample cell, 9-sample outlet of sample injector, 10-negative pressure cavity and 11-air extraction port.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention will be described in further detail with reference to the accompanying drawings in conjunction with the following detailed description. It should be understood that the description is intended to be exemplary only, and is not intended to limit the scope of the present invention.
The embodiment is as follows:
the PDMS chip has a serious problem in that bubbles are easily generated when the PDMS chip is heated due to gas storage and permeability. The inventor of the invention has found out the bubble formation mechanism and the water evaporation principle when the PDMS chip is heated for a long time. FIG. 1 is a schematic diagram of the bubble formation mechanism and water evaporation principle of PDMS chip. As can be seen from fig. 1, when the PDMS chip 1 is exposed to air, the degree of evaporation of water in the PCR solution 2 is significantly affected by temperature. For the PDMS chip 1, in the temperature rising process, water is evaporated to form water vapor which enters the micropores of the PDMS layer of the PDMS chip 1, and in addition, the self thermal expansion effect of air causes the obvious increase of the volume and pressure of the gas in the micropores, and most of the gas can overflow out of the PDMS layer. However, since the flow resistance of the micro-pores is large, part of the gas cannot be released into the air through the PDMS layer, the gas will form small bubbles 3 in the micro-reaction cell against the liquid pressure, and water molecules in the liquid around the bubbles 3 are more easily evaporated into the bubbles 3, so that the volume of the bubbles 3 is rapidly increased. The bubbles 3 are expanded to the adjacent PCR micro-reaction units to cause the PCR solution in the adjacent reaction units to be gasified, and the linkage reaction causes the gasification to occur in the micro-reaction units of the whole micro-channel and further to be expanded to the whole chip. In the cooling process, water vapor in the micropores is condensed to form micro liquid drops 4, gas molecules are reduced to form negative pressure, the air outside the PDMS layer is sucked into the micropores, and in each temperature cycle of the PCR reaction, the PDMS layer continuously sucks air like a pump to form bubbles and discharges water to cause dehydration of the PCR solution, and each micro reaction unit is subjected to cross contamination to further cause failure of the PCR reaction.
Currently, glass, parylene, oil-containing PDMS prepolymers, and the like are commonly used to prevent bubble formation and reduce water evaporation. However, the adoption of the method causes great operation difficulty, high cost and long reaction time of the dPCR chip.
In view of the above problems, an embodiment of the present invention provides a high-pressure liquid immersion digital PCR method, which includes the following steps:
s1, immersing the digital PCR chip subjected to sample introduction treatment into liquid in a high-pressure reaction chamber.
In the embodiment of the present invention, as shown in fig. 2 and fig. 3, when the sample injection process is performed on the digital PCR chip, a sample injector needs to be used for sample injection. The sample injector comprises a sample injection pool 8, a sample outlet 9 and a negative pressure cavity 10, wherein an extraction opening 11 is arranged on the negative pressure cavity 10.
As shown in fig. 2 and fig. 4, the digital PCR chip comprises a substrate layer 5, a blank layer 6 and a reaction layer 7, which are sequentially arranged from bottom to top, wherein the reaction layer 7 is provided with a sample inlet 71 and a sample outlet 72, the sample inlet 71 of the reaction layer is communicated with the sample cell 8 of the sample injector, and the sample outlet 72 of the reaction layer is communicated with the sample outlet 9 of the sample injector.
As shown in fig. 2, 4 and 5, the reaction layer 7 includes a main channel 73, branch channels 74 communicating with the main channel 73, and PCR micro-reaction units 75 communicating with the branch channels 74.
Specifically, as shown in fig. 2 to 5, the sample injection treatment may include the following steps:
s11, combining the sample injector and the digital PCR chip, so that a sample inlet 71 and a sample outlet 72 of a reaction layer of the digital PCR chip are respectively aligned with a sample inlet pool 8 and a sample outlet 9 of the sample injector and are tightly attached to each other.
S12, adding the PCR reaction liquid into the sample inlet pool 8 of the sample injector, and pumping air outwards at the sample outlet 9 of the sample injector by using an injector to enable the PCR reaction liquid to flow into the main runner 73 of the reaction layer of the digital PCR chip through the sample inlet 71 of the reaction layer of the digital PCR chip until part of the PCR reaction liquid flows out of the sample outlet 9 of the sample injector through the sample outlet 72 of the reaction layer of the digital PCR chip.
The PCR reaction solution may include an upstream primer, a downstream primer, a fluorescent-labeled DNA probe, a DNA template, a dNTP mixture, DNA polymerase, and the like. The types, concentrations and sequence order of the components added into the PCR reaction solution can be set according to the requirement of the PCR reaction.
In a specific embodiment, the step S11 may include: and (3) dripping the PCR reaction liquid into the sample pool 8 of the sample injector by using a pipette, exhausting air outwards from the sample outlet 9 of the sample injector through a hose connector by using a syringe, guiding the PCR reaction liquid to flow into a main runner 73 of the reaction layer of the digital PCR chip through a sample inlet 71 of the reaction layer of the digital PCR chip, and as shown in FIG. 6, until part of the PCR reaction liquid flows out from the sample outlet 9 of the sample injector through a sample outlet 72 of the reaction layer of the digital PCR chip.
And S13, using an injector to suck air outwards from the air suction port 11 of the negative pressure cavity 10 of the sample injector until the PCR reaction solution is filled in each branch flow channel 74 communicated with the main flow channel 73 and each PCR micro-reaction unit 75 communicated with each branch flow channel 74.
In the embodiment of the present invention, the negative pressure chamber 10 of the sample injector completely covers the main flow channel 73 of the reaction layer, the branch flow channels 74 communicated with the main flow channel 73, and the PCR micro-reaction units 75 communicated with the branch flow channels 74.
In the embodiment of the present invention, the PCR reaction solution is firstly dropped into the sample cell 8 of the sample injector, then the sample outlet 9 of the sample injector is pumped out, and the pumping port 11 of the negative pressure cavity 10 of the sample injector is pumped out, so that the reaction layer 7 on the digital PCR chip can rapidly generate negative pressure in each PCR micro-reaction unit 75, and the PCR reaction solution is guided to fill each branch flow channel 74 communicated with the main flow channel 73 and enter each PCR micro-reaction unit 75, as shown in fig. 7. And gas residue in the digital PCR chip can be reduced by adopting a negative pressure sample injection mode.
S14, sucking off the redundant PCR reaction liquid at the sample injection pool 8 of the sample injector, and dripping isolation oil.
Wherein the barrier oil may be FC-40.
S15, using an injector to extract air at the sample outlet 9 of the sample injector, so that the main flow channel 73 of the reaction layer is filled with the isolation oil, and the isolation of each PCR micro-reaction unit 75 is completed, as shown in FIG. 8.
In the embodiment of the invention, the pressure of the high-pressure reaction chamber is 210-500KPa. The liquid of the high pressure reaction chamber may be a neutral solution, such as an aqueous solution.
And S2, exhausting the air in the high-pressure reaction chamber, and pressurizing the high-pressure reaction chamber after the air is exhausted.
Specifically, in the step S2, the high-pressure reaction chamber is evacuated by injecting a neutral solution. Further, after the air in the high pressure reaction chamber is exhausted, the pressure of the high pressure reaction chamber needs to be increased, and step S3 is performed again.
And S3, placing the pressurized high-pressure reaction chamber on a PCR instrument for PCR reaction.
Specifically, the reaction parameters of the PCR reaction in step S3 may be set according to actual needs.
And S4, cooling the high-pressure reaction chamber after the PCR reaction.
And S5, reducing the pressure of the cooled high-pressure reaction chamber.
And S6, taking out the digital PCR chip in the high-pressure reaction chamber, and carrying out fluorescence signal analysis on the digital PCR chip.
Specifically, the step S6 may be: and opening the high-pressure reaction chamber, taking out the digital PCR chip in the high-pressure reaction chamber, and carrying out fluorescence signal analysis on the digital PCR chip.
The embodiment of the invention adopts the high-pressure liquid immersion type digital PCR method, so that the digital PCR chip is immersed in the liquid of the high-pressure reaction chamber during the PCR reaction, air does not exist around the digital PCR chip, the air in the PDMS micropores cannot overflow the PDMS due to the high pressure outside the digital PCR chip under the high temperature condition, and no more air enters the PDMS under the low temperature condition to damage the air balance inside, thereby preventing the breathing behavior of the PDMS like a pump, further preventing the generation of bubbles in each PCR micro-reaction unit, and enabling the PCR reaction liquid to complete the thermal cycle reaction under the stable environment. In addition, water vapor can enter the PDMS from the outer surface of the digital PCR chip, so that the water loss of the PCR reaction solution is reduced.
The embodiment of the invention also provides a digital PCR chip for executing the high-pressure liquid immersion type digital PCR method. As shown in fig. 2, the digital PCR chip includes a substrate layer 5, a blank layer 6, and a reaction layer 7.
Preferably, the reaction layer 7 is made of Polydimethylsiloxane (PDMS). The reaction layer 7 is provided above the blank layer 6. In some embodiments, the reactive layer 7 and the blank layer 6 may be bonded together by irreversible plasma bonding.
As shown in fig. 4 and 5, the reaction layer 7 includes a main channel 73, branch channels 74 communicating with the main channel 73, and PCR micro-reaction units 75 communicating with the branch channels 74.
As shown in fig. 2, the reaction layer 7 further has a sample inlet 71 and a sample outlet 72, the sample inlet 71 of the reaction layer is communicated with the sample cell 8 of the sample injector, and the sample outlet 72 of the reaction layer is communicated with the sample outlet 9 of the sample injector.
Preferably, the blank layer 6 is made of Polydimethylsiloxane (PDMS). The blank layer 6 is disposed above the base layer 5. In some embodiments, the blank layer 6 may be spin-coated on a silicon wafer.
Preferably, the substrate layer 5 is made of a supporting material such as glass. The substrate layer 5 is located below the blank layer 6 and the reaction layer 7, and is used for supporting the blank layer 6 and the reaction layer 7.
Accordingly, an embodiment of the present invention further provides a method for preparing the digital PCR chip shown in fig. 2 and 5, where the method includes the following steps:
1) And cleaning a silicon wafer, and manufacturing a reaction layer die on the silicon wafer.
In a specific embodiment, the step 1) may include: cleaning the silicon wafer by using a Phiranha solution, washing the silicon wafer by using deionized water, drying the silicon wafer by using nitrogen, and baking the silicon wafer for 20 to 30min on a hot plate at the temperature of between 170 and 180 ℃; performing plasma treatment for 1-2min, then spin-coating SU83005 (10 μm), and performing photolithography and development to prepare branch flow channels 74 of the reaction layer, which are communicated with the main flow channel; baking on a hot plate at 160-170 deg.C for 20-30min, performing plasma treatment for 1-2min, spin-coating SU83025 (25 μm), performing photolithography, and developing to obtain a main flow channel 73 of the reaction layer and PCR micro-reaction units 75 communicated with branch flow channels 74, thereby forming a reaction layer mold.
2) Uniformly mixing polydimethylsiloxane and a curing agent, degassing, pouring the mixture onto the reaction layer mold, and performing thermocuring to form a polydimethylsiloxane membrane with a main runner 73, branch runners 74 and PCR micro-reaction units 75; and taking the polydimethylsiloxane membrane from the reaction layer die, perforating to form a sample inlet 71 and a sample outlet 72, and manufacturing the reaction layer 7 with the sample inlet 71, the sample outlet 72, the main flow channel 73, the branch flow channels 74 and the PCR micro-reaction units 75.
In a specific embodiment, the step 2) may include: adopting Dow Corning Sylgard 184PDM, uniformly mixing a prepolymer and a curing agent according to the mass ratio of 5 to 1, removing air bubbles in the mixture by using a vacuum degassing method, then pouring a prepolymer on a reaction layer mould with a graphic structure, baking the prepolymer on a hot plate at 60-80 ℃ for 20-30min to form a polydimethylsiloxane membrane with a main flow channel 73, branch flow channels 74 and PCR micro-reaction units 75, removing the polydimethylsiloxane membrane from the reaction layer mould, cutting and punching to form a sample inlet 71 and a sample outlet 72, and thus preparing the reaction layer 7 with the sample inlet 71, the sample outlet 72, the main flow channel 73, the branch flow channels 74 and the PCR micro-reaction units 75.
3) And uniformly mixing polydimethylsiloxane and a curing agent, spin-coating on a silicon wafer, and curing to form a blank layer 6.
In a specific embodiment, the step 3) may include: spin coating Sylgard 184PDMS prepolymer on silicon chip under 3000-5000rpm, baking on hot plate at 60-80 deg.C for 10-20min to form blank layer 6.
4) The reaction layer 7 obtained in step 2) and the blank layer 6 obtained in step 3) are bonded together after plasma activation treatment.
In a specific embodiment, the step 4) may include:
the surfaces of the reaction layer 7 obtained in the step 2) and the blank layer 6 obtained in the step 3) are firstly treated by plasma and then bonded together, and the aligned chips are baked on a hot plate at the temperature of 80 ℃ for 1 to 2 hours to realize the permanent bonding between the reaction layer 7 and the blank layer 6.
5) And (5) bonding the two layers of polydimethylsiloxane bonding structures obtained in the step (4) with the glass slide after plasma activation treatment to obtain the digital PCR chip.
The technical scheme provided by the embodiment of the invention has the following advantages:
1. the chip sample introduction does not depend on complex equipment such as a pump, a valve and the like, high-viscosity thermal polymerization separation oil is not needed, the chip does not need to be sealed after the sample introduction is finished, and the operation is simple;
2. the sample injector is used for assisting in rapid sample injection, and the negative pressure sample injection mode is used for reducing gas residue in the chip, so that the sample injection is rapid;
3. the chip completes the reaction in water environment, the water loss in the PCR micro-reaction unit can be ignored, the volume of the micro-reaction unit can be made smaller, more micro-reaction units can be made on the chip in unit area, and the overall flux of the chip is higher;
4. each micro-reaction unit of the chip has accurate volume and uniform array, and is easy for imaging analysis;
5. the high-pressure reaction chamber is easily compatible with the existing PCR instrument and is also easily integrated on a metal temperature control module of the PCR instrument;
6. the chip has small thickness, fast heat conduction and fast reaction;
7. the chip has simple structure, easy manufacture, low cost and high automation degree;
8. reduce water evaporation and is compatible with other dPCR chips.
It should be noted that the above examples are only for illustrative purposes and should not be construed as limiting the scope of the present invention. While the invention has been described with reference to exemplary embodiments, as will be apparent to those skilled in the art, the invention is not to be limited to the details given herein, and it is to be understood that various changes and modifications can be made without departing from the spirit and scope of the invention, all such changes and modifications being within the scope and meaning equivalent to the terms in which the invention is defined.
Claims (9)
1. A high-pressure liquid immersion type digital PCR method is characterized by comprising the following steps:
s1, immersing a digital PCR chip subjected to sample injection treatment into liquid in a high-pressure reaction chamber; each PCR micro-reaction unit (75) in the digital PCR chip subjected to sample injection treatment is in an isolated state;
s2, exhausting air in the high-pressure reaction chamber, and pressurizing the high-pressure reaction chamber after the air is exhausted; the pressurization is used for inhibiting the generation of air bubbles in the digital PCR chip under the condition of high temperature;
s3, placing the pressurized high-pressure reaction chamber on a PCR instrument for PCR reaction;
s4, cooling the high-pressure reaction chamber after the PCR reaction;
s5, reducing the pressure of the cooled high-pressure reaction chamber;
and S6, taking out the digital PCR chip in the high-pressure reaction chamber, and carrying out fluorescence signal analysis on the digital PCR chip.
2. The high pressure submerged digital PCR method according to claim 1, wherein the sample injection process in step S1 comprises the steps of:
s11, combining a sample injector with a digital PCR chip to ensure that a sample inlet (71) and a sample outlet (72) of a reaction layer of the digital PCR chip are aligned and tightly attached to a sample inlet pool (8) and a sample outlet (9) of the sample injector respectively;
s12, adding a PCR reaction solution into a sample inlet pool (8) of the sample injector, and exhausting air outwards at a sample outlet (9) of the sample injector by using an injector to enable the PCR reaction solution to flow into a main flow channel (73) of a reaction layer of the digital PCR chip through a sample inlet (71) of the reaction layer of the digital PCR chip until part of the PCR reaction solution flows out of the sample outlet (9) of the sample injector through a sample outlet (72) of the reaction layer of the digital PCR chip;
s13, using an injector to extract air from an air extraction port (11) of a negative pressure cavity (10) of the sample injector outwards until the PCR reaction solution is filled in each branch flow channel (74) communicated with the main flow channel (73) and each PCR micro-reaction unit (75) communicated with each branch flow channel (74);
s14, sucking off redundant PCR reaction liquid at a sample inlet pool (8) of the sample injector, and dripping isolation oil;
s15, exhausting air at a sample outlet (9) of the sample injector by using an injector to enable the main runner (73) of the reaction layer to be filled with the isolation oil, and completing isolation of each PCR micro-reaction unit (75).
3. The high pressure liquid immersion digital PCR method according to claim 1, wherein the high pressure reaction chamber is evacuated by injecting a neutral solution in step S2.
4. The high pressure liquid immersion digital PCR method of claim 1, wherein the pressure of the high pressure reaction chamber is 210-500KPa.
5. A digital PCR chip, wherein the digital PCR chip is used for performing the high pressure liquid immersion digital PCR method according to any one of claims 1 to 4.
6. The digital PCR chip according to claim 5, comprising a substrate layer (5), a blank layer (6) and a reaction layer (7) arranged in this order,
wherein the reaction layer (7) has a main flow channel (73), branch flow channels (74) communicating with the main flow channel (73), and PCR micro-reaction units (75) communicating with the branch flow channels (74); the reaction layer (7) is also provided with a sample inlet (71) and a sample outlet (72), the sample inlet (71) of the reaction layer is communicated with a sample cell (8) of a sample injector, and the sample outlet (72) of the reaction layer is communicated with a sample outlet (9) of the sample injector.
7. The digital PCR chip according to claim 6, wherein the substrate layer (5) is made of a support material for supporting the blank layer (6) and the reaction layer (7).
8. The digital PCR chip according to claim 6, wherein the blank layer (6) and the reaction layer (7) are made of polydimethylsiloxane.
9. A method of preparing the digital PCR chip of any one of claims 5 to 8, comprising the steps of:
1) Cleaning a silicon wafer, and manufacturing a reaction layer die on the silicon wafer;
2) Uniformly mixing polydimethylsiloxane and a curing agent, degassing, pouring the mixture onto the reaction layer mold, and performing thermocuring to form a polydimethylsiloxane membrane with a main runner (73), branch runners (74) and PCR micro-reaction units; taking the polydimethylsiloxane membrane off the reaction layer die, perforating to form a sample inlet (71) and a sample outlet (72), and manufacturing a reaction layer (7) with the sample inlet (71), the sample outlet (72), a main flow channel (73), branch flow channels (74) and PCR micro-reaction units (75);
3) Uniformly mixing polydimethylsiloxane and a curing agent, spin-coating on a silicon wafer, and curing to form a blank layer (6);
4) Carrying out plasma activation treatment on the reaction layer (7) obtained in the step 2) and the blank layer (6) obtained in the step 3) and then bonding the reaction layer and the blank layer together;
5) And (3) bonding the two-layer polydimethylsiloxane bonding structure obtained in the step (4) and the substrate layer (5) together after plasma activation treatment, thus obtaining the digital PCR chip.
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