CN110804650A - Circulating type digital PCR method, circulating system, digital PCR chip and preparation method thereof - Google Patents
Circulating type digital PCR method, circulating system, digital PCR chip and preparation method thereof Download PDFInfo
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- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
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Abstract
The invention discloses a circulating digital PCR method, a circulating system, a digital PCR chip and a preparation method thereof, wherein the digital PCR method comprises the following steps: s1, placing a digital PCR chip subjected to sample introduction treatment in a reaction chamber of a circulating system; s2, exhausting air in the reaction chamber, and pressurizing the circulating system; s3, starting a circulating system to perform PCR reaction; s4, decompressing a circulating system after the PCR reaction; and S5, taking out the digital PCR chip in the 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 needed, 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 constant-temperature liquid storage tank is large in volume, rapid in heat exchange, capable of rapidly heating or cooling the chip, good in temperature control and free of temperature overshoot; the chip has simple structure and low cost.
Description
Technical Field
The invention relates to the technical field of nucleic acid detection, in particular to a circulating type digital PCR method, a circulating system, 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 be amplified by millions of times within tens of minutes, and the content of the original PCR template is difficult to determine 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, 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, 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 can cause great operation difficulty, high cost and long reaction time of the dPCR chip.
Disclosure of Invention
The invention aims to provide a circulating type digital PCR method, a circulating system, 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 achieve the purpose, the technical scheme adopted by the invention is as follows:
in a first aspect, the present invention provides a circular digital PCR method, comprising the steps of:
s1, placing a digital PCR chip subjected to sample introduction treatment in a reaction chamber of a circulating system;
s2, exhausting air in the reaction chamber, and pressurizing the circulating system;
s3, starting the circulating system to perform PCR reaction;
s4, decompressing the circulating system after the PCR reaction;
and S5, taking out the digital PCR chip in the reaction chamber, and carrying out fluorescence signal analysis on the digital PCR chip.
Preferably, the sample injection processing in step S1 includes the following steps:
s11, combining a sample injector and a digital PCR chip, so that a sample inlet and a sample outlet of a reaction layer of the digital PCR chip are respectively aligned with a sample inlet pool and a sample outlet of the sample injector and are tightly attached to the sample inlet 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 dropwise adding 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 pressure of the pressurization process in step S2 is 210 — 500 KPa.
In a second aspect of the present invention, there is provided a circulation system for performing the above-mentioned circulating digital PCR method, the circulation system comprising a reaction chamber, a liquid circulation system, a temperature regulation system, a pressure regulation system, and a controller,
the reaction chamber is connected with the liquid circulation system and is used for placing a digital PCR chip;
the controller is respectively connected with the liquid circulation system, the temperature regulation system and the pressure regulation system and is used for automatically controlling the liquid circulation system to convey liquid to the reaction chamber to regulate the temperature of the reaction chamber, automatically controlling the temperature regulation system to regulate the temperature of the liquid in the liquid circulation system and automatically controlling the pressure regulation system to regulate the pressure of the circulation system.
Preferably, the liquid circulating system comprises a constant-temperature liquid storage tank, a circulating pump, a valve and a pipeline,
the constant-temperature liquid storage tank is connected with the reaction chamber through the pipeline and is used for conveying constant-temperature liquid to the reaction chamber to adjust the temperature of the reaction chamber;
the circulating pump and the valve are both arranged on the pipeline and used for driving constant-temperature liquid to circularly flow in the pipeline.
Preferably, the temperature regulation system includes a first temperature sensor, a second temperature sensor, a heater, and a cooler,
the first temperature sensor is arranged in the reaction chamber and used for detecting the temperature in the reaction chamber;
the second temperature sensor is arranged in the constant-temperature liquid storage tank and is used for detecting the temperature in the constant-temperature liquid storage tank;
the heater with the cooler all sets up in the constant temperature liquid reserve tank for to the liquid in the constant temperature liquid reserve tank heats or cools off.
Preferably, the pressure regulating system comprises a pressure sensor, a booster pump and a pressure relief valve,
the pressure sensor is arranged in the circulating system and used for detecting the pressure of the circulating system;
the booster pump and the pressure relief valve are arranged in the circulating system and used for increasing or reducing the pressure of the circulating system.
Preferably, the controller is respectively connected with the circulating pump and the valve, and is used for automatically controlling the operation of the circulating pump and the valve according to a PCR program to deliver liquid with constant temperature to the reaction chamber;
the controller is respectively connected with the first temperature sensor, the second temperature sensor, the heater and the cooler and is used for automatically controlling the operation of the heater and the cooler to heat or cool the liquid in the constant-temperature liquid storage tank according to the temperatures detected by the first temperature sensor and the second temperature sensor;
the controller is respectively connected with the pressure sensor, the booster pump and the pressure relief valve and is used for automatically controlling the work of the booster pump and the pressure relief valve to increase or decrease the pressure of the circulating system according to the pressure detected by the pressure sensor.
Preferably, the constant-temperature liquid storage tanks are 2-4.
The third aspect of the present invention provides a digital PCR chip, which is used to be placed in the reaction chamber of the above-mentioned circulation system, the digital PCR chip comprises a substrate layer and a reaction layer 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 and is used for sealing and supporting the reaction layer.
Preferably, the reaction layer is made of polydimethylsiloxane.
The fourth 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) and (3) bonding the polydimethylsiloxane reaction layer structure obtained in the step 2) and the substrate layer together after plasma activation treatment, thus preparing the digital PCR chip.
Preferably, the bonding in step 2) 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. compared with a reaction chamber, the constant-temperature liquid storage tank is larger in volume, rapid in heat exchange and capable of rapidly heating or cooling the chip;
4. the temperature of the constant-temperature liquid storage tank can be independently controlled, the temperature control effect is excellent, and the inevitable temperature overshoot phenomenon of a metal bath system is avoided;
5. the reaction chamber is easy to integrate with a fluorescence detection system, can realize the on-line detection of the chip, shortens the detection time and is suitable for rapid clinical detection;
6. 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;
7. each micro-reaction unit of the chip has accurate volume and uniform array, and is easy for imaging analysis;
8. the chip has small thickness, fast heat conduction and fast reaction;
9. the chip has the advantages of simple structure, easy manufacture, low cost and high automation degree.
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 description of the embodiments or the prior art 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 of a PDMS chip when it is heated;
FIG. 2 is a schematic structural diagram of a circulation system according to an embodiment of the present invention;
FIG. 3 is a temperature profile of a circulation system in an embodiment of the present invention;
FIG. 4 is an enlarged view of A in FIG. 3;
FIG. 5 is a schematic cross-sectional view of a digital PCR chip and an injector according to an embodiment of the present invention;
FIG. 6 is a schematic diagram of an exemplary injector;
FIG. 7 is a schematic diagram of a reaction layer of a digital PCR chip according to an embodiment of the present invention;
FIG. 8 is an enlarged view of B in FIG. 7;
FIG. 9 is a schematic diagram of a PCR reaction solution entering a main channel of a reaction layer of a digital PCR chip according to an embodiment of the present invention;
FIG. 10 is a schematic diagram of PCR reaction solution entering into 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. 11 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: 1-PDMS chip, 2-PCR solution, 3-bubbles, 4-micro-droplets, 5-reaction chamber, 6-constant temperature liquid storage tank, 7-circulating pump, 8-valve, 9-pipeline, 10-substrate layer, 11-reaction layer, 111-sample inlet of reaction layer, 112-sample outlet of reaction layer, 113-main flow channel, 114-branch flow channel, 115-micro-reaction unit, 12-sample tank, 13-sample outlet of sample injector, 14-negative pressure cavity, 15-air extraction port, 16-digital PCR chip.
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.
Example (b):
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 the thermal expansion effect of air itself causes the volume and pressure of the gas in the micropores to be obviously increased, 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 can cause great operation difficulty, high cost and long reaction time of the dPCR chip.
In view of the above problems, embodiments of the present invention provide a cycling system for a cycling digital PCR method. The circulation system includes a reaction chamber, a liquid circulation system, a temperature regulation system (not shown), a pressure regulation system (not shown), and a controller (not shown).
The reaction chamber 5 is connected to the liquid circulation system for placing a digital PCR chip 16 for PCR reaction, as shown in FIG. 2.
The controller is connected with the liquid circulation system and is used for automatically controlling the liquid circulation system to convey liquid to the reaction chamber 5 so as to adjust the temperature of the reaction chamber 5; the controller is also connected with the temperature adjusting system and is used for automatically controlling the temperature adjusting system to adjust the temperature of the liquid in the liquid circulating system; the controller is also connected with the pressure regulating system and is used for automatically controlling the pressure regulating system to regulate the pressure of the circulating system.
In a particular embodiment, the controller may be a Programmable Logic Controller (PLC).
Specifically, as shown in fig. 2, the liquid circulation system includes a constant temperature liquid storage tank 6, a circulation pump 7, a valve 8 and a pipeline 9. The constant-temperature liquid storage tank 6 is connected with the reaction chamber 5 through the pipeline 9 and is used for conveying constant-temperature liquid for the reaction chamber 5 to adjust the temperature of the reaction chamber 5. The liquid in the constant temperature liquid storage tank 6 is preferably an aqueous solution, and may be other solutions, which is not limited in the embodiment of the present invention. In the embodiment of the invention, the number of the constant-temperature liquid storage tanks 6 can be set according to actual needs. Preferably, 2-4 constant-temperature liquid storage tanks 6 are arranged. Each of the constant temperature reservoirs 6 may be set to a corresponding temperature, such as 94 ℃, 72 ℃, 55 ℃, 0 ℃ and the like, according to the PCR reaction requirement. The circulating pump 7 and the valve 8 are both arranged on the pipeline 9 and are used for driving the liquid with constant temperature in the constant-temperature liquid storage tank 6 to circularly flow in the pipeline 9. In the embodiment of the present invention, the number of the circulating pump 7 and the number of the valves 8 may also be set according to actual requirements.
Specifically, the temperature regulation system includes a first temperature sensor, a second temperature sensor, a heater, and a cooler. The first temperature sensor is disposed in the reaction chamber 5, and is configured to detect a temperature in the reaction chamber 5. The second temperature sensor is arranged in the constant-temperature liquid storage tank 6 and used for detecting the temperature in the constant-temperature liquid storage tank 6. The heater with the cooler all sets up in the constant temperature liquid reserve tank 6 for to the liquid in the constant temperature liquid reserve tank 6 heats or cools off.
Specifically, the pressure regulating system comprises a pressure sensor, a booster pump and a pressure relief valve. The pressure sensor is arranged in the circulating system and used for detecting the pressure of the circulating system. The booster pump and the pressure relief valve are arranged in the circulating system and used for increasing or reducing the pressure of the circulating system according to needs.
Specifically, the circulating pump 7 and the valve 8 are respectively connected to the controller, and the controller is configured to automatically control the opening or closing of the circulating pump 7 and the valve 8 according to a PCR program to deliver a constant temperature liquid to the reaction chamber 5. The first temperature sensor, the second temperature sensor, the heater and the cooler are respectively connected with the controller, and the controller is used for automatically controlling the heater and the cooler to be turned on or off to heat or cool the liquid in the constant-temperature liquid storage box 6 according to the temperatures detected by the first temperature sensor and the second temperature sensor. The pressure sensor, the booster pump and the pressure relief valve are respectively connected with the controller, and the controller is used for automatically controlling the booster pump and the pressure relief valve to be opened or closed according to the pressure detected by the pressure sensor so as to increase or decrease the pressure of the circulating system.
In the embodiment of the invention, the digital PCR chip is placed in the liquid in the reaction chamber of the circulating system, and compared with the reaction chamber, the constant-temperature liquid storage tank has larger volume and rapid heat exchange, and can rapidly heat or cool the digital PCR chip. In addition, the embodiment of the present invention (two-step dPCR, denaturation temperature set to 94 degrees, primer binding and extension temperature set to 60 degrees) can independently control the temperature of the constant-temperature tanks (e.g., high-temperature tank and low-temperature tank), and the temperature control effect is excellent (as shown in fig. 3 and 4), without the temperature overshoot phenomenon inevitable in the metal bath system.
Correspondingly, the embodiment of the invention provides a circulating digital PCR method, which comprises the following steps:
s1, placing the digital PCR chip subjected to sample introduction treatment in a reaction chamber of a circulating system.
In the embodiment of the present invention, as shown in fig. 5 and fig. 6, 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 12, a sample outlet 13 and a negative pressure cavity 14, wherein an extraction opening 15 is arranged on the negative pressure cavity 14.
As shown in fig. 5 and 7, the digital PCR chip includes a substrate layer 10 and a reaction layer 11 sequentially disposed from bottom to top, wherein the reaction layer 11 is provided with a sample inlet 111 and a sample outlet 112, the sample inlet 111 of the reaction layer is communicated with the sample cell 12 of the sample injector, and the sample outlet 112 of the reaction layer is communicated with the sample outlet 13 of the sample injector.
As shown in fig. 5, 7 and 8, the reaction layer 11 includes a main flow channel 113, branch flow channels 114 communicating with the main flow channel 113, and PCR micro-reaction cells 115 communicating with the branch flow channels 114.
Specifically, as shown in fig. 5 to 8, the sample injection treatment may include the following steps:
s11, combining the sample injector and the digital PCR chip, so that a sample inlet 111 and a sample outlet 112 of a reaction layer of the digital PCR chip are respectively aligned with a sample inlet pool 12 and a sample outlet 13 of the sample injector and are tightly attached to each other.
S12, adding the PCR reaction liquid into a sample inlet pool 12 of the sample injector, and pumping air outwards at a sample outlet 13 of the sample injector by using an injector to enable the PCR reaction liquid to flow into a main runner 113 of a reaction layer of the digital PCR chip through a sample inlet 111 of the reaction layer of the digital PCR chip until part of the PCR reaction liquid flows out of the sample outlet 13 of the sample injector through a sample outlet 112 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: using a pipette to drip the PCR reaction solution in the sample cell 12 of the sample injector, using a syringe to pump air out through the hose connector at the sample outlet 13 of the sample injector, and guiding the PCR reaction solution to flow into the main channel 113 of the reaction layer of the digital PCR chip through the sample inlet 111 of the reaction layer of the digital PCR chip, as shown in FIG. 9, until part of the PCR reaction solution flows out from the sample outlet 13 of the sample injector through the sample outlet 112 of the reaction layer of the digital PCR chip.
S13, using an injector to extract air from the air extraction port 15 of the negative pressure cavity 14 of the sample injector outwards until the PCR reaction solution fills the branch flow channels 114 communicated with the main flow channel 113 and the PCR micro-reaction units 115 communicated with the branch flow channels 114.
In the embodiment of the present invention, the negative pressure chamber 14 of the sample injector completely covers the main flow channel 113 of the reaction layer, the branch flow channels 114 communicated with the main flow channel, and the PCR micro-reaction units 115 communicated with the branch flow channels 114.
In the embodiment of the present invention, the PCR reaction solution is firstly dropped into the sample cell 12 of the sample injector, then the sample outlet 13 of the sample injector is pumped out, and the pumping port 15 of the negative pressure cavity 14 of the sample injector is pumped out, so that the reaction layer 11 on the digital PCR chip can rapidly generate negative pressure in each PCR micro-reaction unit 115, and the PCR reaction solution is guided to fill each branch channel 114 communicated with the main channel 113 and enter each PCR micro-reaction unit 115, as shown in fig. 10. 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 position of the sample inlet pool 12 of the sample injector, and dripping isolation oil.
Wherein the spacer oil may be FC-40.
S15, exhausting air at the sample outlet 13 of the sample injector by using an injector, so that the main flow channel 113 of the reaction layer is filled with the isolation oil, and the isolation of each PCR micro-reaction unit 115 is completed, as shown in FIG. 11.
And S2, exhausting air in the reaction chamber, and pressurizing the circulating system.
Specifically, in step S2, the air in the reaction chamber is evacuated by injecting a neutral solution. For example, the neutral solution may be an aqueous solution. Further, after the air in the reaction chamber is exhausted, the circulating system needs to be pressurized, and the pressure of the circulating system is increased. Preferably, the pressure of the pressurization treatment is 210-500 KPa.
And S3, starting the circulating system to perform PCR reaction.
Specifically, the reaction parameters of the PCR reaction in step S3 can be set according to actual needs.
And S4, decompressing the circulating system after the PCR reaction.
Specifically, in step S4, the circulation system may be reduced to a certain pressure, and the pressure may be set according to actual needs.
And S5, taking out the digital PCR chip in the reaction chamber, and carrying out fluorescence signal analysis on the digital PCR chip.
Specifically, the step S5 may be: and opening the reaction chamber, taking out the digital PCR chip in the reaction chamber, and carrying out fluorescence signal analysis on the digital PCR chip. The reaction chamber is integrated with a fluorescence detection system, and can realize on-line detection of the chip.
The embodiment of the invention adopts the circulating digital PCR method, so that the digital PCR chip is immersed in the liquid in the reaction chamber of the circulating system during the PCR reaction, and because no air exists around the digital PCR chip, the gas in the PDMS micropores can not 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 gas 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 which is arranged in the reaction chamber of the circulating system. As shown in fig. 5, the digital PCR chip includes a substrate layer 10 and a reaction layer 11.
Preferably, the reaction layer 11 is made of Polydimethylsiloxane (PDMS). As shown in fig. 7 and 8, the reaction layer 11 includes a main flow channel 113, branch flow channels 114 communicating with the main flow channel 113, and PCR micro-reaction units 115 communicating with the branch flow channels 114.
As shown in fig. 5, the reaction layer further has a sample inlet 111 and a sample outlet 112, the sample inlet 111 of the reaction layer is communicated with the sample cell 12 of the sample injector, and the sample outlet 112 of the reaction layer is communicated with the sample outlet 13 of the sample injector.
Preferably, the substrate layer 10 is made of a supporting material such as glass. The substrate layer 10 is located below the reaction layer 11 and is used for supporting the reaction layer 11.
Accordingly, an embodiment of the present invention further provides a method for preparing the digital PCR chip shown in fig. 5 and 8, which 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 180 ℃ of 170-; treating with plasma for 1-2min, spin-coating SU83005(10 μm), and performing photolithography and development to obtain branch flow channels 114 communicated with the main flow channel; baking on a hot plate at 170 ℃ of 160-.
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 113, branch runners 114 and PCR micro-reaction units 115; and taking the polydimethylsiloxane membrane off the reaction layer die, and punching to form a sample inlet 111 and a sample outlet 112, so as to prepare the reaction layer with the sample inlet 111, the sample outlet 112, the main flow channel 113, the branch flow channels 114 and the PCR micro-reaction units 115.
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 5:1-10:1, removing 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 113, branch flow channels 114 and PCR micro-reaction units 115, removing the polydimethylsiloxane membrane from the reaction layer mould, cutting and punching to form a sample inlet 111 and a sample outlet 112, and thus preparing the reaction layer 11 with the sample inlet 111, the sample outlet 112, the main flow channel 113, the branch flow channels 114 and the PCR micro-reaction units 115.
3) And (3) bonding the polydimethylsiloxane reaction layer structure obtained in the step 2) and the substrate layer 10 together after plasma activation treatment, thus obtaining the digital PCR chip.
In a particular embodiment, the substrate layer 10 may be a glass slide.
The technical scheme provided by the embodiment of the invention has the following advantages:
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. compared with a reaction chamber, the constant-temperature liquid storage tank is larger in volume, rapid in heat exchange and capable of rapidly heating or cooling the chip;
4. the temperature of the constant-temperature liquid storage tank can be independently controlled, the temperature control effect is excellent, and the inevitable temperature overshoot phenomenon of a metal bath system is avoided;
5. the reaction chamber is easy to integrate with a fluorescence detection system, can realize the on-line detection of the chip, shortens the detection time and is suitable for rapid clinical detection;
6. 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;
7. each micro-reaction unit of the chip has accurate volume and uniform array, and is easy for imaging analysis;
8. the chip has small thickness, fast heat conduction and fast reaction;
9. the chip has the advantages of simple structure, easy manufacture, low cost and high automation degree.
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 a preferred embodiment, those skilled in the art will appreciate that various changes can be made in the invention without departing from the spirit and scope of the invention, and all such changes are intended to be within the scope of the invention as defined and equivalents thereof.
Claims (13)
1. A cyclic digital PCR method is characterized by comprising the following steps:
s1, placing a digital PCR chip (16) subjected to sample injection treatment in a reaction chamber (5) of a circulating system;
s2, exhausting air in the reaction chamber (5) and pressurizing the circulating system;
s3, starting the circulating system to perform PCR reaction;
s4, decompressing the circulating system after the PCR reaction;
s5, taking out the digital PCR chip (16) in the reaction chamber (5), and carrying out fluorescence signal analysis on the digital PCR chip (16).
2. The cyclic digital PCR method of claim 1, wherein the sample injection processing in step S1 comprises the following steps:
s11, combining a sample injector and a digital PCR chip (16) so that a sample inlet (111) and a sample outlet (112) of a reaction layer of the digital PCR chip (16) are respectively aligned with and closely attached to a sample inlet pool (12) and a sample outlet (13) of the sample injector;
s12, adding a PCR reaction solution into a sample cell (12) of the sample injector, and exhausting air outwards at a sample outlet (13) of the sample injector by using an injector to enable the PCR reaction solution to flow into a main runner (113) of a reaction layer of the digital PCR chip (16) through a sample inlet (111) of the reaction layer of the digital PCR chip (16) until part of the PCR reaction solution flows out of the sample outlet (13) of the sample injector through a sample outlet (112) of the reaction layer of the digital PCR chip (16);
s13, using an injector to extract air from an extraction opening (15) of a negative pressure cavity (14) of the sample injector outwards until the PCR reaction solution is filled in each branch flow channel (114) communicated with the main flow channel (113) and each PCR micro-reaction unit (115) communicated with each branch flow channel (114);
s14, sucking off redundant PCR reaction liquid at a sample inlet pool (12) of the sample injector, and dropwise adding isolation oil;
s15, exhausting air at the sample outlet (13) of the sample injector by using an injector to enable the main flow channel (113) of the reaction layer to be filled with the isolation oil, and completing isolation of each PCR micro-reaction unit (115).
3. The cyclic digital PCR method as claimed in claim 1, wherein the pressure of the pressurization process in step S2 is 210 KPa and 500 KPa.
4. A cycling system for performing the cycling digital PCR method according to any one of claims 1-3, characterized in that the cycling system comprises a reaction chamber (5), a liquid cycling system, a temperature regulating system, a pressure regulating system and a controller,
the reaction chamber (5) is connected with the liquid circulation system and is used for placing a digital PCR chip (16);
the controller is respectively connected with the liquid circulation system, the temperature regulation system and the pressure regulation system and is used for automatically controlling the liquid circulation system to convey liquid to the reaction chamber (5) to regulate the temperature of the reaction chamber (5), automatically controlling the temperature regulation system to regulate the temperature of the liquid in the liquid circulation system and automatically controlling the pressure regulation system to regulate the pressure of the circulation system.
5. Circulation system according to claim 4, characterized in that it comprises a thermostatic liquid tank (6), a circulation pump (7), a valve (8) and a pipe (9),
the constant-temperature liquid storage tank (6) is connected with the reaction chamber (5) through the pipeline (9) and is used for conveying constant-temperature liquid to the reaction chamber (5) to adjust the temperature of the reaction chamber (5);
the circulating pump (7) and the valve (8) are both arranged on the pipeline (9) and are used for driving constant-temperature liquid to circularly flow in the pipeline (9).
6. The circulation system of claim 5, wherein the temperature regulation system comprises a first temperature sensor, a second temperature sensor, a heater, and a cooler,
the first temperature sensor is arranged in the reaction chamber (5) and is used for detecting the temperature in the reaction chamber (5);
the second temperature sensor is arranged in the constant-temperature liquid storage tank (6) and is used for detecting the temperature in the constant-temperature liquid storage tank (6);
the heater and the cooler are arranged in the constant-temperature liquid storage box (6) and used for heating or cooling liquid in the constant-temperature liquid storage box (6).
7. The circulation system of claim 6, wherein the pressure regulation system comprises a pressure sensor, a booster pump, and a pressure relief valve,
the pressure sensor is arranged in the circulating system and used for detecting the pressure of the circulating system;
the booster pump and the pressure relief valve are arranged in the circulating system and used for increasing or reducing the pressure of the circulating system.
8. The circulation system according to claim 7, wherein the controller is connected to the circulation pump (7) and the valve (8), respectively, for automatically controlling the operation of the circulation pump (7) and the valve (8) according to a PCR program to deliver a constant temperature liquid to the reaction chamber (5);
the controller is respectively connected with the first temperature sensor, the second temperature sensor, the heater and the cooler and is used for automatically controlling the operation of the heater and the cooler according to the temperature detected by the first temperature sensor and the second temperature sensor so as to heat or cool the liquid in the constant-temperature liquid storage tank (6);
the controller is respectively connected with the pressure sensor, the booster pump and the pressure relief valve and is used for automatically controlling the work of the booster pump and the pressure relief valve to increase or decrease the pressure of the circulating system according to the pressure detected by the pressure sensor.
9. A circulation system according to claim 5, characterized in that the thermostatic liquid tank (6) is provided with 2-4.
10. A digital PCR chip, characterized in that the digital PCR chip (16) is intended to be placed in a reaction chamber (5) of a circulation system according to any of claims 5 to 9, the digital PCR chip (16) comprising a substrate layer (10) and a reaction layer (11) arranged in succession,
wherein the reaction layer (11) has a main channel (113), branch channels (114) communicating with the main channel (113), and PCR micro-reaction units (115) communicating with the branch channels (114); the reaction layer (11) is also provided with a sample inlet (111) and a sample outlet (112), the sample inlet (111) of the reaction layer is communicated with a sample cell (12) of the sample injector, and the sample outlet (112) of the reaction layer is communicated with a sample outlet (13) of the sample injector.
11. The digital PCR chip according to claim 10, wherein the substrate layer (10) is made of a support material for sealing and supporting the reaction layer (11).
12. The digital PCR chip according to claim 10, wherein the reaction layer (11) is made of polydimethylsiloxane.
13. A method of preparing the digital PCR chip of any one of claims 10-12, 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 (113), branch runners (114) and PCR micro-reaction units (115); taking the polydimethylsiloxane membrane off the reaction layer die and punching to form a sample inlet and a sample outlet, so as to manufacture a reaction layer (11) with the sample inlet (111), the sample outlet (112), a main flow channel (113), branch flow channels (114) and PCR micro-reaction units (115);
3) and (3) bonding the polydimethylsiloxane reaction layer structure obtained in the step (2) and the substrate layer (10) together after plasma activation treatment, thus obtaining the digital PCR chip (16).
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| CN201911031703.XA CN110804650B (en) | 2019-10-28 | 2019-10-28 | Circulating digital PCR method, circulating system, digital PCR chip and preparation method thereof |
| PCT/CN2020/121493 WO2021082951A1 (en) | 2019-10-28 | 2020-10-16 | Digital pcr method, chip, preparation method and circulation system |
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