CN112108195A - PCR device and control method - Google Patents
PCR device and control method Download PDFInfo
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- CN112108195A CN112108195A CN202011028188.2A CN202011028188A CN112108195A CN 112108195 A CN112108195 A CN 112108195A CN 202011028188 A CN202011028188 A CN 202011028188A CN 112108195 A CN112108195 A CN 112108195A
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
- B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
- B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
- B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L7/00—Heating or cooling apparatus; Heat insulating devices
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- 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
- C12Q3/00—Condition responsive control processes
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L2300/00—Additional constructional details
- B01L2300/18—Means for temperature control
- B01L2300/1805—Conductive heating, heat from thermostatted solids is conducted to receptacles, e.g. heating plates, blocks
- B01L2300/1811—Conductive heating, heat from thermostatted solids is conducted to receptacles, e.g. heating plates, blocks using electromagnetic induction heating
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L2300/00—Additional constructional details
- B01L2300/18—Means for temperature control
- B01L2300/1838—Means for temperature control using fluid heat transfer medium
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L2300/00—Additional constructional details
- B01L2300/18—Means for temperature control
- B01L2300/1838—Means for temperature control using fluid heat transfer medium
- B01L2300/185—Means for temperature control using fluid heat transfer medium using a liquid as fluid
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L2300/00—Additional constructional details
- B01L2300/18—Means for temperature control
- B01L2300/1894—Cooling means; Cryo cooling
Abstract
The invention discloses a PCR device, comprising: the PCR chip is provided with more than two reaction cavities and comprises a heat insulating layer arranged between every two adjacent reaction cavities; the temperature control unit forms a low magnetic induction zone and an air cooling zone at the periphery of the reaction cavity and comprises a temperature probe extending into the reaction cavity; and the output end of the temperature control driving unit is connected with the temperature control unit, and the periphery of the reaction cavity is controlled to form a low magnetic induction zone and an air cooling zone. The PCR device provided by the invention can control the temperature of each reaction cavity respectively, has small mutual thermal interference and has outstanding advantages in high-throughput PCR reaction; the technology for generating the eddy current through low magnetic induction has the advantages of small heat loss, high heating speed and high heat conversion efficiency.
Description
Technical Field
The invention relates to the field of biomedical equipment, in particular to a PCR device and a control method.
Background
Polymerase Chain Reaction (PCR) is a molecular biology Reaction for DNA amplification. One of the key factors for the success of temperature-variable PCR is that the temperature cycling conditions, i.e., high temperature denaturation, low temperature annealing, and medium temperature primer extension, required by the reaction entity must be satisfied, so that temperature cycling is the basis, and the "temperature-variable" rate in temperature cycling will determine the specificity of the PCR reaction and the overall speed of PCR operation. The conventional PCR thermal cycle system is roughly divided into a water bath type, an air flow type and a semiconductor type, wherein the heating mode is heating by a resistance wire, a resistance sheet or a semiconductor combined heat cover, and the like, the heat of a heating source is transferred to a heating device through heat conduction, and then a test tube is heated. However, these heating methods not only have many heat transfer stages, low conversion efficiency and poor utilization rate; in the high-throughput detection and screening process, a plurality of sets of PCR reactions need to be carried out simultaneously, and when the temperature change problem in the reaction process is solved by the conventional PCR chip, the rapid temperature change control cannot be carried out on each single reaction, so that the high-throughput detection and screening efficiency is severely restricted.
Disclosure of Invention
The invention aims to solve the problems of the prior art and provides the following technical scheme.
In a first aspect of the present invention, there is provided a PCR device comprising:
the PCR chip is provided with more than two reaction cavities and comprises a heat insulating layer arranged between every two adjacent reaction cavities;
the temperature control unit is used for forming a low magnetic induction area and an air cooling area on the periphery of each reaction cavity and comprises a temperature probe extending into the reaction cavity;
and the output end of the temperature control driving unit is connected with the temperature control unit and used for controlling the reaction cavity to form the low magnetic induction area and the air cooling area.
Furthermore, the PCR chip is also provided with a temperature control cavity which is wrapped on the periphery of each reaction cavity, and the temperature control unit comprises a temperature control element which extends into the temperature control cavity or is wrapped on the periphery of the reaction cavity and a cooling component which is communicated with the temperature control cavity;
the PCR chip also comprises a temperature control layer positioned between the temperature control cavity and the reaction cavity, the temperature control layer is made of a high-thermal-conductivity rust-resistant material, and the temperature probe is provided with one end extending to the surface of the temperature control layer;
the temperature control cavity is in a near vacuum state.
Specifically, the control by temperature change drive unit includes magnetic induction heating the control unit and control by temperature change spare, the control by temperature change spare around in the reaction chamber periphery, the control by temperature change spare with the magnetic induction heating the control unit is connected, the control by magnetic induction heating the control unit control the current change in the control by temperature change spare.
Furthermore, the temperature control driving unit further comprises a cooling medium control unit, the cooling assembly is communicated with an output end of the cooling medium control unit, and the cooling medium control unit controls the cooling assembly to input and recycle cooling medium into the temperature control cavity.
Specifically, the cooling medium is liquid hydrogen, liquid nitrogen or hydraulic carbon dioxide.
Specifically, the cooling assembly includes an air body and an air source, the air source accommodates the cooling medium, the air body is communicated with an output end of the air source, the air body has an air outlet and an air return opening, and the cooling medium control unit controls the cooling medium in the air source to flow into the air body and input into the temperature control cavity from the air outlet, or controls the cooling medium in the temperature control cavity to enter the air body through the air return opening and flow into the air source.
Further, the PCR device provided by the present invention further comprises:
the transfusion mechanism is used for communicating the reaction cavity, inputting reaction liquid and samples required by PCR into the reaction cavity and/or cleaning the reaction cavity;
a detection electrode for detecting a chemical signal, an electrical signal, or an optical signal generated by the binding of the target nucleic acid and the active substance in the reaction chamber;
the signal output end of the temperature control unit, the signal output end of the temperature control driving unit and the signal output end of the infusion mechanism are respectively in communication connection with the microprocessor;
and the upper terminal is in communication connection with the upper terminal.
In another aspect of the present invention, there is provided a control method of the PCR device, including a PCR reaction control method;
the PCR reaction control method comprises the following steps:
s11, acquiring the temperature in the reaction cavity through the temperature probe, converting the temperature into an electric signal and outputting the electric signal to the microprocessor, wherein the microprocessor outputs the electric signal to the upper terminal through conversion and amplification;
s12, the upper terminal judges the stage of the PCR reaction in the reaction cavity and the maintaining time at the stage through a preset PCR reaction control program;
s13, the upper terminal outputs a PCR reaction stage control instruction to the microprocessor, and the microprocessor inputs the PCR reaction stage control instruction to a signal input end of the temperature control driving unit corresponding to the reaction cavity after judgment;
s14, the temperature control driving unit executes the control instruction of the PCR reaction stage, and outputs a magnetic induction control signal through the magnetic induction heating control unit to control the current in the temperature control piece so as to control the temperature of the temperature control layer on the surface of the reaction cavity; or the temperature control driving unit executes the control instruction of the PCR reaction stage, and the cooling medium output control unit is output through the cooling medium control unit to control the amount of the cooling medium output by the cooling assembly, so as to control the temperature of the temperature control layer on the surface of the reaction cavity;
and S15, the temperature probe acquires the temperatures of the reaction cavity and the surface of the temperature control layer, converts the temperatures into electric signals and outputs the electric signals to the microprocessor, the microprocessor outputs the electric signals to the upper terminal through conversion and amplification, and the upper terminal determines whether to output the control instruction of the PCR reaction stage again through a PCR reaction control program.
Further, the control method of the PCR device further comprises the PCR chip cleaning method, and the detection electrode further comprises a nucleic acid probe for detecting the cleanliness in the reaction cavity;
the PCR chip cleaning method comprises the following steps:
s21, acquiring the gene cleanliness in the reaction cavity by the nucleic acid probe, converting the gene cleanliness into an electric signal, inputting the electric signal to the microprocessor, and outputting the electric signal to the upper terminal by the microprocessor through conversion and amplification;
s22, the upper terminal outputs a reaction cavity cleaning control instruction to the microprocessor through a preset reaction cavity cleaning program, and the microprocessor inputs the instruction into a signal input end of the infusion mechanism corresponding to the reaction cavity to be cleaned after judgment;
s14, the infusion mechanism executes the reaction cavity cleaning control instruction to clean the reaction cavity;
and S15, the nucleic acid probe acquires the amount of nucleic acid on the surface of the reaction cavity, converts the nucleic acid into an electric signal and outputs the electric signal to the microprocessor, the microprocessor outputs the electric signal to the upper terminal through conversion and amplification, and the upper terminal determines whether to output a reaction cavity cleaning control instruction again through a reaction cavity cleaning program.
Further, the control method of the PCR apparatus further includes a PCR reactant signal method, and the PCR reactant signal method includes obtaining a chemical signal, an electrical signal, or an optical signal generated by binding of the target gene, the active substance, and the molecular probe on the surface of the detection electrode in the reaction cavity.
Compared with the prior art, the invention has the beneficial effects that:
1. according to the PCR device provided by the invention, the low magnetic induction area and the air cooling area are formed on the periphery of each reaction cavity, so that the PCR device can directly act on each reaction cavity, can respectively control the temperature of each reaction cavity, has small mutual thermal interference, and has outstanding advantages in high-throughput PCR reaction; the technology for generating the eddy current through low magnetic induction has small heat loss, high heating speed and high thermal conversion efficiency; the temperature of the reaction cavity can be reduced by air cooling, so that the requirement of PCR reaction circulation is met; because the low magnetic induction extraction and the air cooling zone both indirectly control the temperature of the reaction cavity, the temperature control is mild, and the influence on PCR reactants in the reaction cavity is small.
2. The technical scheme of the invention can simultaneously and independently carry out hybridization detection on a plurality of reaction cavities by adopting electrochemical genes of electrochemical technology at most, and can carry out independent temperature control on a reaction system in each reaction cavity so as to meet the temperature requirements of different hybridization detection stages and different detection items.
3. The control method of the PCR device provided by the invention has the advantages that each reaction cavity is provided with an independent closed-loop temperature control unit, the system comprises a comparison element based on an ARM9 processor, a control element based on PWM output control, an execution element based on a heating power tube and a feedback element based on high-precision sensing amplification and ADC, the control program is simple, the size of a PCR chip can be greatly reduced, the amount of a sample is reduced, the PCR time is greatly shortened, PCR of a large number of samples can be carried out simultaneously, and the problems of the existing PCR device are effectively improved.
Drawings
FIG. 1 is a schematic cross-sectional view of an alternative PCR chip provided in an embodiment of the present invention.
FIG. 2 is a schematic cross-sectional view of an alternative PCR chip provided in an embodiment of the present invention.
FIG. 3 is a cross-sectional view of an alternative PCR chip provided in an embodiment of the present invention.
FIG. 4 is a cross-sectional view of an alternative PCR chip provided in an embodiment of the present invention.
Fig. 5 is an enlarged view at a in fig. 1.
Fig. 6 is an enlarged view at B in fig. 1.
Fig. 7 is a schematic structural diagram of an alternative cooling assembly provided by an embodiment of the present invention.
FIG. 8 is a schematic diagram of the structure of the connection infusion mechanism of FIG. 5.
FIG. 9 is a schematic diagram of the structure of the connection infusion mechanism of FIG. 6.
Fig. 10 is a logic block diagram of a PCR device according to an embodiment of the present invention.
FIG. 11 is a flowchart of a PCR reaction control method of the PCR apparatus according to the embodiment of the present invention.
FIG. 12 is a flowchart of a method for washing a PCR chip of a PCR apparatus according to an embodiment of the present invention.
1PCR chip, 10 reaction chambers, 100 heat insulating layers, 101 magnetic shielding parts, 11 temperature control chambers, 12 temperature control layers, 13 communication ports, 130 transfusion ports, 131 sample ports, 132 cleaning ports,
2 temperature control units, 20 temperature probes, 200 low-magnetism induction areas, 201 air cooling areas, 21 temperature control pieces, 22 cooling assemblies, 220 air bodies, 2200 air outlets, 2201 air return inlets, 2202 buffer tanks, 2203 air inlet pipelines, 2204 air outlet pipelines, 2205 air return pumps, 2206 air inlet valves, 2207 air inlet flow valves, 2208 air outlet valves, 2209 air outlet flow valves, 221 air sources,
3 a temperature control drive unit, 31 a magnetic induction heating control unit, 32 a cooling medium control unit,
4 transfusion mechanism, 40 raw material box, 41 transfusion pipeline, 42 sample box, 43 sample pipeline, 44 cleaning box, 45 cleaning pipeline, 46 nucleic acid probe,
5a detection electrode,
6 a microprocessor,
And 7, upper terminal.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is described in further detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
PCR device
Referring to fig. 1 to 4, the present invention provides a PCR device including:
the PCR chip 1 is provided with more than two reaction cavities 10, and the PCR chip 1 comprises a heat insulating layer 100 which is arranged between every two adjacent reaction cavities 10;
the temperature control unit 2 is formed in the periphery of each reaction chamber 10 to form a low magnetic induction area 200 and an air cooling area 201, and the temperature control unit 2 comprises a temperature probe 20 extending into the reaction chamber 10;
and the output end of the temperature control driving unit 3 is connected with the temperature control unit 3, and the periphery of the reaction chamber 10 is controlled to form a low magnetic induction zone 200 and an air cooling zone 201.
According to the PCR device provided by the invention, the low magnetic induction area and the air cooling area are formed on the periphery of each reaction cavity, so that the PCR device can directly act on each reaction cavity, can respectively control the temperature of each reaction cavity, has small mutual thermal interference, and has outstanding advantages in high-throughput PCR reaction; the technology for generating the eddy current through low magnetic induction has small heat loss, high heating speed and high thermal conversion efficiency; the temperature of the reaction cavity can be reduced by air cooling, so that the requirement of PCR reaction circulation is met; because the low magnetic induction extraction and the air cooling zone both indirectly control the temperature of the reaction cavity, the temperature control is mild, and the influence on PCR reactants in the reaction cavity is small.
Specifically, the heat insulating layer 100 is made of a low thermal conductive material, or is in a structure in which the low thermal conductive material includes a vacuum cavity, or is in a structure in which the low thermal conductive material includes a vacuum aerogel. The low thermal conductivity material may be selected from foamed silica gel, tungsten diselenide or bismuth selenide halide.
Specifically, the PCR chip 1 further has a temperature control chamber 11 wrapped around each reaction chamber 10, and the temperature control unit 2 includes a temperature control member 21 extending into the temperature control chamber 11 or wrapped around the reaction chamber 10 and a cooling assembly 22 communicating with the temperature control chamber 11.
As shown in fig. 5 and 6, the PCR chip 1 further includes a temperature control layer 12 located between the temperature control chamber 11 and the reaction chamber 10, the temperature control layer 12 is made of a high thermal conductivity rust-resistant material, and the temperature probe 20 has one end extending to the surface of the temperature control layer 12; the temperature control chamber 11 is in a state close to vacuum, so that the influence of the temperature control chamber 11 on the peripheral insulating layer 100 can be further reduced, and the temperature of each reaction chamber 10 can be controlled in a single temperature range without mutual influence. Specifically, temperature control part 21 can heat temperature control chamber 11 according to the PCR needs, and cooling module 22 can cool off temperature control chamber 11 according to the PCR needs, and the two cooperation is controlled the temperature in temperature control chamber 11 to the temperature in indirect control reaction chamber 10. Since the reaction chamber 10 and the temperature control chamber 11 are separated by only one temperature control layer 12, and the temperature control layer 12 is made of a high thermal conductivity rust-resistant material selected from copper, copper alloy, aluminum nitride, copper nitride or aluminum copper nitride; so, can conduct the temperature in control by temperature change chamber 11 to reaction chamber 10 in through control by temperature change layer 12 fast, reduce energy loss, also can realize the rapid cooling to control by temperature change layer 12 through controlling by temperature change chamber 11 rapid cooling simultaneously to let reaction chamber 10 rapid cooling, satisfy the needs of PCR reaction.
Specifically, the temperature control driving unit 3 includes a magnetic induction heating control unit 31, the temperature control member 21 is a coil, the temperature control member 21 surrounds the periphery of the reaction chamber 10, the temperature control member 21 is connected with the magnetic induction heating control unit 31, and the magnetic induction heating control unit 31 controls the current change in the temperature control member 21, so that an alternating magnetic field is generated in the temperature control member, an eddy current generated in the temperature control layer 12 wrapped by the temperature control member is smaller than that generated in the temperature control layer, and the temperature control member generates self-heating. Specifically, each reaction chamber 1 corresponds to one group of temperature control members 21, and each group of temperature control members 21 is independently connected with the magnetic induction heating control unit 31 respectively to control the current change in each temperature control member 21 respectively.
In an optional embodiment, the temperature control element 21 is suspended in the temperature control cavity 11 and is wound around the reaction cavity 10, so that in order to energize the temperature control element 21, two ends of the temperature control element 21 need to penetrate out of the temperature control cavity 11 and be connected to the external magnetic induction heating control unit 31. Specifically, as shown in fig. 1, the temperature control member 21 is provided in the temperature control chamber 11.
In an optional embodiment, the temperature control member 21 is wrapped at a corresponding portion of the reaction chamber 10 outside the PCR chip 1, so that the temperature control member 21 directly generates an eddy current effect by magnetic induction without damaging the temperature control chamber 11. As shown in fig. 2, the temperature control member 21 is wrapped outside the temperature control chamber 11 and embedded in the heat insulating layer 100. Or as shown in fig. 4, the temperature control member 21 is included outside the temperature control chamber 11 and embedded in the outer surface of the heat insulating layer 100.
In both embodiments, the heat insulating layer 100 has a magnetic shielding function to reduce the eddy current effect caused by magnetic induction between the two reaction chambers 10, and in this case, the heat insulating layer 100 has a magnetic shielding portion 101 extending to the outside of the PCR chip 1.Specifically, as shown in fig. 3 and 4, the magnetic shield 101 is embedded in the heat insulating layer 100 and extends to the outer surface of the chip after passing through the heat insulating layer 100; preferably, the shape of the magnetic shielding part 101 is adapted to the magnetic induction line formed by the temperature control member 21, and the part of the magnetic shielding part 101 inside the heat insulating layer 100 is not in direct contact with the temperature control chambers on both sides. Specifically, the magnetic shield 101 may be formed by machining a commercially available zinc film into the shape of the magnetic shield 101 required in the present invention, and then passivating the magnetic shield 101, wherein a passivating solution is prepared from sulfuric acid (1.8g/L), nitric acid (2.8g/L), hypochlorous acid (1.8g/L), chromium sulfate (0.9g/L), sodium silicate (18g/L), titanium trichloride (10g/L), and sodium fluoride (0.4g/L), and the passivating treatment time is 25 to 30 seconds and the treatment temperature is 20 to 25 ℃. The passivated material is plated with amorphous soft magnetic alloy, plating solutions are nickel sulfate (80-110g/L), ferrous sulfate (100-120g/L), boric acid (50-70g/L), nickel chloride (40-60g/L), zinc sulfate (10-30g/L) and sodium citrate (10-30g/L), after the plating solutions are prepared and placed for 48-72h, the water level and the pH value are adjusted, and a large cathode area and a low current density (0.1-0.5A/dm) are adopted2) The magnetic shield part 101 can be obtained by electroplating for 5-6 h.
Specifically, the temperature control driving unit 3 further includes a cooling medium control unit 32, the cooling module 22 is communicated with an output end of the cooling medium control unit 32, and the cooling medium control unit 32 controls the cooling module 22 to input and recover the cooling medium into the temperature control chamber 11. The cooling medium control unit 32 mainly includes a valve and components for controlling opening and closing of the valve and opening and closing amounts, such as an electric valve, an electromagnetic valve controller, a proportional electromagnetic valve, and a proportional electromagnetic controller, which are all cooling media input or recovered from the temperature control chamber 11 by electric signals.
More specifically, the cooling medium is liquid hydrogen, liquid nitrogen or hydraulic carbon dioxide, so that the temperature in the temperature control chamber 11 can be rapidly reduced through the cooling medium cooled at high pressure, and the temperature in the reaction chamber 10 can be rapidly reduced, thereby achieving the effect of rapid temperature reduction.
More specifically, as shown in fig. 7, the cooling assembly 22 includes a wind body 220 and a wind source 221, the wind source 221 contains a cooling medium, the wind body 220 is communicated with an output end of the wind source 221, the wind body 220 has an air outlet 2200 and an air return opening 2201, the cooling medium control unit 32 controls the cooling medium in the wind source 221 to flow into the wind body 220 and be input into the temperature control cavity 11 from the air outlet 2201, or controls the cooling medium in the temperature control cavity 11 to flow into the wind body 220 and flow into the wind source 221 through the air return opening 2201. Specifically, the air source 221 can hold a high-pressure-resistant and low-temperature-resistant pressure container of the cooling medium in real time, the air body 220 can be a container for releasing pressure of the hydraulic cooling medium, converting the pressure of the hydraulic cooling medium into a gas phase and buffering the pressure, the high-pressure liquid cooling medium of the air source 221 is converted into a proper hydraulic state and a proper temperature state, and the cooling medium with a certain flow and a certain temperature approximately in a gaseous state is released by the air body 220 to enter the temperature control cavity 11, so that the requirement for cooling the temperature control cavity 11 is met.
More specifically, the air body 220 is communicated with the output end of the air source 221 through a pipeline, and a pressure reducing valve or a pressure relief valve is arranged on the pipeline to reduce the pressure of the air in the air source 221 or change the phase state of the air. The air body 220 extends into the temperature control cavity 11 through a pipeline, the tail end of the air body is an air outlet 2200, and when cooling is not needed, the air body further passes through the pipeline extending into the temperature control cavity 11, and the tail end of the pipeline is an air return port 2201, so that cooling medium in the temperature control cavity 11 is pumped away. Thus, specifically, the air body 220 includes a buffer tank 2202, an air inlet pipe 2203, an air outlet pipe 2204, a return air pump 2205, an air inlet valve 2206 and an air inlet flow valve 2207 which are communicated with the air inlet pipe 2203, an air outlet valve 2208 and an air outlet flow valve 2209 which are communicated with the air outlet pipe 2204, and the air outlet pipe 2203 is communicated with the temperature control cavity 11 and the return air pump 2205.
Further, the PCR device provided by the present invention further comprises:
the transfusion mechanism 4 is used for communicating the reaction cavity 10, inputting reaction liquid and samples required by PCR into the reaction cavity 10 and/or cleaning the interior of the reaction cavity 10;
a detection electrode 5 for detecting a chemical signal, an electrical signal, or an optical signal generated by the binding of the target nucleic acid and the active substance in the reaction chamber 10;
the signal output end of the temperature control unit 2, the signal output end of the temperature control driving unit 3 and the signal output end of the infusion mechanism 4 are respectively in communication connection with the microprocessor 6;
the upper terminal 7 is in communication connection with the upper terminal;
the PCR chip 1 includes an immobilization layer, and is surface-treated with a capture probe formed in a region inside the reaction chamber 10 and capable of complementarily binding to a region of the nucleic acid to be amplified.
Therefore, the PCR chip 1 provided by the invention is of an integral chip structure, the reaction cavity 10 in the PCR chip is not disassembled and assembled when the PCR chip is used, the influence on the reaction cavity 10 in the PCR chip is reduced, and the requirement can be met when some special samples with biological safety hazards need to be treated. In view of its closed structure, in a further embodiment, as shown in FIGS. 8, 9 and 10, the PCR chip 1 has a communication port 13 communicating with the reaction chamber 10 and extending to the outside thereof, the communication port 13 including an infusion solution port 130, a sample port 131 and a washing port 132. Infusion port 130 is used for inputing the required raw materials of PCR reaction in the reaction chamber 10, and sample port 131 is used for inputing the sample that awaits measuring in the reaction chamber 10, through wash mouthful 132 and/sample mouth 121 inputed the washing liquid in reaction chamber 10 when wasing, retrieves the washing liquid through infusion port 130, can wash the reaction intracavity cleanly, satisfies the clean requirement of PCR reaction. Specifically, the invention provides a liquid conveying mechanism 4, which comprises a raw material box 40, a liquid conveying pipeline 41, a sample box 42, a sample pipeline 43, a cleaning box 44 and a cleaning pipeline 45, wherein the raw material box 40 is used for containing reaction raw materials, the raw material box 40 is connected to a liquid conveying port 130 through the liquid conveying pipeline 41 so as to be communicated with a reaction cavity 10, the sample box 42 is connected to a sample port 131 through the sample pipeline 43 so as to be communicated with the reaction cavity 10, the cleaning box 43 is communicated with the liquid conveying pipeline 41 through the cleaning pipeline 44, and valves are arranged on the liquid conveying pipeline 41, the sample pipeline 43 and the cleaning pipeline 45 so as to control the flow direction and the flow rate of fluid in the pipelines.
The detection electrode 5 is used for labeling molecules by adopting an electrochemical technology, or performing fluorescent molecular labeling, or coupling a molecular probe and the like on the surface of the electrode according to a molecular hybridization reaction, a molecular coupling reaction or specific molecular coordination, and scanning a labeled detection signal or an optical signal generated by fluorescent scanning detection by an alternating current voltammetry so as to realize the detection of a reaction process and a reactant.
The microprocessor 6 controls the detection motor to carry out gene detection, receives the scanning data of the detection mechanism, carries out result analysis, and transmits the analysis result to the upper terminal 7, and the upper terminal 7 adjusts the microprocessor in real time by sending a control instruction.
PCR device control method
In another embodiment of the present invention, as shown in FIGS. 10 and 11, the present invention provides a method for controlling a PCR device in the above embodiments, including a PCR reaction control method;
the PCR reaction control method comprises the following steps:
s11, acquiring the temperature in the reaction cavity through the temperature probe, converting the temperature into an electric signal and outputting the electric signal to the microprocessor, and outputting the electric signal to an upper terminal through conversion and amplification processing by the microprocessor;
s12, the upper terminal judges the stage of the PCR reaction in the reaction cavity and the maintaining time at the stage through a preset PCR reaction control program;
s13, the upper terminal outputs a control instruction of the PCR reaction stage to the microprocessor, and the microprocessor inputs the control instruction into a signal input end of a temperature control driving unit corresponding to the reaction cavity after judgment;
s14, the temperature control driving unit executes a control instruction of a PCR reaction stage, outputs a magnetic induction control signal through the magnetic induction heating control unit, controls the current in the temperature control piece and further controls the temperature of the temperature control layer on the surface of the reaction cavity; or the temperature control driving unit executes a control instruction of the PCR reaction stage, the cooling medium output control unit is output through the cooling medium control unit, the quantity of the cooling medium output by the cooling assembly is controlled, and the temperature of the temperature control layer on the surface of the reaction cavity is further controlled;
s15, the temperature probe acquires the temperature of the reaction cavity and the surface of the temperature control layer, and converts the temperature into an electric signal to be output to the microprocessor, the microprocessor outputs the electric signal to the upper terminal through conversion and amplification, and the upper terminal determines whether to output the control instruction of the PCR reaction stage again through the PCR reaction control program.
Further, as shown in fig. 12, the present invention provides the method for controlling the PCR apparatus in the above embodiment, further comprising a PCR chip cleaning method, the transfusion mechanism 4 further comprises a nucleic acid probe 46 for detecting the cleanliness in the reaction chamber;
the PCR chip cleaning method comprises the following steps:
s21, acquiring the cleanliness of the gene in the reaction cavity through the nucleic acid probe, converting the cleanliness of the gene into an electric signal, inputting the electric signal to the microprocessor, and outputting the electric signal to an upper terminal through conversion and amplification by the microprocessor;
s22, the upper terminal outputs a reaction cavity cleaning control instruction to the microprocessor through a preset reaction cavity cleaning program, and the microprocessor inputs the instruction into a signal input end of the infusion mechanism corresponding to the reaction cavity to be cleaned after judgment;
s13, the infusion mechanism executes a reaction cavity cleaning control instruction to clean the reaction cavity;
s14, the nucleic acid probe acquires the nucleic acid amount on the surface of the reaction cavity, converts the nucleic acid amount into an electric signal and outputs the electric signal to the microprocessor, the microprocessor outputs the electric signal to the upper terminal through conversion and amplification, and the upper terminal determines whether to output the reaction cavity cleaning control instruction again through the reaction cavity cleaning program.
Further, the present invention provides the method for controlling a PCR device in the above embodiment, further comprising a PCR reactant signal method, wherein the PCR reactant signal method comprises obtaining a chemical signal, an electrical signal, or an optical signal generated by binding of the target gene, the active substance, and the molecular probe on the surface of the detection electrode in the reaction cavity. For example, when a PCR raw material and a reagent solution are injected into the reaction chamber 10 by the infusion means 4 and the above-mentioned PCR reaction control method is continuously performed to perform the denaturation step and the annealing/elongation step in the reaction chamber, the amplified nucleic acid is sequentially detected and measured in real time at the detection electrode 5 for an electrochemical signal (current change) generated by complementary binding between the amplified nucleic acid and the capture probe and the signal probe of the complex. Therefore, the reaction result according to the amplification of the nucleic acid is monitored in real time (real-time) in the reaction chamber (without fluorescent substance and light detection system) during the progress of each cycle of PCR, thereby enabling real-time detection and measurement of the amount of the target nucleic acid.
Therefore, in the reaction chamber 10 of the PCR chip 1, the electrochemical signals repeatedly generated according to the sequential nucleic acid amplification can be respectively detected by the detection electrodes 5 of the PCR chip 1, and the detected signals are transmitted to the microprocessor, specifically, the signals are measured by the electrochemical signal measuring module included in the microprocessor, and then processed or analyzed. The electrochemical signal measuring module may be embodied in various forms, and may be selected from the group consisting of Anodic Stripping Voltammetry (ASV), Chronoamperometry (CA), cyclic voltammetry (cyclovoltammetry), Square Wave Voltammetry (SWV), square wave voltammetry (DPV), and impedance (impedance).
The technical scheme of the invention can independently perform hybridization detection on a plurality of reaction cavities by adopting electrochemical genes of electrochemical technology at most simultaneously, can perform independent temperature control on a reaction system in each reaction cavity so as to meet the temperature requirements of different hybridization detection stages and different detection items, and the temperature control of each module is required to be accurate and stable. To achieve this requirement, each reaction chamber is formed as a separate closed-loop temperature control unit, and the system comprises a comparison element based on an ARM9 processor, a control element based on PWM output control, an actuator based on a heating power tube and a feedback element based on high-precision sensing amplification and ADC.
The detection items of each element are processed and analyzed based on ARM9, the current target temperature of each module is obtained through the chip protocol and the hybridization stage of the item, the current temperature of a temperature control layer is obtained through a temperature feedback element (comprising a high-precision temperature sensor, a high-precision amplifier and a precision ADC), the temperature deviation and the control quantity are obtained through comparison and calculation, the execution element controls power to be output to a heating pipe, and the copper substrate is subjected to temperature control to obtain accurate and stable temperature. The copper substrate is in full contact with a chip inserted into the detection module, and the temperature is transmitted to the chip to provide a suitable temperature environment for chip hybridization.
The above-described embodiments of the present invention should not be construed as limiting the scope of the present invention. Any other corresponding changes and modifications made according to the technical idea of the present invention should be included in the protection scope of the claims of the present invention.
Claims (10)
1. A PCR device, comprising:
the PCR chip is provided with more than two reaction cavities and comprises a heat insulating layer arranged between every two adjacent reaction cavities;
the temperature control unit is used for forming a low magnetic induction area and an air cooling area on the periphery of each reaction cavity and comprises a temperature probe extending into the reaction cavity;
and the output end of the temperature control driving unit is connected with the temperature control unit and controls the periphery of the reaction cavity to form the low magnetic induction area and the air cooling area.
2. The PCR device of claim 1, wherein the PCR chip further comprises a temperature control chamber surrounding each reaction chamber, and the temperature control unit comprises a temperature control member extending into the temperature control chamber or surrounding the reaction chamber and a cooling member communicating with the temperature control chamber;
the PCR chip also comprises a temperature control layer positioned between the temperature control cavity and the reaction cavity, the temperature control layer is made of a high-thermal-conductivity rust-resistant material, and the temperature probe is provided with one end extending to the surface of the temperature control layer;
the temperature control cavity is in a near vacuum state.
3. The PCR device as claimed in claim 1, wherein the temperature control driving unit comprises a magnetic induction heating control unit, the temperature control member surrounds the periphery of the reaction chamber, the temperature control member is connected with the magnetic induction heating control unit, and the magnetic induction heating control unit controls the current change in the temperature control member.
4. The PCR device of claim 1, wherein the temperature-controlled driving unit further comprises a cooling medium control unit, the cooling module is communicated with an output end of the cooling medium control unit, and the cooling medium control unit controls the cooling module to input and recover a cooling medium into the temperature-controlled chamber.
5. The PCR device according to claim 4, wherein the cooling medium is liquid hydrogen, liquid nitrogen or hydraulic carbon dioxide.
6. The PCR device as claimed in claim 5, wherein the cooling module comprises a wind body and a wind source, the wind source contains the cooling medium, the wind body is communicated with the output end of the wind source, the wind body has a wind outlet and a wind return opening, the cooling medium control unit controls the cooling medium in the wind source to flow into the wind body and be input into the temperature control cavity from the wind outlet, or controls the cooling medium in the temperature control cavity to enter into the wind body through the wind return opening and flow into the wind source.
7. The PCR device of claim 6, further comprising:
the transfusion mechanism is used for communicating the reaction cavity, inputting reaction liquid and samples required by PCR into the reaction cavity and/or cleaning the reaction cavity;
a detection electrode for detecting a chemical signal, an electrical signal, or an optical signal generated by the binding of the target nucleic acid and the active substance in the reaction chamber;
the signal output end of the temperature control unit, the signal output end of the temperature control driving unit and the signal output end of the infusion mechanism are respectively in communication connection with the microprocessor;
and the upper terminal is in communication connection with the upper terminal.
8. A method for controlling a PCR device according to any one of claims 1 to 7, comprising a PCR reaction control method;
the PCR reaction control method comprises the following steps:
s11, acquiring the temperature in the reaction cavity through the temperature probe, converting the temperature into an electric signal and outputting the electric signal to the microprocessor, wherein the microprocessor outputs the electric signal to the upper terminal through conversion and amplification;
s12, the upper terminal judges the stage of the PCR reaction in the reaction cavity and the maintaining time at the stage through a preset PCR reaction control program;
s13, the upper terminal outputs a PCR reaction stage control instruction to the microprocessor, and the microprocessor inputs the PCR reaction stage control instruction to a signal input end of the temperature control driving unit corresponding to the reaction cavity after judgment;
s14, the temperature control driving unit executes the control instruction of the PCR reaction stage, and outputs a magnetic induction control signal through the magnetic induction heating control unit to control the current in the temperature control piece so as to control the temperature of the temperature control layer on the surface of the reaction cavity; or the temperature control driving unit executes the control instruction of the PCR reaction stage, and the cooling medium output control unit is output through the cooling medium control unit to control the amount of the cooling medium output by the cooling assembly, so as to control the temperature of the temperature control layer on the surface of the reaction cavity;
and S15, the temperature probe acquires the temperatures of the reaction cavity and the surface of the temperature control layer, converts the temperatures into electric signals and outputs the electric signals to the microprocessor, the microprocessor outputs the electric signals to the upper terminal through conversion and amplification, and the upper terminal determines whether to output the control instruction of the PCR reaction stage again through a PCR reaction control program.
9. The method for controlling a PCR apparatus according to claim 8, further comprising the PCR chip washing method, wherein the detection electrode further comprises a nucleic acid probe for detecting cleanliness in the reaction chamber;
the PCR chip cleaning method comprises the following steps:
s21, acquiring the gene cleanliness in the reaction cavity by the nucleic acid probe, converting the gene cleanliness into an electric signal, inputting the electric signal to the microprocessor, and outputting the electric signal to the upper terminal by the microprocessor through conversion and amplification;
s22, the upper terminal outputs a reaction cavity cleaning control instruction to the microprocessor through a preset reaction cavity cleaning program, and the microprocessor inputs the instruction into a signal input end of the infusion mechanism corresponding to the reaction cavity to be cleaned after judgment;
s23, the infusion mechanism executes the reaction cavity cleaning control instruction to clean the reaction cavity;
and S24, the nucleic acid probe acquires the amount of nucleic acid on the surface of the reaction cavity, converts the nucleic acid into an electric signal and outputs the electric signal to the microprocessor, the microprocessor outputs the electric signal to the upper terminal through conversion and amplification, and the upper terminal determines whether to output a reaction cavity cleaning control instruction again through a reaction cavity cleaning program.
10. The method for controlling a PCR device according to claim 8 or 9, further comprising a PCR reactant signal method including obtaining a chemical signal, an electrical signal, or an optical signal generated by binding of a target gene, an active substance, and a molecular probe on the surface of the detection electrode in the reaction chamber.
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