CN111349555B - Digital PCR amplification device based on micropore array chip and use method thereof - Google Patents

Digital PCR amplification device based on micropore array chip and use method thereof Download PDF

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CN111349555B
CN111349555B CN201811570499.4A CN201811570499A CN111349555B CN 111349555 B CN111349555 B CN 111349555B CN 201811570499 A CN201811570499 A CN 201811570499A CN 111349555 B CN111349555 B CN 111349555B
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array chip
micro
pcr amplification
microwell
chip body
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CN111349555A (en
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岳耀斐
王晓飞
张志峰
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Chengdu Wanzhong One Core Biotechnology Co ltd
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Chengdu Wanzhong One Core Biotechnology Co ltd
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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
    • B01L7/52—Heating or cooling apparatus; Heat insulating devices with provision for submitting samples to a predetermined sequence of different temperatures, e.g. for treating nucleic acid samples
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
    • B01L3/502769—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by multiphase flow arrangements
    • B01L3/502784—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by multiphase flow arrangements specially adapted for droplet or plug flow, e.g. digital microfluidics
    • 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
    • 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
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6851—Quantitative amplification
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/12—Specific details about manufacturing devices
    • 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
    • B01L2300/12—Specific details about materials
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Abstract

The invention provides a digital PCR amplification device based on a micro-pore array chip and a use method thereof. The device comprises an upper sealing cover, a middle micropore array and a lower biochemical sensor chip. When in use, the liquid to be detected injected into the micropores contacts the sensor below the inside of the micropores, and the liquids in different micropores are sealed by the sealing cover to be isolated from each other. The invention can separate the PCR reaction into tens of thousands of tiny units, carry out the PCR expansion reaction of a single molecule sample in the micropore, and detect the number of positive reaction units by detecting the concentration change of hydrogen ions, phosphate ions or pyrophosphate ions released in the PCR process, thereby accurately quantifying the DNA sample. The invention adopts the highly integrated semiconductor chip to finish the detection of the digital PCR, not only greatly reduces the reaction system and miniaturizes the instrument, but also overcomes the dependence on fluorescence excitation and detection devices, avoids the use of fluorescent dye or fluorescent probe, and greatly reduces the instrument and reagent cost of the digital PCR.

Description

Digital PCR amplification device based on micropore array chip and use method thereof
Technical Field
The invention belongs to the field of nucleic acid detection, and particularly relates to a digital PCR detection method based on a micro-pore array chip.
Background
Modern biological research, particularly medical research, often involves the need for quantitative analysis of nucleic acids. For example, detection of certain free DNA and its content in blood can guide clinical diagnosis of certain cancers, as well as monitor the therapeutic effects of cancers. In the past, the relative quantification is mainly carried out by adopting fluorescent quantitative PCR, namely, a transcript of a gene with stable expression is selected as a reference to judge the quantity of target nucleic acid. However, this method is susceptible to interference by non-target nucleic acid molecules (background noise), is only suitable for qualitative or low-precision detection, and cannot meet the detection of some target nucleic acid molecules with particularly rare contents.
Digital PCR (dPCR) can then be amplified by large-scale parallel PCR, extracting the weak amplified signal from the background noise, counting the number of amplified molecules with or without an "endpoint signal". So far, a wide variety of dPCR devices have been developed, mainly well plate and droplet.
The orifice plate dPCR is the implementation of digital amplification by discretizing nucleic acid molecules on a sufficient number of plates arranged in an array of microwells of the same size. For example: this is the case with the Quantum 3D dPCR system from ThermoFisher. Etching up to 20000 hexagonal micropores with a volume of 0.8nL on a square plate with a side length of 10mm, realizing isolation among the micropores, and applying a sample by simple coating, wherein each micropore has about 1 target nucleic acid molecule; after PCR, the fluorescent signal in the well can be detected, and the number of target molecules is calculated through poisson distribution.
Drop-type dPCR is to divide a sample by tens of thousands of monodisperse droplets, which disperse DNA with each water-in-oil droplet by means of a hydrophobic tube and an oil phase liquid, one droplet theoretically containing only one DNA molecule; the amplified DNA can be transferred to a fluorescence detection device for final detection. The disadvantage of this method is that droplets are prone to merge with each other during the PCR process or transfer process, resulting in inaccurate results.
The above techniques of dPCR all use fluorescent signals to read the results, which requires expensive fluorescence excitation devices and consumable, fluorescent high-resolution detection devices in the set-up. In addition, the difficulty of judging the result is also increased by the mutual pollution of the optical signals.
Disclosure of Invention
In order to solve the problems, the invention provides a digital PCR amplification method based on a microwell array chip.
In order to solve the technical problems, the invention provides a digital PCR amplification device based on a micro-pore array chip, which comprises a micro-pore array chip body, wherein the micro-pore array chip body comprises a sealing cover, a micro-pore array and a biochemical sensor, the sealing cover is arranged above the micro-pore array and is used for sealing micro-pores of the micro-pore array and isolating the micro-pores from each other, the biochemical sensor is arranged at the bottom of the micro-pores, and the sealing cover or the biochemical sensor is provided with an electrode for providing voltage for a solution in the micro-pores.
Further, the micropore array contains 0.1 to 1000 tens of thousands of micropores, and the volume of each micropore is 1fL to 10pL.
Further, the surface of the biochemical sensor is coated with a hydrogen ion sensitive material, phosphate ion or pyrophosphate ion sensitive material.
Further, the hydrogen ion sensitive material is SiO 2 、Al 2 O 3 、HfO 2 、TiO 2 、Ta 2 O 5 One or more of (3-aminopropyl) triethoxysilane and triethoxysilylundecanal.
Further, the phosphate ion or pyrophosphate ion sensitive material is phosphate ion organic membrane ISM or molecular probe.
Further, the biochemical sensor is one of an ion sensitive field effect transistor, a nanowire, graphene or molybdenum disulfide transistor sensor, or a miniature electrochemical sensor.
Further, the device also comprises a reading device which is electrically connected with the computer to read the electric signals of the micro-pore biosensor after the micro-pore array chip body PCR amplification.
The reading device comprises a base and an upper cover, wherein a groove for accommodating the micro-hole array chip body is formed in the base, one side edge of the upper cover is hinged to the edge of the base, a probe which is electrically connected with a biochemical sensor pin pad of the micro-hole array chip body is arranged in the base, and a circuit board is arranged below the base and used for connecting the probe, so that power supply to the micro-hole array chip body and data reading of biochemical sensor electric signals are realized;
the circuit board comprises a data acquisition module, an ADC chip and a processor, wherein the data acquisition module acquires an electric signal of the biochemical sensor at the bottom of the micropore, and the electric signal is subjected to analog-to-digital conversion by the ADC chip and then is transmitted to the processor for digital signal processing and calculating the concentration of amplified DNA; the processor is an FPGA, a DSP or an ARM.
As another technical scheme, the reading device comprises a card inserting circuit board and a data processing circuit board;
the card inserting circuit board comprises a containing bin for containing the micro-pore chips, a gland arranged at the mouth of the containing bin, a signal amplifier chip for amplifying the signals of the biochemical sensors on the micro-pore chips and a plug for communicating with the data processing circuit board;
the data processing circuit board comprises a slot, an ADC analog-to-digital conversion chip, a microprocessor MCU and a communication port, wherein the slot is matched with the plug, the communication between the card inserting circuit board and the data processing circuit board is realized through the connection between the plug and the slot, the ADC analog-to-digital conversion chip is respectively connected with the signal amplifier chip and the microprocessor MCU, and the microprocessor MCU is connected with the terminal equipment through the communication port.
The invention also provides a using method of the digital PCR amplification device based on the micro-pore array chip, which comprises the following steps:
step one, sample adding: uniformly mixing the DNA reaction system, dripping the mixture onto a micropore array chip body without a sealing cover, and sealing the micropore array chip body with the sealing cover;
step two, amplification: placing the micropore array chip body on a constant temperature PCR instrument, a variable temperature PCR instrument or a thermal circulator for amplification reaction;
step three, signal reading: and placing the microwell array chip body after the amplification reaction in a reading device, reading the electric signal of the biochemical sensor at the bottom of the microwell, and obtaining the initial concentration of the DNA sample after data processing and displaying the initial concentration on a computer or a display screen.
The third step specifically comprises the following steps:
3.1 histogram analysis: firstly, obtaining a plurality of frames of two-dimensional thermodynamic diagram data of DNA samples with various concentrations corresponding to a micropore array chip body; then, respectively obtaining a frame of mean value thermodynamic diagram for the data of each micro-pore array chip body, and carrying out Histogram analysis on each frame of data so as to obtain a distribution peak: if the PCR amplification reaction does not occur in the microwells, the pH value in the microwells is unchanged, the output value of the sensor is 0, and the microwells without the PCR amplification reaction are negative reaction units; if a single DNA sample amplification reaction occurs in the microwells, the pH value in the wells changes, and the microwells with the single DNA sample amplification reaction are positive reaction units; if the pH value change in the microwell is larger than the pH value change caused by the amplification reaction of a single DNA sample in the microwell, carrying out PCR amplification reaction on a plurality of DNA samples in the microwell;
3.2 Calculation of initial concentration of DNA sample: the initial concentration of the DNA sample is calculated from the number of positive reaction units or the number of negative reaction units obtained in step 3.1.
Further, the specific process of step 3.2 is:
drawing a standard curve: drawing a standard curve C=f (N) according to the concentration of each group of DNA samples and the corresponding number N of positive units;
and (3) data calculation: after the DNA sample with the concentration to be detected is subjected to PCR amplification reaction, firstly, the number of all positive cells on the micro-pore array chip body is calculated according to the step 3.1, and then, the initial concentration of the DNA sample is calculated according to a standard curve drawn in advance in the step 3.2.
Alternatively, the specific process of step 3.2 is: and carrying the number of positive reaction units or the number of negative reaction units obtained by the histogram analysis into a poisson equation to calculate the initial concentration of the DNA sample.
Further, the thermal cycler comprises an end cover and a base, wherein the end cover is movably arranged on the top of the base; the base comprises a placing cavity for accommodating the micropore array chip body, a heat conducting block arranged close to the bottom of the placing cavity, a temperature sensor arranged at the upper end part of the heat conducting block and close to the placing cavity, a heater arranged at the bottom of the heat conducting block, and a radiator arranged at the bottom of the heater; the two sides of the heat conducting block are fixed in the base through limiting plates, the heater is fixed at the bottom of the heat conducting block through a positioning plate, and the temperature sensor and the heater are connected with the main control board through a control circuit.
The invention achieves the beneficial technical effects that:
the invention directly senses the concentration of hydrogen ions, phosphate ions or pyrophosphoric acid ions released in the amplification reaction process through the electric signal without fluorescent staining of the reagent, thereby overcoming the defect that the current mainstream digital PCR utilizes a fluorescent method to detect, namely: the optical signals pollute each other, resulting in poor signal reading accuracy. And by employing the number of large-scale sensor arrays (e.g., hundreds of thousands, even millions), the accuracy, sensitivity, and dynamic range of the digital PCR method is improved.
The invention not only greatly reduces the reaction system and greatly reduces the usage amount of reagents, but also eliminates a fluorescence excitation device and a high-resolution fluorescence detection device which are necessary to be used in mainstream digital PCR, thereby realizing the miniaturization of the instrument; and the use of fluorescent dye or fluorescent probe is avoided, so that the cost of digital PCR is greatly reduced.
After overcoming the above-mentioned drawbacks of the prior art, the present invention actually achieves a very high absolute quantitative accuracy and dynamic range of DNA, while at the same time greatly reducing the cost and operating time.
In summary, the PCR amplification device provided by the invention is provided with the same DNA fragment in each microwell when in use, namely, the single PCR amplification is realized. Secondly, when data are read, DNA copy number can be obtained by carrying out static test on the PCR amplification for two times.
Drawings
FIG. 1 is a schematic diagram of a micro-hole array chip body according to the present invention; wherein h is a general structure schematic diagram, i is a cross-sectional view along a line B-B in the diagram h;
FIG. 2 is a schematic diagram of a chip-holder type reader according to the present invention; wherein a is an open side view, b is an open perspective view, C is a closed schematic view, d is a sectional view along A-A in the view C, e is a closed perspective view, f is a closed top view, and g is a partially enlarged schematic view of C in the view d;
FIG. 3 is a block diagram of a chip-holder reader according to the present invention;
FIG. 4 is a schematic diagram of a PCR amplification reaction according to the present invention; wherein j is a schematic diagram before amplification, and k is a schematic diagram of an amplification process;
FIG. 5 is an exemplary data digital two-dimensional thermodynamic diagram of embodiment 1 of the present invention;
FIG. 6 shows a histogram of a sensor signal history of embodiment 1 of the present invention;
FIG. 7 is a graph of the number of positive units versus DNA concentration standard according to the present invention;
FIG. 8 is a schematic diagram of a thermal cycler structure of the present invention;
FIG. 9 is a schematic perspective view of a thermal cycler of the present invention;
FIG. 10 is a schematic diagram of a card reader according to the present invention; wherein 7 is a general structural schematic diagram, and 71 is a schematic diagram of a card circuit board structure;
FIG. 11 is a block diagram of a card reader of the present invention.
Wherein: 1 a micropore array chip body; 11 micropores; 12 sealing covers; 13 micro-hole walls; a 14 biochemical sensor; 2 a chip-holder type reading device; 21 an upper cover; 22 probes; a 23 base; 24 circuit boards; 3 a target DNA fragment; 4 PCR amplification enzyme; 5 primer pairs; 6 a thermal cycler; 61 end caps; 62 a heat conducting block; 63 heaters; 64 heat sinks; 65 main control board; 66 limiting plates; 67 positioning plates; 68 placing the cavity; 69 temperature sensor. 7, a card-inserting type reading device; 71 a card circuit board; 72 a data processing circuit board; 711 capping; 712 a holding bin; 713 signal amplifier chips; 714 a plug; 721 slots; 722 A USB port; 723 serial port; 724 An ADC analog-to-digital conversion chip; 725 a microprocessor MCU;726 parallel ports.
Detailed Description
The invention is further described below in connection with specific embodiments. The following examples are only for more clearly illustrating the technical aspects of the present invention, and are not intended to limit the scope of the present invention.
The patent of the invention is further described below with reference to the drawings and examples.
As shown in fig. 1, the invention provides a digital PCR amplification device based on a microwell array chip, comprising a microwell array chip body 1, wherein the microwell array chip body 1 comprises a sealing cover 12, a microwell array 11 and a biochemical sensor 14, the sealing cover 12 is arranged above the microwell array for sealing the microwells 11 of the microwell array and isolating each microwell, the biochemical sensor 14 is arranged at the bottom of the microwell, and the sealing cover 12 or the biochemical sensor 14 is provided with an electrode for providing voltage to a solution in the microwell 11. The microwell array contains 0.1-1000 ten thousand microwells 11, the volume of each microwell 11 is 1 fl-10 pL, after DNA sample and amplification liquid are added into microwells 11, the microwells 11 are tightly pressed and sealed by a sealing cover 12, so that the liquids in different microwells 11 are isolated from each other, and each microwell 11 is internally provided with a target cost-effective molecule, thereby realizing the single-molecule PCR amplification of DNA samples
The biosensor 14 is coated with a hydrogen ion sensitive material, such as SiO 2 、Al 2 O 3 、HfO 2 、TiO 2 、Ta 2 O 5 Or (3-aminopropyl) triethoxysilane, or phosphate ion or pyrophosphate ion sensitive materials, e.g. phosphate ion organic membranes ISM or C4- [3]]Molecular probes such as rotaxane and cyanostar.
As a preferred embodiment, the biosensor 14 is an ISFET, i.e., an ion sensitive field effect transistor, produced by standard CMOS semiconductor processes. The metal floating gate of the ISFET has deposited thereon a hydrogen ion sensitive material, such as SiO by Atomic Layer Deposition atomic layer deposition or sputtering 2 、Al 2 O 3 、HfO 2 、TiO 2 Or Ta 2 O 5 . The deposition of the organic molecule (3-Aminoopyl) triethoxycilane, etc. by solution or vapor methods is also possible. The sensitive materials can detect hydrogen ions released in the PCR amplification reaction process or the transient reduced pH value of the reaction solution. The change of the pH value changes the current in the channel of the ISFET sensor or the voltage between the source and the drain, and then the signal is transmitted through the integrated circuit on the micro-pore array chip body. The proper operation of ISFETs also requires the supply of a voltage to the reaction solution, also known as the solution gate voltage. In this example, the solution electrode is formed using the upper metal layer of the CMOS chip at the gap location between the microwells. Conductive sealing caps, such as conductive silicone, may also be used to provide electrodes to the solution.
In addition, the upper surface of the gate electrode of ISFET may be coated with a probe or a film, such as phosphate organic film ISM, molecular probe C4-3 rotaxane or cyanostar, with selectivity to or through phosphate ions. These materials can detect phosphate ions released during the PCR amplification reaction, resulting in a current change in the channel of the ISFET sensor, or a voltage change between the source and drain.
As another preferred aspect of the present invention, the biosensor 14 samples field effect transistor nano FETs with emerging nanowires as channels or FETs with two-dimensional semiconductor materials such as graphene or molybdenum disulfide as channels, which can provide higher sensitivity than ISFETs, thereby providing smaller reaction cavities, higher integration and accuracy;
as another preferred embodiment of the present invention, the biochemical sensor 14 may also employ conventional electrodes to detect hydrogen ions or phosphate ions, i.e., a micro electrochemical sensor, which may greatly reduce the cost of the present invention.
In addition, the amplification device provided by the invention further comprises a reading device which is electrically connected with the computer to read the electric signals of the micro-pore biological sensor after the micro-pore array chip body PCR amplification.
As a preferred technical solution, as shown in fig. 2-3, the reading device is a chip-base-based chip-base reading device 2, and specifically includes a base 23 and an upper cover 21, a groove for accommodating the micro-hole array chip body 1 is provided in the base 23, one side edge of the upper cover 21 is hinged at the edge of the base 23, a probe 22 electrically connected with a pin pad of the biochemical sensor 12 of the micro-hole array chip body 1 is provided in the base 23, and a circuit board 24 is provided below the base 23 for connecting the probe 22, so as to realize power supply to the micro-hole array chip body 1 and data reading of an electric signal of the biochemical sensor 14;
the circuit board 24 comprises a data acquisition module, an ADC chip and a processor, wherein the data acquisition module acquires the electric signals of the biochemical sensor at the bottom of the micropore, and the electric signals are transmitted to the processor for digital signal processing after analog-to-digital conversion by the ADC chip, and the concentration of amplified DNA is calculated; the processor is an FPGA, a DSP or an ARM.
As another technical solution, as shown in fig. 10, the reading device is a card-inserting type reading device 7 based on a card-inserting type chip PCB circuit board, and specifically includes a card-inserting circuit board 71 and a data processing circuit board 72;
the card-inserting circuit board 71 comprises a holding bin 712 for holding the micro-porous chip 1, a gland 711 arranged at the bin opening of the holding bin 712, a signal amplifier chip 713 for amplifying the signal of the biochemical sensor 14 on the micro-porous chip, and a plug 714 for communicating with the data processing circuit board 72; when in use, the pins of the biochemical sensor 14 are welded on the card-inserting circuit board 71 to realize circuit connection of the biochemical sensor 14, the signal amplifier chip 713 amplifies the electric signal output by the biochemical sensor 14, and the electric signal is transmitted to the ADC analog-to-digital conversion chip 724 of the data processing circuit board 72 after the signal-to-noise ratio is improved;
the data processing circuit board 72 includes a slot 721, an ADC analog-to-digital conversion chip 724, a microprocessor MCU725 and a communication port, where the slot 721 is matched with the plug 714, the connection between the plug 714 and the slot 721 is used to implement communication between the card inserting circuit board 71 and the data processing circuit board 72, the ADC analog-to-digital conversion chip 724 is respectively connected to the signal amplifier chip 713 and the microprocessor MCU725, and the microprocessor MCU725 is connected to a terminal device, which may be a computer or a smart phone, through a communication port such as a USB port 722, a serial port 723 or a parallel port 726; when in use, the plug 714 of the card circuit board 71 is only required to be inserted into the slot 721 of the data processing circuit board 72, so that the card circuit board 71 and the data processing circuit board 72 can be in communication connection, and further the data processing circuit board is used for carrying out data processing on the signals received by the biochemical sensor 14, so that the concentration of amplified DNA is obtained and calculated.
The invention also provides a using method of the digital PCR amplification device based on the micro-pore array chip, which comprises the following steps:
step one, sample adding: the DNA reaction system is evenly mixed and then is dripped on the micro-pore array chip body 1, which is specifically as follows: diluting the target DNA fragment 3 by a certain proportion, uniformly mixing the target DNA fragment 3 with buffer solution containing a primer 5 pair and an amplifying enzyme 4, adding the mixture into micropores 11 on a chip, compacting and sealing the mixture by a sealing cover, and forming a PCR reaction unit isolated from each other by each micropore 11; as shown in fig. 4 j;
step two, amplification: the micro-pore array chip body 1 after the sample is added in the first step can carry out constant temperature PCR and variable temperature PCR amplification reaction, for example, the micro-pore array chip body 1 is placed on a variable temperature PCR instrument or a thermal circulator 6 to realize variable temperature PCR amplification reaction, and the micro-pore array chip body 1 is placed on a constant temperature PCR instrument or a constant temperature table to realize constant temperature PCR amplification reaction; placing the sealed micropore array chip body 1 into a PCR instrument or a thermal circulator for amplification reaction; the target DNA fragment 3 in each microwell 11 undergoes a chain amplification reaction under the action of the primer pair 5 and the amplifying enzyme 4, the copy number is continuously increased, and hydrogen ions and phosphate ions are released, as shown in FIG. 4 k;
wherein, the PCR instrument is plate PCR or is matched with a PCR adapter to amplify in a traditional PCR instrument, such as a 96-well plate;
as shown in fig. 8 to 9, the thermal cycler 6 includes an end cap 61 and a base, the end cap 61 being movably mounted on top of the base; the base comprises a placing cavity 68 for accommodating the micro-hole array chip body 1, a heat conducting block 62 arranged close to the bottom of the placing cavity 68, a temperature sensor 69 arranged at the upper end part of the heat conducting block 62 close to the placing cavity 68, a heater 63 arranged at the bottom of the heat conducting block 62, and a radiator 64 arranged at the bottom of the heater 63; the two sides of the heat conducting block 62 are fixed in the base through limiting plates 66, the heater 63 is fixed at the bottom of the heat conducting block 62 through a positioning plate 67, and the temperature sensor 69 and the heater 63 are connected with a main control board 65 through a control circuit. When in use, the main control board 65 sets the temperature in the placing cavity 68 according to the requirement of the PCR amplification reaction, the temperature sensor 69 is used for monitoring the temperature in the placing cavity 68 and transmitting the temperature to the main control board 65, and the main control board 65 sends a heating or heating stopping command to the heater 63 according to the actual temperature in the placing cavity 68; wherein the heater 63 may be a semiconductor heater or a thermal resistor.
Step three, signal reading: placing the microwell array chip body after the amplification reaction in a groove of a reading device, reading an electric signal of a biochemical sensor at the bottom of a microwell, and displaying the concentration of amplified DNA on a computer or a display screen after data processing; the pin pad of the biochemical sensor at the bottom of the micro-hole array chip body is electrically connected with the probe in the base, and then the probe is connected into the circuit board, so that the chip body is powered; then the data acquisition module acquires the electric signals of the biochemical sensor at the bottom of the micropore, the ADC chip carries out analog-to-digital conversion and then generates signals to FPGA, DSP, ARM and other processors for processing and carrying out digital signal calculation processing, and then the initial concentration of the DNA sample obtained by calculation is sent to a display screen for display;
the signal reading specifically comprises the following steps:
3.1 histogram analysis: firstly, obtaining a plurality of frames of two-dimensional thermodynamic diagram data of DNA samples with various concentrations corresponding to a micropore array chip body; then, respectively obtaining a frame of mean value thermodynamic diagram for the data of each micro-pore array chip body, and carrying out Histogram analysis on each frame of data so as to obtain a distribution peak: if the PCR amplification reaction does not occur in the microwells, the pH value in the microwells is unchanged, the output value of the sensor is 0, and the microwells without the PCR amplification reaction are negative reaction units; if a single DNA sample amplification reaction occurs in the microwells, the pH value in the wells changes, and the microwells with the single DNA sample amplification reaction are positive reaction units; if the pH value change in the microwell is larger than the pH value change caused by the amplification reaction of a single DNA sample in the microwell, carrying out PCR amplification reaction on a plurality of DNA samples in the microwell;
3.2 Calculation of initial concentration of DNA sample: the initial concentration of the DNA sample is calculated from the number of positive reaction units or the number of negative reaction units obtained in step 3.1.
As a preferred technical solution in the present application, step 3.2 may be calculated by means of a standard curve:
firstly, drawing a standard curve C=f (N) according to the concentration of each group of DNA samples and the corresponding number N of positive units;
then, after the DNA sample with the concentration to be detected is subjected to PCR amplification reaction, firstly, the number of all positive cells on the micro-pore array chip body is calculated according to the step 3.1, and then, the initial concentration of the DNA sample is calculated according to the standard curve drawn in the step 3.2.
As another preferred embodiment of the present application, step 3.2 may also be calculated using poisson's equation:
and carrying the number of positive reaction units or the number of negative reaction units obtained by the histogram analysis into a poisson equation to calculate the initial concentration of the DNA sample.
Example 1
In order to better illustrate the use method of the amplification device provided by the invention, a specific description will be given by taking a micro-pore array chip body of a CMOS ISFET as a biochemical sensor as an example, and specific indexes are shown in Table 1:
TABLE 1 index of micro-pore array chip body
Sensor type ISFET
Microwell and sensor count 26 ten thousand
Micropore size (length, width, depth) 2 microns, 1.5 microns
Sensor surface sensing material type Ta 2 O 5
Sensor surface sensing material thickness 20nm
Detection of signal type of PCR Hydrogen ion concentration, or pH variation
1. Loading sample
As shown in FIG. 4, 6 target DNA solutions (0.01 PM,0.1PM,1PM,10PM,100PM,1 NM) of different known concentrations were mixed uniformly with PCR amplification solutions containing DNA polymerase, dNTPs, and primers, respectively. The mixing amounts and ratios of the solutions are shown in Table 2, and a 10. Mu.L system can be used for four chips. The chip is put into a PCR conical adapter shown in fig. 8, 2.5uL of the mixed solution is dripped onto the chip (each specific concentration solution corresponds to one chip), so that each micropore contains the mixed solution, then the surface of the semiconductor chip is sealed by a sealing cover, the upper cover of the PCR adapter is closed, and a sealing pressing sheet is tightly sealed with the upper surface of each micropore of the chip, so that each micropore of the chip body is an independent reaction space.
Table 2: preparation of PCR solution
DNA Template 0.01pM to 1nM
PCR MIX 5ul
Primer Proper amount of
Deionization H 2 O To 10ul
PCR amplification reaction
One or more PCR adapters containing a chip body are placed in holes of a 96-well plate on a common PCR instrument, the adapter base is made of metal or heat conducting material, the chip body is subjected to heat conduction, PCR amplification is carried out, and the amplification conditions are shown in table 3:
table 3: PCR amplification conditions
3. Signal reading
After the PCR amplification is finished, the chip with the sealing tablet is taken out from the PCR adapter (kept in a sealing state), then is placed in a socket of a reading device shown in fig. 2-3, the pins of the chip are connected through a circuit of the reading device, and the electric signals of all the sensors in the micropores are read out to a computer or a display screen through data processing.
In the process of data driving of the chip body, 26 ten thousand data are generated at each time point and used as a frame, and 26 biochemical sensors are respectively corresponding to the data. As shown in fig. 5, the data are formed into a two-dimensional thermodynamic diagram by 512 rows by 512 columns, and the color of each pixel corresponds to the magnitude of the output signal of the corresponding sensor, i.e., the pH value in its corresponding microwell. Multiple time points may produce a multi-frame two-dimensional thermodynamic diagram.
The specific process is as follows:
for 6 samples of each initial DNA concentration (c1=0.01 pM to 1 nM), corresponding 6 chip bodies, each of which obtained several frames of two-dimensional thermodynamic diagram data. Firstly, a frame of average value thermodynamic diagram is respectively obtained for the data of each chip body. Taking one frame of data as an example, as shown in fig. 6, histogram analysis is performed on each frame of data to obtain a plurality of distribution peaks: the pH in the microwells without PCR is not changed, the sensor output is 0, and the corresponding microwells are negative reaction units, namely negative sensors; secondly, the pH in the micropores with the single DNA sample subjected to PCR reaction changes, and the corresponding micropores are positive reaction units, namely positive sensors; there are also small peaks with a larger pH change (greater sensor signal), i.e.a plurality of DNA samples in the microwells where PCR reactions occur, and the number of interfering units is small if the dilution ratio of the solution is appropriate.
Each initial DNA concentration (c1=0.01 pM to 1 nM) and the corresponding number N of positive sensors were plotted as a standard curve c=f (N) as shown in fig. 7.
And then carrying out standard curve equation according to the number of positive sensors calculated by the signal of the sample to be detected on the chip body, and calculating to obtain the initial copy concentration (C2) of the DNA sample. And comparing the result of the Poisson's formula calculation, wherein the measured DNA copy concentration C2 is closer to the actual copy concentration C1, so that the obtained standard curve is verified to be consistent with the Poisson's distribution formula.
Example 2
The embodiment uses Ta 2 O 5 As a biochemical sensor.
The DNA solution at a concentration of about 5pM was mixed with a PCR amplification solution containing DNA polymerase, dNTPs and primers uniformly. And the mixed solution is dripped into the belt Ta 2 O 5 In the sample loading process, the solution to be measured is diluted appropriately to make the number of DNA fragments in the DNA solution to be measured smaller than or equal to the number of the microwells (26 ten thousand) of the chip, so that only one DNA fragment is present in the microwells as much as possible.
The whole process of digital PCR was then completed according to the procedure of example 1, giving a positive sensor number N of 13672, corresponding to a concentration of about 4.8pM on the standard curve in FIG. 7, close to the actual concentration. The present invention has been described to provide a good absolute quantification of DNA.
The present invention has been disclosed in the preferred embodiments, but the invention is not limited thereto, and the technical solutions obtained by adopting equivalent substitution or equivalent transformation fall within the protection scope of the present invention.

Claims (12)

1. A method of using a microwell array chip-based digital PCR amplification device, the device comprising: the micro-pore array chip body comprises a sealing cover, a micro-pore array and a biochemical sensor, wherein the sealing cover is arranged above the micro-pore array and is used for sealing micro-pores of the micro-pore array and isolating the micro-pores from each other, the biochemical sensor is arranged at the bottom of the micro-pores, and an electrode for providing voltage for a solution in the micro-pores is arranged on the sealing cover or the biochemical sensor;
the method comprises the following steps:
step one, sample adding: uniformly mixing the DNA reaction system, dripping the mixture onto a micropore array chip body without a sealing cover, and sealing the micropore array chip body with the sealing cover;
step two, amplification: placing the micropore array chip body on a constant temperature PCR instrument, a variable temperature PCR instrument or a thermal circulator for amplification reaction;
step three, signal reading: placing the amplified microwell array chip body in a reading device, reading the electric signal of a biochemical sensor at the bottom of the microwell, and obtaining the initial concentration of a DNA sample after data processing and displaying the initial concentration on a computer or a display screen;
the third step specifically comprises the following steps:
3.1 histogram analysis: firstly, obtaining a plurality of frames of two-dimensional thermodynamic diagram data of DNA samples with various concentrations corresponding to a micropore array chip body; then, respectively obtaining a frame of mean value thermodynamic diagram for the data of each micro-pore array chip body, and carrying out Histogram analysis on each frame of data so as to obtain a distribution peak: if the PCR amplification reaction does not occur in the microwells, the pH value in the microwells is unchanged, the output value of the sensor is 0, and the microwells without the PCR amplification reaction are negative reaction units; if a single DNA sample amplification reaction occurs in the microwells, the pH value in the wells changes, and the microwells with the single DNA sample amplification reaction are positive reaction units; if the pH value change in the microwell is larger than the pH value change caused by the amplification reaction of a single DNA sample in the microwell, carrying out PCR amplification reaction on a plurality of DNA samples in the microwell;
3.2 Calculation of initial concentration of DNA sample: the initial concentration of the DNA sample is calculated from the number of positive reaction units or the number of negative reaction units obtained in step 3.1.
2. The method of using a microwell array chip based digital PCR amplification device as set forth in claim 1, wherein: the micropore array contains 0.1-1000 ten thousand micropores, and the volume of each micropore is 1 fL-10 pL.
3. The method of using a microwell array chip based digital PCR amplification device as set forth in claim 1, wherein: the surface of the biochemical sensor is coated with a hydrogen ion sensitive material, phosphate ion or pyrophosphate ion sensitive material.
4. The method of using a microwell array chip based digital PCR amplification apparatus as set forth in claim 3, wherein: the hydrogen ion sensitive material is SiO 2 、Al 2 O 3 、HfO 2 、TiO 2 、Ta 2 O 5 One or more of (3-aminopropyl) triethoxysilane and triethoxysilylundecanal.
5. The method of using a microwell array chip based digital PCR amplification apparatus as set forth in claim 3, wherein: the phosphate ion or pyrophosphate ion sensitive material is phosphate ion organic membrane ISM or molecular probe.
6. The method of using a microwell array chip based digital PCR amplification device as set forth in claim 1, wherein: the biochemical sensor is one of an ion sensitive field effect transistor, a nanowire, graphene or molybdenum disulfide transistor sensor, or a miniature electrochemical sensor.
7. The method of using a microwell array chip based digital PCR amplification device as set forth in claim 1, wherein: the device also comprises a reading device which is electrically connected with the computer to read the electric signals of the micro-pore biosensor after the micro-pore array chip body PCR amplification.
8. The method of using a microwell array chip based digital PCR amplification device as set forth in claim 7, wherein: the reading device comprises a base and an upper cover, wherein a groove for accommodating the micro-hole array chip body is formed in the base, one side edge of the upper cover is hinged to the edge of the base, a probe which is electrically connected with a biochemical sensor pin pad of the micro-hole array chip body is arranged in the base, and a circuit board is arranged below the base and used for connecting the probe, so that power supply to the micro-hole array chip body and data reading of biochemical sensor electric signals are realized;
the circuit board comprises a data acquisition module, an ADC chip and a processor, wherein the data acquisition module acquires an electric signal of the biochemical sensor at the bottom of the micropore, and the electric signal is subjected to analog-to-digital conversion by the ADC chip and then is transmitted to the processor for digital signal processing and calculating the concentration of amplified DNA; the processor is an FPGA, a DSP or an ARM.
9. The method of using a microwell array chip based digital PCR amplification device as set forth in claim 7, wherein: the reading device comprises a card inserting circuit board and a data processing circuit board;
the card inserting circuit board comprises a containing bin for containing the micro-pore chips, a gland arranged at the mouth of the containing bin, a signal amplifier chip for amplifying the signals of the biochemical sensors on the micro-pore chips and a plug for communicating with the data processing circuit board;
the data processing circuit board comprises a slot, an ADC analog-to-digital conversion chip, a microprocessor MCU and a communication port, wherein the slot is matched with the plug, the communication between the card inserting circuit board and the data processing circuit board is realized through the connection between the plug and the slot, the ADC analog-to-digital conversion chip is respectively connected with the signal amplifier chip and the microprocessor MCU, and the microprocessor MCU is connected with the terminal equipment through the communication port.
10. The method of using a microwell array chip based digital PCR amplification device as set forth in claim 1, wherein the specific process of step 3.2 is:
drawing a standard curve: drawing a standard curve C=f (N) according to the concentration of each group of DNA samples and the corresponding number N of positive units;
and (3) data calculation: after the DNA sample with the concentration to be detected is subjected to PCR amplification reaction, firstly, the number of all positive cells on the micro-pore array chip body is calculated according to the step 3.1, and then, the initial concentration of the DNA sample is calculated according to a standard curve drawn in advance in the step 3.2.
11. The method of using a microwell array chip based digital PCR amplification device as set forth in claim 1, wherein the specific process of step 3.2 is: and carrying the number of positive reaction units or the number of negative reaction units obtained by the histogram analysis into a poisson equation to calculate the initial concentration of the DNA sample.
12. The method of using a microwell array chip based digital PCR amplification device as set forth in claim 1, wherein: the thermal cycler comprises an end cover and a base, wherein the end cover is movably arranged at the top of the base; the base comprises a placing cavity for accommodating the micropore array chip body, a heat conducting block arranged close to the bottom of the placing cavity, a temperature sensor arranged at the upper end part of the heat conducting block and close to the placing cavity, a heater arranged at the bottom of the heat conducting block, and a radiator arranged at the bottom of the heater; the two sides of the heat conducting block are fixed in the base through limiting plates, the heater is fixed at the bottom of the heat conducting block through a positioning plate, and the temperature sensor and the heater are connected with the main control board through a control circuit.
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CN112239719A (en) * 2019-07-19 2021-01-19 成都万众壹芯生物科技有限公司 Digital PCR amplification device based on micropore array chip and method for amplifying by using same
CN114606297A (en) * 2020-12-09 2022-06-10 成都万众壹芯生物科技有限公司 Nucleic acid detection chip and application thereof
CN113846149A (en) * 2021-09-28 2021-12-28 领航基因科技（杭州）有限公司 Digital PCR real-time analysis method of micropore array chip
CN114015560A (en) * 2021-10-20 2022-02-08 深圳市中科先见医疗科技有限公司 Molecular detection chip
CN114058686A (en) * 2021-11-17 2022-02-18 东南大学 Digital PCR chip based on impedance detection and detection method thereof

Family Cites Families (14)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2000233487A (en) * 1999-02-15 2000-08-29 Riso Kagaku Corp Plate-making device for thermosensitive stencil plate
US20020177135A1 (en) * 1999-07-27 2002-11-28 Doung Hau H. Devices and methods for biochip multiplexing
CN100453651C (en) * 2003-07-02 2009-01-21 松下电器产业株式会社 Pyrophosphoric acid detection sensor, method of detecting nucleic acid and method of discriminating base variety
US20060166223A1 (en) * 2005-01-26 2006-07-27 Reed Michael W DNA purification and analysis on nanoengineered surfaces
US9487822B2 (en) * 2008-03-19 2016-11-08 Fluidigm Corporation Method and apparatus for determining copy number variation using digital PCR
US8470164B2 (en) * 2008-06-25 2013-06-25 Life Technologies Corporation Methods and apparatus for measuring analytes using large scale FET arrays
WO2013138767A1 (en) * 2012-03-16 2013-09-19 Life Technologies Corporation Systems and methods for biological analysis
EP2839029B1 (en) * 2012-04-19 2017-11-15 Life Technologies Corporation Method of performing digital pcr
CN203269943U (en) * 2013-05-08 2013-11-06 中山大学达安基因股份有限公司 Detection device based on gene chip
CN203422367U (en) * 2013-09-17 2014-02-05 徐云鹏 System detector capable of detecting food components rapidly based on micro electrode array
CN106164295B (en) * 2014-02-25 2020-08-11 生物纳米基因公司 Reducing bias in genome coverage measurements
CN107460233A (en) * 2016-06-06 2017-12-12 张家港万众芯生物科技有限公司 A kind of gene order surveying method by detecting pyrophosphoric acid electric charge
CN112239719A (en) * 2019-07-19 2021-01-19 成都万众壹芯生物科技有限公司 Digital PCR amplification device based on micropore array chip and method for amplifying by using same
TWM614600U (en) * 2021-01-12 2021-07-21 史丹佛國際有限公司 6D spectrum analyzer equipment

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