CN110295109B - Digital PCR detection method based on microfluidic droplet printing system and application thereof - Google Patents
Digital PCR detection method based on microfluidic droplet printing system and application thereof Download PDFInfo
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- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/6851—Quantitative amplification
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Abstract
The invention provides a digital PCR detection method based on a microfluidic droplet printing system and application thereof. The method comprises the following steps: printing a sample solution to be detected on a substrate from a microfluidic printing chip nozzle in a pneumatic mode by utilizing a microfluidic printing system in a non-closed environment, and forming a preset liquid drop array on the substrate; placing a substrate bearing the liquid drop array on a heating plate of a PCR instrument for thermal circulation; and taking a fluorescent photo of the liquid drop array on the substrate, analyzing and identifying bright and dark liquid drops and counting the positive liquid drop ratio. The method can generate liquid drops in the non-closed space, and the liquid drops are not contacted with each other and are not interfered with each other, and the industrialization of digital PCR detection is facilitated by automatically controlling the liquid drop printing process, the printing effect and the PCR detection.
Description
Technical Field
The invention relates to the technical field of PCR (polymerase chain reaction), in particular to a digital PCR detection method based on a microfluidic droplet printing system and application thereof.
Background
The digital PCR (polymerase chain reaction) technology is a nucleic acid molecule absolute quantification technology based on the poisson distribution principle, and has wide application prospect in the field of absolute counting/quantification of nucleic acid molecules. Dispersion of nucleic acid samples is the most important step in digital PCR. Ideally, the DNA template should be completely dispersed in a separate reaction system, such as microwells or droplets. These tiny reaction systems do not interfere with each other and the instrument detects the change in fluorescence of each of the microsystems to determine whether a PCR reaction is present therein. At present, there are two main sample dispersion modes, one is a microfluidic chip method, such as Thermo Quant Studio TM A 3d digital PCR system, comprising a plurality of dna probes, which is prepared by biological treatment,The basic operation units of sample preparation, reaction, separation, detection and the like in the chemical and medical analysis process are integrated on a micron-scale chip, and the whole analysis process is automatically completed. The method has the advantages of complex working flow, complex chip manufacturing process, high requirements on manufacturing process and the problems of different droplet sizes and overlapping among droplets, thereby increasing the detection cost. Another is a droplet method, such as BIO-RAD QX200 TM The digital PCR system adopts a two-phase flow technology to disperse a sample in a large number of liquid drops, the number and the size of the liquid drops are controllable, but in the liquid drop type digital PCR system, three functions of sample division, amplification and detection are independent, the integration and automation degree are not high, the problems of liquid drop fusion, breakage, cross contamination and the like are easily caused in the liquid transferring process, the liquid drop generation speed is not constant, and the liquid drop generation time cannot be accurately mastered.
Furthermore, both of the above methods are performed in closed centrifuge tubes, and there is no report of directly separating the template solution into small droplets in an open environment. In addition, the number of the generated liquid drops is about 1000-6000, the aerosol pollution problem exists in the liquid drop generating process, and the biological safety is low.
Therefore, there is a need for improvements in the art to provide a digital PCR detection method that is precisely controllable in droplets, does not interfere with each other, and is low in detection cost.
Disclosure of Invention
The invention aims to overcome the defects of the prior art, and provides a digital PCR detection method based on a microfluidic liquid drop printing system and application thereof, wherein the microfluidic printing system is utilized to automatically control processes such as liquid drop printing, digital PCR detection and the like.
According to a first aspect of the present invention, there is provided a digital PCR detection method based on a microfluidic droplet printing system, the method comprising:
step S1: printing a sample solution to be detected on a substrate from a microfluidic printing chip nozzle in a pneumatic mode by utilizing a microfluidic printing system in a non-closed environment, and forming a preset liquid drop array on the substrate;
step S2: placing a substrate bearing the liquid drop array on a heating plate of a PCR instrument for thermal circulation;
step S3: and taking a fluorescent photo of the liquid drop array on the substrate, analyzing and identifying bright and dark liquid drops and counting the positive liquid drop ratio.
In one embodiment, the microfluidic printing chip is provided with an air pressure channel for receiving air and a fluid channel for receiving a sample solution to be detected, the air pressure shears the sample solution to be detected into liquid drops, and the liquid drops are printed on a substrate below the microfluidic printing chip through a nozzle arranged on the microfluidic printing chip.
In one embodiment, the microfluidic printing system presets a droplet printing-related parameter and controls relative movement of the microfluidic printing chip with respect to the substrate based on the set printing-related parameter, thereby forming a preset array of droplets on the substrate.
In one embodiment, the microfluidic droplet printing system comprises a driving control module, an electric translation stage, a gas valve device, a camera microscope system and a data analysis processing module, wherein the gas valve device is used for providing gas pressure for the microfluidic printing chip; the electric translation stage is used for placing the microfluidic printing chip; the image pickup microscope system is used for taking a fluorescent picture of liquid drops, the data analysis processing module is used for identifying bright and dark liquid drops and counting positive liquid drop proportion, and the driving control module is used for controlling the electric translation table, the air valve device and the image pickup microscope system based on the set printing related parameters.
In one embodiment, the print-related parameters set by the drive control module include one or more of a number of print drops, a number of print drop rows, a number of print drop columns, a print mode, a channel number of the air valve device, a pitch between print drops, a print frequency, a print pulse width, and a distance between the print head and the motorized translation stage.
In one embodiment, the denaturation parameter in step S2 is set to 90 ℃,60S; the annealing parameters are set to 95 ℃,5s,60 ℃ and 15s; the extension parameter is set to 72 ℃ for 120s; the number of thermal cycles was set to 40.
In one embodiment, the substrate is bonded by plasma cleaning from 75 x 50 x 1mm plain glass and polydimethylsiloxane walls.
In one embodiment, the microfluidic print chip is fabricated using the steps of:
printing the design drawing of the microfluidic printing chip on a transparent film;
taking the transparent film as a photoetching mask and transferring the photoetching mask to a silicon wafer coated with photoresist;
and manufacturing a polydimethylsiloxane chip by adopting a soft etching method to form the microfluidic printing chip.
In one embodiment, the microfluidic print chip orifice is a sealing membrane.
According to a second aspect of the present invention there is provided the use of the method of the present invention in digital PCR detection, for example in genetic engineering, medical diagnostics, environmental engineering and the like.
Compared with the existing PCR technology, the invention has the advantages that: the liquid drops can be generated in the non-closed space, the liquid drops are not contacted with each other and are not interfered with each other, the liquid drop array, the liquid drop size and the liquid drop quantity are all accurately controllable, and the liquid drop printing process can be stopped and started at any time; in addition, the microfluidic printing chip is simple to manufacture, low in cost and less in sample solution loss in the printing process.
Drawings
The following drawings are illustrative of the invention and are not intended to limit the scope of the invention, in which:
FIG. 1 is a flow chart of a digital PCR detection method based on a microfluidic droplet printing system according to one embodiment of the present invention;
fig. 2 is an effect diagram of a microfluidic print chip according to one embodiment of the present invention;
FIG. 3 is a process schematic of a digital PCR detection method of a microfluidic droplet printing system according to one embodiment of the invention;
FIG. 4 is a graph of the fluorescence effect of an array of droplets according to one embodiment of the invention;
FIG. 5 is a schematic diagram of positive drop duty cycle according to one embodiment of the invention;
fig. 6 is a simulated diagram of a microfluidic printing platform according to one embodiment of the invention;
fig. 7 is a physical diagram of a microfluidic drop printing platform according to one embodiment of the invention.
Detailed Description
The present invention will be further described in detail with reference to the following specific examples, which are given by way of illustration, in order to make the objects, technical solutions, design methods and advantages of the present invention more apparent. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the invention.
In all examples shown and discussed herein, any specific values should be construed as merely illustrative, and not a limitation. Thus, other examples of exemplary embodiments may have different values.
Techniques, methods, and apparatus known to one of ordinary skill in the relevant art may not be discussed in detail, but are intended to be part of the specification where appropriate. According to one embodiment of the present invention, there is provided a digital PCR detection method, in short, using a microfluidic droplet printing system (or printing platform) to print a sample solution to be detected directly on a substrate, where droplets can form an array of droplets of different sizes; then, carrying out digital PCR reaction to obtain a liquid drop array fluorescence effect diagram; and further analyzing the relationship between the initial concentration of the sample solution and the positive droplet ratio.
Specifically, referring to fig. 1 and 3, the digital PCR detection method based on the microfluidic droplet printing system according to the embodiment of the present invention includes the following steps:
in step S110, a microfluidic printing chip having a fluid channel and an air pressure channel is prepared.
In the invention, the microfluidic printing chip is used for generating liquid drops, and is provided with a fluid channel for receiving a sample solution to be detected, a gas pressure channel for accessing gas and a nozzle for conveying the generated liquid drops.
In one embodiment, the microfluidic printing chip is divided into a thick layer channel and a thin layer channel, wherein the thick layer is a fluid channel and a pneumatic channel, the thin layer provides a droplet ejection opening, a sample solution is accessed from one end of the thick layer channel, the pneumatic pressure is accessed from the other end of the thick layer channel, the sample solution and air cross-meet in the thick layer channel to form droplets under the action of pressure and shearing force, and the generated droplets can be output through the ejection opening, see the structure of the microfluidic printing chip illustrated in fig. 3 (b) and the effect of the microfluidic printing chip illustrated in fig. 2. The microfluidic printing chip can be provided with a plurality of channels, for example 12 channels, and 12 air valve channels can be simultaneously connected for simultaneous printing.
Optionally, the thin layer of the microfluidic printed chip is a sealing film.
In one embodiment, the preparation process of the microfluidic printing chip comprises: firstly, designing a chip pattern by utilizing computer commercial software AutoCAD, and printing a chip design drawing on a transparent film; then, using the transparent film as a photoetching mask, and transferring a mask pattern to a silicon wafer coated with photoresist by utilizing a photoetching technology; then adopting a soft etching method to manufacture a Polydimethylsiloxane (PDMS) chip; and pasting the prepared thick layer and thin layer by a PLASMA cleaning method (PLASMA), and baking for more than 2 hours in an oven at 80 ℃ to finish the manufacturing of the microfluidic printing chip.
Step S120, preparing a substrate for carrying an array of microfluidic droplets.
In the present invention, the substrate is used to carry droplets output from the orifice of the microfluidic printing chip. For example, the substrate may be formed by pasting 75 x 50 x 1mm plain glass and PDMS walls via PLASMA.
In step S130, a PCR sample solution to be detected is prepared.
In this step, a PCR sample solution to be detected is prepared, placed in a centrifuge tube, and for example, as shown in FIG. 3 (a), a mixture solution of a human c-DNA template, PCR MIX, a probe and water is prepared in a proportion of 20ul, and the mixture solution is subjected to shaking, uniform mixing and centrifugation, and placed in a centrifuge tube for standby.
And step S140, printing the sample solution to be detected onto the substrate from the jet of the microfluidic printing chip in an air pressure mode by utilizing the microfluidic liquid drop printing system.
Referring to fig. 6 and 7, in an embodiment of the present invention, the microfluidic droplet printing system includes a driving control module, an electric translation stage, a gas valve device, a camera microscope system, and a data analysis processing module.
The air valve device is used for providing air pressure for the microfluidic printing chip, for example, an air pressure channel connecting the air pump and the microfluidic printing chip, so that proper air pressure is provided for the microfluidic printing chip.
The electric translation stage is used for placing the microfluidic printing chip and the substrate, wherein the substrate is positioned below the microfluidic printing chip.
The image microscope system is used for taking fluorescent pictures of the liquid drops and comprises a camera, a microscope lens, a light source controller and the like.
The data analysis processing module is used for identifying bright and dark liquid drops and counting positive liquid drop duty ratio, for example, the scanned images are automatically spliced to form liquid drop images and further identifying images.
The driving control module is used for setting printing related parameters and correspondingly controlling the electric translation stage, the air valve device, the camera microscope system and the like based on the set parameters.
For example, the driving control module controls the movement speed of the electric translation stage, single step length, automatic movement to different positions of the microfluidic printing chip and the like, so that the free movement of the electric translation stage in the X-Y axis and the Z axis is realized.
For example, the driving control module is further used for realizing control functions of the camera, including controlling exposure time, brightness, contrast, white balance, photographing and the like.
For example, the drive control module may also control the passage and air pressure level of the air valve assembly, etc.
In the embodiment of the invention, the driving control module and the data analysis processing module can be realized by adopting software and provide a man-machine interaction interface so as to facilitate the setting of printing parameters, the visual display of data analysis results and the like by a user. It should be noted that the driving control module and the data analysis processing module may be integrated into one software module.
In one embodiment, a process of microfluidic droplet printing includes: before use, the microfluidic droplet printing system detects the connection status with the device (e.g., motorized translation stage, air valve device, camera, etc.), then opens the software, automatically tests the connection status with the device, and initializes the position of the translation stage. Specifically, firstly, a printing template (comprising the number of printing liquid drops, the number of rows, the number of columns and the like) is selected, and then parameters such as a printing mode (such as intermittent printing or continuous printing), the number of a gas valve channel, the distance between the printing liquid drops, the printing frequency, the printing pulse width and the like are set according to different properties of liquid; then, adjusting the air pressure of the air pump and pressing a button to automatically position the printing head (the distance between the printing head and the translation stage can be adjusted through a control panel in software); finally, a manual rocker can be added, and printing can be started after liquid adding by selecting a proper initial printing position through the rocker. During printing, the printing can be paused at any time, restarted and ended.
The microfluidic printing system can control the relative movement of the microfluidic printing chip and the substrate, the reaction unit is separated in a translation mode, a droplet array is formed on the substrate, the microfluidic printing system can regulate the size of generated droplets, generate stable and uniform droplets, has high regulating speed and droplet generating speed, does not influence the generation of droplets in the regulating process, and can be continuously regulated.
It should be understood that the air pump, camera, microscope head, etc. of embodiments of the present invention may be manufactured in commercially available or custom products, and that the connection of these devices to the computer may be accomplished using existing interfaces and circuit boxes.
In step S150, the substrate is placed on a heating plate of a PCR instrument for thermal cycling.
As shown in fig. 3 (c), 3 (d), 2 to 3 drops of mineral oil were dropped on a heating plate to reduce drop evaporation during PCR amplification; then, a substrate (or substrate) carrying the array of droplets is placed on a plate PCR instrument heating plate to perform a thermal cycling reaction, wherein the reaction parameters are set as follows: denaturation: 90 ℃ for 60s; annealing: 95 ℃,5s,60 ℃,15s, extension: 72 ℃,120s;40 thermal cycles.
Through thermal cycling reaction, the properties of the sample liquid drop are changed, so that the stimulated luminescence intensity of the detected sample is changed.
Step S160, taking a fluorescence photograph of the generated droplet array.
After running the PCR thermal cycle, the glass substrate with the well-spread droplets was placed on a fluorescent inverted microscope and a partial fluorescence photograph of the droplets was taken by an electric translation stage until all of the droplet arrays were completely taken, as shown in fig. 3 (e).
And S170, analyzing and identifying the fluorescent photo of the liquid drop to obtain the positive liquid drop ratio of the sample solution to be tested.
As shown in fig. 3 (f), the partial droplet fluorescent photographs are spliced by a data analysis processing module (such as computer software) to form a complete droplet fluorescent photograph, and the computer software identifies bright and dark droplets and counts the positive droplet ratio, so as to calculate the initial concentration of the sample.
For example, the fluorescence photograph is spliced by direct splicing, i.e., a portion where two images are not overlapped is directly connected edge to edge.
For example, the process of recognizing a bright and dark droplet is: the captured image of the liquid drops is converted into gray level images, and the adhesion between the liquid drops, between the liquid drops and the background and between the liquid drops and impurities are separated, so that the liquid drops are identified.
As shown in FIG. 4, seven concentration gradient human-derived c-DNA samples are adopted as a PCR reaction system in the embodiment of the invention, the PCR reaction is carried out after the printing of a microfluidic digital printing platform, and then the complete image of each system droplet array is obtained by splicing fluorescent images, wherein white spots are positive droplets, black spots are negative droplets, and the concentration of bright droplets is low. Positive liquid drop duty ratio and concentration gradient form positive correlation, as shown in figure 5, thus proving that the liquid drop printing platform can achieve high-efficiency stable and accurate printing effect, and the liquid drop array can present digital PCR effect.
The invention generates liquid drops with controllable size through liquid drop printing, and then the substrate full of the liquid drops is placed on a microscope for detection and counting of fluorescent signals. The method can avoid sample pollution, improves the simplicity of experimental operation, and provides a new direction for full-automatic digital PCR.
In summary, the invention utilizes the microfluidic droplet printing system to carry out digital PCR detection, can generate the droplet with controllable and adjustable droplet size and array in a non-closed environment by precisely controlling the movement of the electric moving table, the air valve pressure and the like in the droplet generation process, can realize the integrated operation of droplet generation, amplification and detection without an additional digital PCR detection device, and avoids the problems of sample pollution and droplet fusion caused by the existing split type multi-step operation; in addition, because the on-line detection is realized, the fluorescent signal can be timely and effectively detected, and the problem that the fluorescent signal cannot be normally detected due to time lapse is avoided. By automating the digital PCR detection to realize droplet generation and droplet identification, the identification time and error rate are reduced, and the automation control and processing is beneficial to the industrialization of the digital PCR detection.
The digital PCR detection method can be applied to genetic engineering, medical diagnosis, environmental engineering and the like, such as tumor research, single cell analysis, virus microorganism analysis, gene sequencing verification and the like.
It should be noted that, although the steps are described above in a specific order, it is not meant to necessarily be performed in the specific order, and in fact, some of the steps may be performed concurrently or even in a changed order, as long as the required functions are achieved.
The foregoing description of embodiments of the invention has been presented for purposes of illustration and description, and is not intended to be exhaustive or limited to the embodiments disclosed. Many modifications and variations will be apparent to those of ordinary skill in the art without departing from the scope and spirit of the various embodiments described. The terminology used herein was chosen in order to best explain the principles of the embodiments, the practical application, or the technical improvements in the marketplace, or to enable others of ordinary skill in the art to understand the embodiments disclosed herein.
Claims (9)
1. A digital PCR detection method based on a microfluidic droplet printing system comprises the following steps:
step S1: in a non-closed environment, printing a sample solution to be detected onto a substrate from a microfluidic printing chip nozzle in an air pressure mode by utilizing a microfluidic printing system control, and forming a liquid drop array on the substrate;
step S2: adding mineral oil to the substrate on which the array of droplets is formed, such that the array of droplets is covered by the mineral oil;
step S3: performing thermal cycling on the substrate placed thereon on a PCR instrument heating plate;
step S4: taking a fluorescent photo of the liquid drop array on the substrate, analyzing and identifying bright and dark liquid drops and counting the positive liquid drop ratio;
the microfluidic printing chip is divided into a thick layer and a thin layer, wherein the thick layer and the thin layer enclose a thick layer channel which is horizontally arranged, the thick layer further comprises a first channel communicated with one end of the thick layer channel and a second channel communicated with the other end of the thick layer channel, the first channel and the second channel are in vertical directions, an access end of the first channel is a gas access port, a communication end of the first channel is communicated with the first end of the thick layer channel, an access end of the second channel is a sample access port, and a communication end of the second channel is communicated with the second end of the thick layer channel;
the thin layer comprises a thin layer channel which is vertically arranged, the thin layer channel penetrates through the thin layer, the lowest end of the thin layer channel is used as a liquid drop nozzle, and the projection of the thin layer channel on the horizontal plane falls into the projection of the thick layer channel on the horizontal plane;
the substrate is formed by sticking common glass and a polydimethylsiloxane wall positioned at the periphery of the glass through a plasma cleaning method;
step S1, introducing a sample solution from an access end of the second channel, and introducing gas from an access end of the first channel; and shearing the sample solution into liquid drops through the air pressure of the air, and enabling the liquid drops to fly to the substrate below the microfluidic printing chip after being separated from the liquid drop nozzle.
2. The method of claim 1, wherein the microfluidic printing system presets droplet printing-related parameters and controls relative movement of the microfluidic printing chip with respect to the substrate based on the set printing-related parameters to form a preset array of droplets on the substrate.
3. The method of claim 1, wherein the microfluidic droplet printing system comprises a drive control module, an electric translation stage, a gas valve device, a camera microscope system, and a data analysis processing module, wherein the gas valve device is configured to provide gas pressure to the microfluidic printing chip; the electric translation stage is used for placing the microfluidic printing chip; the shooting microscope system is used for shooting fluorescent pictures of liquid drops; the data analysis processing module is used for identifying bright and dark liquid drops and counting positive liquid drop proportion, and the driving control module is used for controlling the electric translation table, the air valve device and the camera microscope system based on the set printing related parameters.
4. A method according to claim 3, wherein the print related parameters set by the drive control module include one or more of a number of print drops, a number of print drop rows, a number of print drop columns, a print mode, a channel number of the air valve device, a spacing between print drops, a print frequency, a print pulse width, a distance between the print head and the motorized translation stage.
5. The method according to claim 1, wherein in step S2, the denaturation parameter is set at 90 ℃,60S; the annealing parameters are set to 95 ℃,5s,60 ℃ and 15s; the extension parameter is set to 72 ℃ for 120s; the number of thermal cycles was set to 40.
6. The method of claim 1, wherein the substrate is bonded by plasma cleaning from 75 x 50 x 1mm plain glass and polydimethylsiloxane walls.
7. The method of claim 1, wherein the microfluidic print chip is fabricated by: printing the design drawing of the microfluidic printing chip on a transparent film; taking the transparent film as a photoetching mask and transferring the photoetching mask to a silicon wafer coated with photoresist; and manufacturing a polydimethylsiloxane chip by adopting a soft etching method to form the microfluidic printing chip.
8. The method of claim 1, wherein the microfluidic print chip orifice is a sealing membrane.
9. Use of a method according to any one of claims 1 to 8 in digital PCR detection.
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| CN111951294A (en) * | 2019-12-04 | 2020-11-17 | 上海孚清生物科技有限公司 | Micropore traversing method and device of PCR chip and storage medium |
| CN110835599A (en) * | 2019-12-11 | 2020-02-25 | 中国科学技术大学 | Biological detection device and method based on microfluidic impact printing |
| CN111748466B (en) * | 2020-05-28 | 2023-10-13 | 湖南工业大学 | Detection device based on digital micro-fluidic control, application and detection method thereof |
| CN112816706B (en) * | 2021-01-06 | 2023-08-29 | 上海理工大学 | Digital ELISA system and use method thereof |
| CN114958566A (en) * | 2022-06-10 | 2022-08-30 | 珠海大略科技有限公司 | Printing device matched with microfluidic chip and printing method |
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