CN110295109A - Based on the digital pcr detection method of Microfluidic droplet print system and application - Google Patents
Based on the digital pcr detection method of Microfluidic droplet print system and application Download PDFInfo
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- CN110295109A CN110295109A CN201910608487.4A CN201910608487A CN110295109A CN 110295109 A CN110295109 A CN 110295109A CN 201910608487 A CN201910608487 A CN 201910608487A CN 110295109 A CN110295109 A CN 110295109A
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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
- 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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- 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 present invention provides a kind of based on the digital pcr detection method of Microfluidic droplet print system and application.This method comprises: being printed in substrate in such a way that micro-fluidic print system is controlled sample solution to be detected through air pressure from micro-fluidic printing chip spout in non-close environment and forming preset droplet array on this substrate；The substrate for carrying droplet array is placed in PCR instrument heating plate and carries out thermal cycle；The fluorescence photo of droplet array in substrate is shot, analysis identification light and shade drop simultaneously counts positive drop accounting.Method of the invention can generate drop in non-close space, and drop is not in contact with each other, and does not interfere with each other, and detected by automatically controlling drop print progress, printing effect and PCR, be conducive to the industrialization of digital pcr detection.
Description
Technical field
The present invention relates to round pcr field more particularly to a kind of digital pcr detections based on Microfluidic droplet print system
Method and application.
Background technique
Digital pcr (polymerase chain reaction) technology is a kind of nucleic acid molecules absolute quantitation skill based on Poisson distribution principle
Art has broad application prospects in absolute counting/quantitative field of nucleic acid molecules.The dispersion of nucleic acid samples is digital pcr
A most important step.Ideally, DNA profiling should be thoroughly dispersed in individual reaction system, such as micropore or drop.This
Slightly small reaction system is not interfere with each other, and instrument detects the change in fluorescence of each micro-system, to determine whether that it is anti-that there are PCR
It answers.Currently, there are mainly two types of sample dispersion modes, one is micro-fluidic chip methods, such as Thermo Quant StudioTM3d number
Word PCR system is by basic operation units such as biology, chemistry, the sample preparation of medical analysis process, reaction, separation, detections
It is integrated on the chip of one piece of micro-meter scale, is automatically performed analysis overall process.This mode workflow is cumbersome, chip manufacturing mistake
Journey is complicated, more demanding to manufacture craft, there are problems that droplet size is different, has overlapping between drop, to increase detection
Cost.Another kind is sessile drop method, such as BIO-RAD QX200TMDigital pcr system, this system use two-phase Flow Technique by sample
It is dispersed in a large amount of drop, amount of droplets and size are controllable, but in this drop formula digital pcr system, and sample divides, expands
Increase and three functional independences of detection, integrated and the degree of automation is not high, easily caused in pipetting processes droplet coalescence, it is broken and
The problems such as cross contamination, and drop generates speed and is not constant between, and cannot accurately hold drop generation time.
In addition, above two method is carried out in closed centrifuge tube, currently without direct in open environment
Template solution is separated into the report of droplet.Also, drop number 1000-6000 generated at present or so, need to be improved,
In drop formation process there are Aerosol, biological safety is lower.
Therefore, it is necessary to be improved to the prior art, with provide drop controllable precise, do not interfere with each other, testing cost it is low
Digital pcr detection method.
Summary of the invention
It is an object of the invention to overcome the defect of the above-mentioned prior art, provide a kind of based on Microfluidic droplet print system
Digital pcr detection method and application, using micro-fluidic print system automatically control drop printing, digital pcr detection etc.
Process.
According to the first aspect of the invention, a kind of digital pcr detection side based on Microfluidic droplet print system is provided
Method, this method comprises:
Step S1: sample solution to be detected is passed through into air pressure side using the control of micro-fluidic print system in non-close environment
Formula prints in substrate from micro-fluidic printing chip spout and forms preset droplet array on this substrate；
Step S2: the substrate for carrying droplet array is placed in PCR instrument heating plate and carries out thermal cycle；
Step S3: the fluorescence photo of droplet array in shooting substrate, analysis identify light and shade drop and count positive drop and account for
Than.
In one embodiment, the micro-fluidic printing chip is equipped with for receiving the pressure channel of gas and for connecing
The fluid channel of sample solution to be detected is received, sample solution to be tested is cut into drop by air pressure, passes through the micro-fluidic printing
The spout being arranged on chip prints in the substrate of the miniflow console keyboard beneath chips.
In one embodiment, the micro-fluidic print system presets drop printing relevant parameter, and based on set
The printing relevant parameter set controls relative movement of the micro-fluidic printing chip relative to the substrate, thus in the base
Preset droplet array is formed on bottom.
In one embodiment, the Microfluidic droplet print system includes drive control module, motorized precision translation stage, air valve
Device, photographic microscope system and data analysis and processing module, wherein the air-valve device is used for the micro-fluidic printing core
Piece provides air pressure；The motorized precision translation stage is for placing the micro-fluidic printing chip；The photographic microscope system is for clapping
Take the photograph the fluorescence photo of drop, the Data Analysis Services module light and shade drop and counts positive drop accounting for identification, described
Drive control module is used to control the motorized precision translation stage, the air-valve device and described based on set printing relevant parameter
Photographic microscope system.
In one embodiment, the printing relevant parameter of drive control module setting include printed droplets number,
Printed droplets line number, printed droplets columns, printing model, the channel number of air-valve device, the spacing between printed droplets, printing
It is one or more in distance between frequency, printing pulsewidth, print head and motorized precision translation stage.
In one embodiment, the denaturation parameter in step S2 is set as 90 DEG C, 60s；Annealing parameter is set as 95 DEG C,
5s, 60 DEG C, 15s；Extend parameter and is set as 72 DEG C, 120s；Times of thermal cycle is set as 40.
In one embodiment, the substrate is by 75*50*1mm simple glass and dimethyl silicone polymer wall through plasma
Cleaning method is pasted.
In one embodiment, the micro-fluidic printing chip is manufactured using following steps:
The design drawing of the micro-fluidic printing chip is printed upon on transparent film；
It is transferred on the silicon wafer for being coated with photoresist using transparent film as photo etched mask and by mask；
Polydimethylsiloxanechip chip is made using Soft lithograph method, forms the micro-fluidic printing chip.
In one embodiment, the micro-fluidic printing chip spout is sealed membrane.
According to the second aspect of the invention, provide method of the invention digital pcr detection in application, such as using
In genetic engineering, medical diagnosis and environmental project etc..
Compared with existing round pcr, the present invention has the advantages that drop can be generated in non-close space, and
Drop is not in contact with each other, and does not interfere with each other, droplet array, droplet size, amount of droplets all controllable precises, and drop print progress can be with
Stop with opening；In addition, micro-fluidic printing chip is simple to manufacture, at low cost, the loss of print procedure sample solution is few.
Detailed description of the invention
The following drawings only makees schematical description and interpretation to the present invention, is not intended to limit the scope of the present invention, in which:
Fig. 1 is the digital pcr detection method according to an embodiment of the invention based on Microfluidic droplet print system
Flow chart；
Fig. 2 is the effect picture of micro-fluidic printing chip according to an embodiment of the invention；
Fig. 3 is the process of the digital pcr detection method of Microfluidic droplet print system according to an embodiment of the invention
Signal；
Fig. 4 is the fluorescent effect figure of droplet array according to an embodiment of the invention；
Fig. 5 is the schematic diagram of positive drop accounting according to an embodiment of the invention；
Fig. 6 is the simulation drawing of micro-fluidic print platform according to an embodiment of the invention；
Fig. 7 is the pictorial diagram of Microfluidic droplet print platform according to an embodiment of the invention.
Specific embodiment
It is logical below in conjunction with attached drawing in order to keep the purpose of the present invention, technical solution, design method and advantage more clear
Crossing specific embodiment, the present invention is described in more detail.It should be appreciated that specific embodiment described herein is only used for explaining
The present invention is not intended to limit the present invention.
It is as shown herein and discuss all examples in, any occurrence should be construed as merely illustratively, without
It is as limitation.Therefore, other examples of exemplary embodiment can have different values.
Technology, method and apparatus known to person of ordinary skill in the relevant may be not discussed in detail, but suitable
In the case of, the technology, method and apparatus should be considered as part of specification.According to one embodiment of present invention,
A kind of digital pcr detection method is provided, in short, this method utilizes Microfluidic droplet print system (or print platform),
Sample solution to be detected is directly printed upon on substrate, drop is capable of forming different size of droplet array on substrate；Then,
It carries out digital pcr reaction and obtains droplet array fluorescent effect figure；And then the initial concentration of analysis sample solution and positive drop account for
Relationship than between.
Specifically, in conjunction with shown in Fig. 1 and Fig. 3, the digital pcr based on Microfluidic droplet print system of the embodiment of the present invention
Detection method includes the following steps:
Step S110 prepares the micro-fluidic printing chip with fluid channel and pressure channel.
In the present invention, micro-fluidic printing chip which is provided with for generating drop for receiving sample to be tested solution
Fluid channel, the pressure channel for accessing gas and the spout for conveying generated drop.
In one embodiment, micro-fluidic printing chip is divided into thick-layer channel and thin layer channel, and medium bed is that fluid is logical
Road and pressure channel, thin layer provide drop spout, and sample solution is accessed from the one end in thick-layer channel, and air pressure is another from thick-layer channel
One end access, sample solution and air intersect in thick-layer channel to meet, and under the action of pressure and shearing force, forms drop, institute
The drop of generation can be exported by spout, referring to the structure of the micro-fluidic printing chip of Fig. 3 (b) signal and the miniflow of Fig. 2 signal
Control printing chip effect.Settable multiple channels on micro-fluidic printing chip, such as 12 channels of setting, can access 12 simultaneously
A air valve channel prints simultaneously.
Optionally, the thin layer of micro-fluidic printing chip is sealed membrane.
In one embodiment, the preparation process of micro-fluidic printing chip includes: firstly, using computer business software
AutoCAD design chips pattern, floor layout is printed upon on transparent film；Then, it is covered using the transparent film as photoetching
Mask pattern is transferred on the silicon wafer for being coated with photoresist by film using photoetching technique；Poly- diformazan is made using Soft lithograph method again
Radical siloxane (PDMS) chip；The thick-layer prepared and thin layer are pasted with plasma cleaning method (PLASMA), and
80 DEG C of baking ovens dry 2h or more, to complete the production of micro-fluidic printing chip.
Step S120 prepares the substrate for carrying Microfluidic droplet array.
In the present invention, substrate is used to carry the drop exported from the spout of micro-fluidic printing chip.For example, substrate can be by
75*50*1mm simple glass and PDMS wall are pasted through PLASMA.
Step S130 prepares PCR sample solution to be detected.
In this step, PCR sample solution to be detected is prepared, is placed in centrifuge tube, for example, with reference to shown in Fig. 3 (a),
20ul mixed solution is made in source of people c-DNA template, PCR MIX, probe, water in proportion, concussion mixes centrifugation, is placed in centrifuge tube
In it is spare.
Step S140, from miniflow console keyboard in such a way that Microfluidic droplet print system is by sample solution to be detected air pressure
Print chip spout prints in substrate.
Referring to shown in Fig. 6 and Fig. 7, in embodiments of the present invention, Microfluidic droplet print system include drive control module,
Motorized precision translation stage, air-valve device, photographic microscope system and data analysis and processing module.
Air-valve device is used to provide air pressure, such as the gas of connection air pump and micro-fluidic printing chip to micro-fluidic printing chip
Pressure passageway, to provide air pressure appropriate for micro-fluidic printing chip.
Motorized precision translation stage is for placing micro-fluidic printing chip and substrate, and wherein substrate is located under micro-fluidic printing chip
Side.
Photographic microscope system is used to shoot the fluorescence photo of drop, for example including camera, microlens, light source and light
Source controller etc..
Data Analysis Services module light and shade drop and counts positive drop accounting for identification, for example, by scan image from
It is dynamic to be spliced to form Liquid particle image and further identify image.
Drive control module is for being arranged printing relevant parameter and correspondingly controlling electric translation based on set parameter
Platform, air-valve device and photographic microscope system etc..
For example, drive control module controls the movement velocity of motorized precision translation stage, single step step-length, is automatically moved to miniflow console keyboard
The different location etc. of chip is printed, realizes motorized precision translation stage in the free movement of X-Y axis and Z axis.
For example, drive control module is also used to realize the control function to camera, including the control time for exposure, brightness,
It contrast, white balance and takes pictures.
For example, drive control module can also control channel and air pressure size of air-valve device etc..
In embodiments of the present invention, drive control module and data analysis and processing module can be used software realization and provide people
Machine interactive interface, in order to user setting print parameters and intuitive display data analysis result etc..It should be noted that driving control
Molding block and data analysis and processing module also can integrate as a software module.
In one embodiment, the process of Microfluidic droplet printing includes: before use, Microfluidic droplet print system detects
With the connection state of equipment (such as motorized precision translation stage, air-valve device, video camera etc.), software is then opened, system is first automatic to be surveyed
The connection state of examination and equipment, and initialize the position of translation stage.Specifically, firstly, selection printing template (including printing liquid
Number, line number and columns of drop etc.), printing model is set (as being interrupted printing or continuously beating further according to the heterogeneity of liquid
Print), the number of air valve channel channel, the parameters such as frequency, the pulsewidth of the spacing between printed droplets and drop and printing；
Then, adjusting the air pressure of air pump and touching the button (can also adjust print head by the control panel in software in place automatically
Print head is at a distance from translation stage)；Finally, manual rocker can also be increased, suitable Loading position is selected by rocking bar, is added
It can start to print after liquid.During printing, it can suspend at any time, restart, terminate printing.
The relative movement that micro-fluidic printing chip and substrate can be controlled using micro-fluidic print system, passes through translation mode
Separate reaction member, be formed on the substrate droplet array, and micro-fluidic print system can adjust the drop of generation size,
The stable and higher drop of uniformity is generated, and there is faster adjustment speed and drop formation speed, and adjustment process will not
The generation of drop is influenced, it can serialization adjustment.
It should be understood that the air pump of the embodiment of the present invention, camera, microscope are first-class to can be used commercially available or customization production
The connection of product, these equipment and computer can be realized using existing interface and circuit box.
Step S150 is placed the substrate above and is carried out thermal cycle in PCR instrument heating plate.
As shown in Fig. 3 (c), Fig. 3 (d), on hot plate by 2 to 3 drop mineral oil droplets, to reduce the liquid during PCR amplification
Drop evaporation；Then, by the substrate (or substrate) for carrying droplet array be placed in plate PCR instrument heating plate carry out thermal cycle it is anti-
It answers, wherein response parameter is arranged are as follows: denaturation: 90 DEG C, 60s；Annealing: 95 DEG C, 5s, 60 DEG C, 15s, extend: 72 DEG C, 120s；40
Thermal cycle.
By thermal cycle reaction, sample drop character is made to change, to change the light intensity that is stimulated for being detected sample
Degree.
Step S160 shoots the fluorescence photo of droplet array generated.
As shown in Fig. 3 (e), after running through PCR thermal cycle, the substrate of glass for being paved with drop is placed in fluorescence inverted microscope
On, drop localized fluorescence photo is shot by motorized precision translation stage, until all shooting finishes all droplet arrays.
Step S170, analysis identification drop fluorescence photo, obtains the positive drop accounting of testing sample solution.
As shown in Fig. 3 (f), local droplet fluorescence photo is carried out by Data Analysis Services module (such as computer software)
Splicing forms a complete drop fluorescence photo, and computer software identification light and shade drop simultaneously counts positive drop accounting, with this
Calculate sample initial concentration.
For example, the splicing of fluorescence photo use direct splicing mode, i.e., by part unfolded between two sub-pictures into
Row while with while be directly connected to.
For example, the identification process of light and shade drop is: the Liquid particle image clapped is converted into gray level image, by between drop, liquid
Drop between background, drop separates with being adhered between impurity, and then identifies drop.
As shown in figure 4, the embodiment of the present invention using the source of people c-DNA sample of seven concentration gradients as PCR reaction system,
PCR reaction is carried out after the printing of micro-fluidic figure punch platform, then obtains each system drop battle array by splicing fluorescent image
The complete image of column, wherein white point is positive drop, and stain is negative drop, and the dilute bright drop of concentration is few.Positive drop accounting
With being positively correlated property of concentration gradient, as shown in figure 5, thus prove drop print platform can accomplish that efficient stable accurately prints
Digital pcr effect can be presented in effect, droplet array.
The present invention generates the drop of controllable size by drop printing, and then the substrate for being paved with drop is placed on microscope
Carry out the detection and counting of fluorescence signal.This mode, which can be avoided, occurs sample pollution, improves the simplification of experimental implementation,
New direction is provided for full-automatic digital pcr.
To sum up, the present invention carries out digital pcr detection using Microfluidic droplet print system, passes through essence in drop formation process
Really control that movement, air valve pressure of electronic mobile station etc. can generate droplet size under non-close environment and array controllably may be used
The drop of tune, and do not need additional digital pcr detection device, that is, it is able to achieve drop formation, amplification and the integration of detection
Operation avoids existing split type many more manipulations bring sample pollution and droplet coalescence problem；Further, since realizing online
Detection, can timely and effectively detect fluorescence signal, avoid normally detecting fluorescence caused by elapsing due to the time
The problem of signal.The automation that drop formation and drop identification are realized by detecting to digital pcr, reduces recognition time and mistake
Accidentally rate, this automation control and processing are conducive to the industrialization of digital pcr detection.
Digital pcr detection method of the invention can be applied to genetic engineering, medical diagnosis and environmental project etc., such as tumour
Research, single cell analysis, viral micro-organisms analysis, gene sequencing verifying etc..
It should be noted that, although each step is described according to particular order above, it is not intended that must press
Each step is executed according to above-mentioned particular order, in fact, some in these steps can concurrently execute, or even is changed suitable
Sequence, as long as can be realized required function.
Various embodiments of the present invention are described above, above description is exemplary, and non-exclusive, and
It is not limited to disclosed each embodiment.Without departing from the scope and spirit of illustrated each embodiment, for this skill
Many modifications and changes are obvious for the those of ordinary skill in art field.The selection of term used herein, purport
In principle, the practical application or to the technological improvement in market for best explaining each embodiment, or make the art its
Its those of ordinary skill can understand each embodiment disclosed herein.
Claims (10)
1. a kind of digital pcr detection method based on Microfluidic droplet print system, comprising the following steps:
Step S1: in non-close environment by micro-fluidic print system control by sample solution to be detected by air pressure in the way of from
Micro-fluidic printing chip spout prints in substrate and forms preset droplet array on this substrate；
Step S2: the substrate for carrying droplet array is placed in PCR instrument heating plate and carries out thermal cycle；
Step S3: the fluorescence photo of droplet array in shooting substrate, analysis identification light and shade drop simultaneously count positive drop accounting.
2. the method according to claim 1, wherein the micro-fluidic printing chip is equipped with for receiving gas
Pressure channel and fluid channel for receiving sample solution to be detected, air pressure sample solution to be tested is cut into drop,
It is printed in the substrate of the miniflow console keyboard beneath chips by the spout being arranged on the micro-fluidic printing chip.
3. the method according to claim 1, wherein the micro-fluidic print system presets drop printing phase
Parameter is closed, and the micro-fluidic printing chip is controlled relative to the opposite of the substrate based on set printing relevant parameter
It is mobile, to form preset droplet array on the substrate.
4. the method according to claim 1, wherein the Microfluidic droplet print system includes drive control mould
Block, motorized precision translation stage, air-valve device, photographic microscope system and data analysis and processing module, wherein the air-valve device is used for
Air pressure is provided to the micro-fluidic printing chip；The motorized precision translation stage is for placing the micro-fluidic printing chip；It is described to take the photograph
As microscopic system is used to shoot the fluorescence photo of drop；The Data Analysis Services module light and shade drop and counts for identification
Positive drop accounting, the drive control module be used to control based on set printing relevant parameter the motorized precision translation stage,
The air-valve device and the photographic microscope system.
5. according to the method described in claim 4, it is characterized in that, the printing relevant parameter packet of drive control module setting
Include number, printed droplets line number, printed droplets columns, printing model, the channel number of air-valve device, printing liquid of printed droplets
It is one or more in distance between spacing, print frequency between drop, printing pulsewidth, print head and motorized precision translation stage.
6. the method according to claim 1, wherein in step s 2, denaturation parameter is set as 90 DEG C, 60s；It moves back
Fiery parameter is set as 95 DEG C, 5s, 60 DEG C, 15s；Extend parameter and is set as 72 DEG C, 120s；Times of thermal cycle is set as 40.
7. method according to any one of claims 1 to 6, which is characterized in that the substrate is by 75*50*1mm simple glass
It is pasted with dimethyl silicone polymer wall through plasma cleaning method.
8. method according to any one of claims 1 to 6, which is characterized in that manufactured using following steps described micro-fluidic
Printing chip:
The design drawing of the micro-fluidic printing chip is printed upon on transparent film；
It is transferred on the silicon wafer for being coated with photoresist using transparent film as photo etched mask and by mask；
Polydimethylsiloxanechip chip is made using Soft lithograph method, forms the micro-fluidic printing chip.
9. method according to any one of claims 1 to 6, which is characterized in that the micro-fluidic printing chip spout is envelope
Membrana oralis.
10. a kind of application of method according to any one of claims 1 to 9 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 |
| CN111748466A (en) * | 2020-05-28 | 2020-10-09 | 湖南工业大学 | Detection device based on digital microfluidic and application and detection method thereof |
| 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 |
| CN112816706A (en) * | 2021-01-06 | 2021-05-18 | 上海理工大学 | Digital ELISA system and using method thereof |
| CN112816706B (en) * | 2021-01-06 | 2023-08-29 | 上海理工大学 | Digital ELISA system and use method thereof |
| WO2023236760A1 (en) * | 2022-06-10 | 2023-12-14 | 珠海大略科技有限公司 | Printing apparatus matched with microfluidic chip and printing method |
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