CN107083326A - A kind of microchamber array digital pcr chip based on PDMS material - Google Patents
A kind of microchamber array digital pcr chip based on PDMS material Download PDFInfo
- Publication number
- CN107083326A CN107083326A CN201710334689.5A CN201710334689A CN107083326A CN 107083326 A CN107083326 A CN 107083326A CN 201710334689 A CN201710334689 A CN 201710334689A CN 107083326 A CN107083326 A CN 107083326A
- Authority
- CN
- China
- Prior art keywords
- chip
- microchamber
- microchamber array
- outlet
- water
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- 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
-
- 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
- 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
Abstract
The present invention relates to a kind of microchamber array digital pcr chip based on PDMS material, the material of the chip is PDMS material, including injection port, microchamber array and outlet, the injection port includes sample feeding mouthful and water injection port, and the outlet includes sample outlet and water outlet；The water injection port connects the water microchamber array positioned at chip periphery with water outlet；The sample feeding mouth connects each reative cell in microchamber array with sample outlet by microchannel.The aquaporin of periphery of the invention can prevent the evaporation of PCR reaction solutions during PCR；Designed automatic Counting software can automatically be counted to chip results；The present invention can reach high detection sensitivity, realize DNA Single Molecule Detection.
Description
Technical field
The invention belongs to digital pcr field, more particularly to a kind of microchamber array digital pcr core based on PDMS material
Piece.
Background technology
Digital pcr be it is a kind of can to DNA molecular copy number carry out absolute quantitation technology.The technology has many excellent
Point：Accurate quantitative analysis can be carried out to initial sample, and not need pre-rendered standard curve；Sensitivity is high, and test limit can reach
To individual molecule；Can be with small change in concentration of quantitative nucleic acid molecule etc..These advantages all make digital pcr compare traditional quantitative methods
It is more accurate, sensitiveer, so as to be more applicable for the quantitative detection to DNA molecular.
Digital pcr chip is also always one of the study hotspot in micro-fluidic chip field.At present, digital pcr miniflow system
System is broadly divided into emulsion droplet formula and the class of array two.It is emulsion droplet formula digital pcr microfluid system first, its principle is by generating number
Ten thousand to millions of Water-In-Oil drops, PCR reaction solutions are uniformly divided into reaction member independent one by one, each reaction member
In PCR processes by independent progress.Initial DNA profiling molecule can be calculated by counting the number of positive reaction unit
Number.
Array digital pcr chip system, tens thousand of of the digital pcr system of the type pre-production on chip is mutually solely
Vertical reative cell, is then divided PCR reaction solutions by the way that PCR reaction solutions to be evenly distributed to realize among these reative cells
Liquid.The PCR processes of each reative cell are independently carried out, and positive reaction room is counted after PCR processes terminate, and pass through Poisson
Distribution finally calculates the number of initial DNA profiling molecule.
At present, emulsion droplet digital pcr needs complicated emulsion droplet generation, manipulation, readout equipment, so based on emulsion droplet digital pcr
Commercial apparatus it is sufficiently expensive.And current array digital pcr is still immature, or complicated or use is cumbersome.
The content of the invention
The technical problems to be solved by the invention are to provide a kind of microchamber array digital pcr core based on PDMS material
Piece, the aquaporin of the chip periphery can prevent the evaporation of PCR reaction solutions during PCR；Designed automatic Counting software energy
It is enough that chip results are counted automatically；High detection sensitivity can be reached, DNA Single Molecule Detection is realized.
A kind of microchamber array digital pcr chip based on PDMS material of the present invention, the material of the chip is PDMS
Material, including injection port, microchamber array and outlet, the injection port include sample feeding mouthful and water injection port, it is described go out sample
Mouth includes sample outlet and water outlet；The water injection port connects the water microchamber battle array positioned at chip periphery with water outlet
Row；The sample feeding mouth connects each reative cell in microchamber array with sample outlet by microchannel.
The water microchamber array of the chip periphery provides wet environment for inside PCR reaction microchamber arrays.
The microchamber array is equally divided into several rectangular areas, and the size correspondence field of microscope of each rectangular area is big
It is small.
Positive reaction room is made a distinction with negative reaction room using k-means clustering algorithms, and to positive reaction room
Number is counted automatically.
Beneficial effect
(1) present invention can realize the Single Molecule Detection of DNA molecular, show high detection sensitivity；
(2) present invention is easy to operate, and anti-evaporating is designed to the moisture loss of reaction warehouse during preferably resistance PCR；
(3) present invention can be entered positive reaction room with negative reaction room using the software based on k-means clustering algorithms
Row is distinguished, and the number of positive reaction room is counted automatically, can directly count DNA molecular number, it is not necessary to paint in advance
Standard curve processed, realizes the absolute quantitation to DNA molecular.
Brief description of the drawings
Fig. 1 is structural representation of the invention；
Fig. 2 is anti-moisture loss ability schematic diagram of the invention；
Fig. 3 (a) is the microphoto of positive reaction room and negative reaction room, (b) be along fluorescence intensity distribution；
Fig. 4 is the average fluorescent strength of positive reaction room and negative reaction room, and error bar represents the mark of 500 duplicate measurements
It is accurate poor；
Fig. 5 is the fluorescence intensity distribution histogram of positive reaction room and negative reaction room；
Fig. 6 is pictorial diagram of the invention；
Fig. 7 is the digital pcr result of various concentrations genomic DNA, wherein (a) is 5 × 104copy/μL；(b) it is
104copy/μL；(c) it is 103copy/μL；(d) it is 102copy/μL；(e) it is 10copy/ μ L；(f) it is not add any gene
Group DNA control group；
Fig. 8 is the DNA target fragments concentration and DNA target fragment actual concentrations linear relationship curves that embodiment is measured, by mistake
Poor rod represents the standard deviation that the multiple experimental calculation of three independences is obtained.
Embodiment
With reference to specific embodiment, the present invention is expanded on further.It should be understood that these embodiments are merely to illustrate the present invention
Rather than limitation the scope of the present invention.In addition, it is to be understood that after the content of the invention lectured has been read, people in the art
Member can make various changes or modifications to the present invention, and these equivalent form of values equally fall within the application appended claims and limited
Scope.
Embodiment 1
(1) chip structure
As shown in figure 1, a kind of microchamber array digital pcr chip based on PDMS material is present embodiments provided, it is described
The material of chip is PDMS material, including injection port, microchamber array and outlet, the injection port include sample feeding mouthful (figure
In indicate Sample injection port) and water injection port (injection port that Water is indicated in figure), the outlet goes out sample including sample
Mouth and water outlet；The water injection port connects the water microchamber array positioned at chip periphery with water outlet；The sample feeding
Mouth connects each reative cell in microchamber array with sample outlet by microchannel.Wherein, sample feeding mouthful is used for PCR reaction solutions
Sample introduction, and water injection port be used for chip periphery water microchamber array reclaimed water sample introduction.
Microchannel is wide about 20-60 μm, high about 10-30 μm, about 100 μm of the diameter of reative cell, about 100 μm of height.Whole core
Piece has reative cell 10368, and PCR reaction solutions can be divided into 10368 parts, can be independent in reative cell per portion reaction solution
Enter performing PCR amplification.Because chip size is excessive, it is impossible to taken pictures by a submicroscope whole chip shooting complete, so will
10368 reative cells of chip are divided into 24 rectangular areas, and the size in each region is exactly 4 powered microscope fields
Size.So, shoot all reative cells of whole chip can just be shot by 24 times and finish.
(2) chip manufacturing
First to the mixture that PDMS performed polymers and crosslinking agent are poured on silicon substrate mould；Then through processes such as standing, heating,
Solidify PDMS；PDMS after solidification is peeled off from mould, and with slide key and, chip just completes.
(3) the anti-moisture evaporation design of chip
Because PDMS material is loose structure, so during PCR, the liquid in reative cell can be heated volatilization, cause
The loss of moisture in reative cell, the reative cell of chip edge part can be completely evaporated because of moisture loss.As shown in Fig. 2 being
Prevent this phenomena impair from being reacted to PCR, anti-moisture evaporation design as depicted is added in chip edge.Figure chips
The water microchamber array of periphery, it, as evaporation sacrifice layer, is that internal PCR microchamber arrays are carried in digital pcr Thermal Cycling
For wet environment, to prevent the evaporation of PCR reaction solutions.As illustrated, using cochineal dye aqueous solution test chip it is anti-
Evaporability.Through 40 PCR Thermal Cyclings, the partial reaction room of only outermost aquaporin is evaporated, and internal PCR is micro-
Room array, without obvious moisture loss.Above-mentioned the results show, the anti-evaporating of the chip is designed to preferable resistance
The moisture loss of reative cell during PCR.
(4) design counted automatically
During digital pcr, the reative cell containing target DNA fragment can send fluorescence signal, referred to as positive reaction room.And
The reative cell that there is no target DNA fragment will not then produce fluorescence signal, referred to as negative reaction room.In order to target DNA piece
Duan Jinhang is quantified it may first have to which the positive reaction number of chambers mesh of chip is counted.
, first should be to positive reaction room and negative reaction room carry out area in order to realize the automatic counting to positive reaction room
Point.It is the fluorescence micrograph on positive reaction room (right side) and negative reaction room (left side) shown in Fig. 3 (a)；It is shown in Fig. 3 (b)
Fluorescence intensity distribution along the line.As illustrated, the fluorescence intensity of the fluorescence intensity ratio negative reaction room of positive reaction room has significantly
Rise.
Using Image-Pro Plus softwares (Media Cybernetics companies of the U.S.) to 500 sun randomly selecting
Property reative cell and the microphoto of 500 negative reaction rooms are analyzed, as a result as shown in Figure 4.Positive reaction room is averaged
Average fluorescent strength of the fluorescence intensity apparently higher than negative reaction room.
Fig. 5 is the fluorescence intensity distribution histogram of positive reaction room and negative reaction room.As illustrated, histogrammic X-axis
The average fluorescent strength of each reative cell is represented, Y-axis represents the number of reative cell.It can be seen that negative reaction room
In the range of fluorescence intensity is distributed mainly on 0 to 30 (unit is relative unit a.u.), and the fluorescence intensity master of positive reaction room
It is distributed in the range of 100 to 140.These data illustrate that both florescent intensity values have very big difference, can be by poly-
Both automatic distinguishings are opened and the number of positive reaction room are counted automatically by class algorithm.
Therefore, according to the above results, positive reaction room and negative reaction room can be carried out using k-means clustering algorithms
Distinguish, and the number of positive reaction room is counted automatically.
(5) actual test of the present embodiment chip
1. reagent and instrument：
Reagent：LightCycler 480Probes Master (German Roche companies)；Experimental water is that DEPC handles water
(Sangon Biotech)；(Hong Kong New England BioLabs are public by CpG Methylated Jurkat Genomic DNA
Department).
Instrument：BX51 fluorescence microscopes (Japanese OLYMPUS companies)；Mastercyclernexus flat In situPCR instrument
(German Eppendorf companies)；Vacuum desiccator (gets over magnetic electronic Science and Technology Ltd. in Shanghai).
DNA sequence dna：DNA sequence dna used is shown in Table 1 in experiment.It is marked with the Taqman probe sequences of fluorophor and quenching group
Row are synthesized by Shanghai Zhan Biao bio tech ltd.PCR primer is synthesized by Sangon Biotech (Shanghai) Co., Ltd.,
And purified by the said firm by polyacrylamide gel electrophoresis (PAGE).
Table 1 tests DNA sequence dna used
2.PCR reaction systems：
In order to test the range of linearity and detection sensitivity of the chip, to CpG Methylated Jurkat genomic DNAs
Gradient dilution is carried out, having prepared the genomic DNA standard items of various concentrations a series of, (concentration is from 5 × 104Copy/ μ L are arrived
10copy/μL).Before experiment, take 1 μ L standard items to react each group with PCR and be distributed into 13 μ L PCR reaction premixed liquids.Premixed liquid is existed
Thermal cycle reaction is carried out on digital pcr chip.The system of digital pcr reaction premixed liquid is as shown in table 2, thermal circulation parameters such as table 3
It is shown.PCR Thermal Cyclings are carried out on Mastercyclernexus flat In situPCR instrument.Selection suppression cancer base in this experiment
The purpose fragment expanded by PCDHGB6 promoter as PCR.
The digital pcr system of table 2
The digital pcr thermal cycle reference value of table 3
3. experimental result：
After the completion of PCR cycle, experimental result as shown in fig. 7, being gradually reduced with added genomic DNA concentration,
The number of positive reaction room is also accordingly reduced.Meanwhile, in the control group for not adding any genomic DNA, it is not observed and appoints
He Yang property reative cell.By the number of positive reaction room, the copy number of DNA molecular in standard items to be detected can be calculated.Tool
Body computational methods are：
If chip total positives reative cell number is n, if chip reative cell number is set to N.Due to DNA molecular in the reaction chamber
Number x meet Poisson distribution, then according to the distribution function of Poisson distribution：
λ is the expectation of Poisson distribution in formula 3.1, and P (x) is general when DNA molecular number is x in each reative cell
Rate.Due to containing 0 target dna molecule, then having in negative reaction room：
The desired value λ of Poisson distribution calculation formula can be released by formula 3.2：
As molecular number x in reative cell >=1, the reative cell will produce fluorescence and as positive reaction room, so having following
Formula：
Formula 3.4 is brought into formula 3.3, you can draw：
λ is the expectation in Poisson distribution, i.e., the desired value of molecule amount occur inside single reative cell, so only needing to use
The number N that λ is multiplied by overall reaction room can draw total copy number copy_number of target DNA molecule：
Copy_number=λ × N (3.6)
By the total copy number copy_number divided by volume v of initial sample, you can obtain the initial dense of target dna molecule
Degree：
So far, by mathematical computations, the copy number and concentration of target DNA molecule in sample have been accurately obtained.
Target dna molecule concentration and the linear relationship curve of its actual concentrations that Fig. 8 measures for experiment, as illustrated, inspection
The range of linearity of survey is 10copy/ μ L to 5 × 104copy/ μ L (R2=0.9999).The result shows that chip is to DNA molecular
It is quantitative accurate, and the linearity is good.Meanwhile, minimum detection limit has reached 10 molecules.Result above proves, the chip can be with
Absolute quantitation for target dna molecule is detected.
SEQUENCE LISTING
<110>Shanghai Inst. of Microsystem and Information Technology, Chinese Academy of Sci
<120>A kind of microchamber array digital pcr chip based on PDMS material
<130> 1
<160> 3
<170> PatentIn version 3.3
<210> 1
<211> 20
<212> DNA
<213>Artificial sequence
<400> 1
gatgtacacc tgcattttcg 20
<210> 2
<211> 20
<212> DNA
<213>Artificial sequence
<400> 2
cgttcgctcg ggttctcgct 20
<210> 3
<211> 25
<212> DNA
<213>Artificial sequence
<400> 3
acgccgggga tccgtcagcc tctgg 25
Claims (4)
1. a kind of microchamber array digital pcr chip based on PDMS material, the material of the chip is PDMS material, including is entered
Sample mouthful, microchamber array and outlet, it is characterised in that：The injection port includes sample feeding mouthful and water injection port, it is described go out sample
Mouth includes sample outlet and water outlet；The water injection port connects the water microchamber battle array positioned at chip periphery with water outlet
Row；The sample feeding mouth connects each reative cell in microchamber array with sample outlet by microchannel.
2. a kind of microchamber array digital pcr chip based on PDMS material according to claim 1, it is characterised in that：
The water microchamber array of the chip periphery provides wet environment for inside PCR reaction microchamber arrays.
3. a kind of microchamber array digital pcr chip based on PDMS material according to claim 1, it is characterised in that：
The microchamber array is equally divided into several rectangular areas, the size correspondence field of microscope size of each rectangular area.
4. a kind of microchamber array digital pcr chip based on PDMS material according to claim 1, it is characterised in that：
Positive reaction room is made a distinction with negative reaction room using k-means clustering algorithms, and the number of positive reaction room is carried out
It is automatic to count.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710334689.5A CN107083326A (en) | 2017-05-12 | 2017-05-12 | A kind of microchamber array digital pcr chip based on PDMS material |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710334689.5A CN107083326A (en) | 2017-05-12 | 2017-05-12 | A kind of microchamber array digital pcr chip based on PDMS material |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN107083326A true CN107083326A (en) | 2017-08-22 |
Family
ID=59607296
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201710334689.5A Pending CN107083326A (en) | 2017-05-12 | 2017-05-12 | A kind of microchamber array digital pcr chip based on PDMS material |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN107083326A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110408533A (en) * | 2018-04-28 | 2019-11-05 | 康码（上海）生物科技有限公司 | The automatic reaction chip of array type integral biology and its application method |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1940949A (en) * | 2005-09-29 | 2007-04-04 | 霍夫曼-拉罗奇有限公司 | Determination of the cycle threshold (CT) value by cluster analysis with variable cluster endpoint |
| US20090045057A1 (en) * | 2005-11-29 | 2009-02-19 | Nec Corporation | Electrophoresis chip, electrophoresis apparatus, and method for analyzing a sample |
| US20120157349A1 (en) * | 2010-12-21 | 2012-06-21 | Samsung Electro-Mechanics Co., Ltd. | Cell chip package |
| CN104946505A (en) * | 2014-03-24 | 2015-09-30 | 中国科学院深圳先进技术研究院 | Microfluidic chip for realizing PCR (polymerase chain reaction) and virus rapid detection device for real-time PCR |
-
2017
- 2017-05-12 CN CN201710334689.5A patent/CN107083326A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1940949A (en) * | 2005-09-29 | 2007-04-04 | 霍夫曼-拉罗奇有限公司 | Determination of the cycle threshold (CT) value by cluster analysis with variable cluster endpoint |
| US20090045057A1 (en) * | 2005-11-29 | 2009-02-19 | Nec Corporation | Electrophoresis chip, electrophoresis apparatus, and method for analyzing a sample |
| US20120157349A1 (en) * | 2010-12-21 | 2012-06-21 | Samsung Electro-Mechanics Co., Ltd. | Cell chip package |
| CN104946505A (en) * | 2014-03-24 | 2015-09-30 | 中国科学院深圳先进技术研究院 | Microfluidic chip for realizing PCR (polymerase chain reaction) and virus rapid detection device for real-time PCR |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110408533A (en) * | 2018-04-28 | 2019-11-05 | 康码（上海）生物科技有限公司 | The automatic reaction chip of array type integral biology and its application method |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN104404134B (en) | Multiplex nucleic acid detection methods and systems | |
| US9988667B2 (en) | Device and methods for epigenetic analysis | |
| Liu et al. | Microarrays and clinical investigations. | |
| CN101501212B (en) | Real-time PCR microarray based on an evanescent wave biosensor | |
| JP2022088444A (en) | Single light source, and two-optical channel sequence determination | |
| ES2701775T3 (en) | Procedure and device to detect microdeletion in the area of the STS chromosome | |
| CN108373971A (en) | Method and apparatus for carrying out real-time digital PCR | |
| CN106148552A (en) | The fluorescence labeling composite amplification test kit of 30 str locus seats of human Y-chromosome and application thereof | |
| CN101111607A (en) | Highly orthogonal universal sequences for use in nucleic acid assays | |
| WO2017096737A1 (en) | Capillary microarray-based high-throughput quick nucleic acid testing method | |
| WO2020261858A1 (en) | Measurement method and measurement device for digital pcr | |
| US20190352699A1 (en) | Pcr measuring method and measurement device | |
| GB2613471A (en) | Flow cell systems and devices | |
| CN105177146A (en) | Fluorescent mark multi-amplification kit of 27 STR loci of human Y-chromosome and application of kit | |
| CN105229168B (en) | Characterization of molecules in nanofluids | |
| CN107083326A (en) | A kind of microchamber array digital pcr chip based on PDMS material | |
| Mao et al. | A digitalized isothermal nucleic acid testing platform based on a pump-free open droplet array microfluidic chip | |
| CN103898226A (en) | Mitochondria SNP (single nucleotide polymorphism) fluorescence-labeling multiple amplification kit and application thereof | |
| CN113782095A (en) | Method for detecting cell state in high-flux real time | |
| Wu et al. | Microfluidic DNA combing for parallel single-molecule analysis | |
| CN106148482B (en) | A kind of sequencing approach suitable for small-sized sequenator | |
| CN108474778A (en) | It carries out being based on reverse transcriptase polymerase chain reaction using integrated approach（RT-PCR）And/or the device of the analysis of DNA/ protein arrays | |
| CN105950755A (en) | Method for detecting microRNA based on split-type recognition mode and cascade signal amplification strategy | |
| Cherney et al. | Theoretical estimation of drag tag lengths for direct quantitative analysis of multiple miRNAs (DQAMmiR) | |
| CN107099596A (en) | Mankind's Y STR29 locus fluorescence labeling composite amplification kits |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |