CN106047677B - The method for detecting the micro-fluidic chip and the unicellular amplifying nucleic acid of detection of unicellular amplifying nucleic acid - Google Patents
The method for detecting the micro-fluidic chip and the unicellular amplifying nucleic acid of detection of unicellular amplifying nucleic acid Download PDFInfo
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- CN106047677B CN106047677B CN201610334016.5A CN201610334016A CN106047677B CN 106047677 B CN106047677 B CN 106047677B CN 201610334016 A CN201610334016 A CN 201610334016A CN 106047677 B CN106047677 B CN 106047677B
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
- B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
- B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
- B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
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- 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
- C12Q1/6813—Hybridisation assays
- C12Q1/6816—Hybridisation assays characterised by the detection means
- C12Q1/682—Signal amplification
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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
- B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
- B01L2200/02—Adapting objects or devices to another
- B01L2200/026—Fluid interfacing between devices or objects, e.g. connectors, inlet details
- B01L2200/027—Fluid interfacing between devices or objects, e.g. connectors, inlet details for microfluidic devices
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L2300/00—Additional constructional details
- B01L2300/08—Geometry, shape and general structure
- B01L2300/0861—Configuration of multiple channels and/or chambers in a single devices
Abstract
The invention belongs to nucleic acid detection assay technical fields, and in particular to a kind of micro-fluidic chip detecting unicellular amplifying nucleic acid using branched chain DNA signal amplification techniques.Multiple cellular constructions are evenly equipped on chip, the chip includes the upper cover being from top to bottom sequentially stacked, cell sieve unit and egative film, cellular construction in cell sieve unit includes fluid passage and the unicellular screening structure that is set in fluid passage, cellular construction on upper lid includes inlet and liquid outlet, and the inlet and liquid outlet are located at the top at fluid passage both ends.The present invention is combined branched chain DNA signal amplification techniques with micro-fluidic chip, the characteristics of chip is to be integrated in unicellular screening on one micro-fluidic chip with functions such as nucleic acid extraction, detections, the nucleic acid released after slender cellular lysate can carry out target acquistion with core on piece microballoon, then carry out the purpose that detection of nucleic acids is realized in the amplification of branched chain DNA signals.
Description
Technical field
The invention belongs to nucleic acid detection assay technical fields, and in particular to a kind of to utilize branched chain DNA signal amplification techniques
The method for detecting the micro-fluidic chip and its unicellular amplifying nucleic acid of detection of unicellular amplifying nucleic acid.
Background technology
With the arrival in accurate medicine (Precision Medicine) epoch, for the accurate detection of nucleic acid, ultramicron
The demand of even unimolecule accuracy detection technology grows to even greater heights.It is big absolutely in the widely used technology of foranalysis of nucleic acids detection field at present
Part is still based on PCR technical principle, has been had 30 years since round pcr is born.From classical PCR, real-time quantitative PCR
Digital pcr till now again, this technology is in continuous our visual field of but never fading out of changing in quality.Although round pcr is by scientific circles
It is described as one of greatest invention of biotechnology, and obtains Nobel chemistry Prize in 1993, but regular-PCR technology also has
Some shortcomings:1. no matter Taq enzyme or reverse transcription AMV enzymes are all without 3 '~5 ' exonuclease activities, it is easy to happen alkali
Base mispairing.The mispairing frequency of Taq enzyme be 1/9 000, AMV reverse transcriptases existing for high dNTP and Mg2+ under the conditions of, every about
500 bases just will appear the mispairing of a base；2. the pairing of 3 ' end part of primer can lead to non-specific amplification；3. exogenous
Pollution easily causes false positive；4. all factors for influencing nucleic acid amplification can influence it, influence factor is too many, reacts poor repeatability；⑤
Since influence factor is too many during PCR amplification, it cannot be guaranteed that amplification efficiency remains unchanged and actual sample during the reaction
Amplification efficiency between standard sample and different samples is identical.It is extremely low for content even if amplification efficiency is identical
Mutator is easily covered by other a large amount of gene signals after amplification.For these reasons, it is detected using round pcr a large amount of
The effect is unsatisfactory for denier nucleic acid in sample, can not even detect.Therefore the present invention proposes to believe using branched chain DNA
Number amplifying technique (branched DNA) combines microflow control technique to carry out the technology of unicellular amplifying nucleic acid detection, and target is to evade
The shortcoming of round pcr is realized to unicellular middle denier even effective detection of monomolecular nucleic acid.
Branched chain DNA signal amplification techniques are that Urdea etc. is developed after synthesizing pectination DNA.This is that one kind is disobeyed
Rely PCR amplification nucleic acid hybridization signal amplification detection technology, the technology overcome the defects of traditional PCR technique with it is uncertain because
Element is not necessarily to reverse transcription, is not necessarily to PCR amplification, as long as mixing the sample with the cracking of specific cleavage liquid without being stripped purifying to sample
Afterwards, gene quantification result can be obtained rapidly after probe hybridization and signal amplification.Cardinal principle be using pectination DNA molecular and
The signal of the alkali phosphatase enzyme mark probe molecule progress signal amplification connected on branch, each DNA or RNA molecule about can
Enough amplify 2400 times.Since technology specificity is high, without carrying out complicated purification step to sample, in terms of transcriptome analysis
Also the favor of researcher has been obtained.Now with the development of detection technique of fluorescence, by single molecular fluorescence to single biology point
Son carries out analysis and has become reality, and what identification can become after the fluorescence signal of individual molecule is amplified thousands of times is more prone to,
Therefore it is feasible to carry out the detection of monokaryon acid molecule using bDNA technologies.But it not yet finds to utilize bDNA technologies so far
The research work of nucleic acid coherent detection is carried out in unicellular single molecules level.
Invention content
Present invention aims at provide a kind of miniflow detecting unicellular amplifying nucleic acid using branched chain DNA signal amplification techniques
The method for controlling chip and its unicellular amplifying nucleic acid of detection.
To achieve the above object, the technical solution adopted by the present invention is：
A kind of micro-fluidic chip detecting unicellular amplifying nucleic acid using branched chain DNA signal amplification techniques, it is uniformly distributed on chip 1
There are multiple cellular constructions, which includes upper cover 2, cell sieve unit 3 and the egative film 4 being from top to bottom sequentially stacked, cell sieve
Cellular construction 31 on subdivision 3 includes fluid passage 312 and the unicellular screening structure 311 being set in fluid passage 312,
Cellular construction 21 in upper cover 2 includes inlet 211 and liquid outlet 212, and the inlet 211 and liquid outlet 212 are located at liquid
The top at 312 both ends of body channel, liquid are entered by inlet 211, are flowed out again by liquid outlet 212 after fluid passage 312；
Unicellular screening structure 311 includes unicellular screening arresting structure A and unicellular screening arresting structure B, which catches
It is fixed single celled space between one end of inlet 211 to obtain structure A and unicellular screening arresting structure B, liquid by
Between unicellular screening arresting structure A and unicellular screening arresting structure B, unicellular screening arresting structure A and fluid passage 312
Between and unicellular screening arresting structure B and fluid passage 312 between flow through, any in liquid unicellular is fixed in list
Cell sieves arresting structure A and unicellular screening arresting structure B between one end of inlet 211；Unit knot on egative film 4
Structure 41 is along the uniformly distributed multiple micropores 411 of liquid flow direction, and the micropore is located at unicellular screening structure 311 and liquid outlet
Between 212.
The distance between the unicellular screening arresting structure A and unicellular screening arresting structure B are less than target cell
The diameter range of diameter, the specially 10-50% of target cell diameter, target cell is 1-50 microns.
The overall structure that the unicellular screening arresting structure A and unicellular screening arresting structure B is combined into can be ellipse
It is round, or splayed.
The unicellular screening arresting structure A and unicellular screening arresting structure B can be it is as shown in the figure abnormally-structured,
Can also be simple triangular prism or quadrangular or other multiple structure combinations that may be implemented to fix individual cells.
411 array of the micropore arranges；A diameter of 0.5-25 microns of micropore 411, depth are 0.7-30 microns.
The upper cover 2 is the transparent material of thickness 0.5-3mm；Cell sieve unit 3 be 10-100 microns of thickness silicon,
PDMS, SU8 photoresist, PMMA or PC plastic；Silicon, quartz glass, PMMA or the PC plastic for the thickness 0.5-3mm that egative film 4 is.
During a kind of micro-fluidic chip detection detecting unicellular amplifying nucleic acid using branched chain DNA signal amplification techniques is unicellular
The method of nucleic acid：
Cell solution to be detected is injected into the chip, liquid is by unicellular screening arresting structure A and unicellular sieve
Between point arresting structure B, between unicellular screening arresting structure A and fluid passage 312 and unicellular screening arresting structure B and
It is flowed through between 312, until individual cells are fixed at unicellular screening structure 311, i.e., unicellular screening arresting structure A and list
Cell sieves arresting structure B between one end of inlet 211, replaces caching liquid and unwanted cell is gone out chip；
Cell pyrolysis liquid is injected after flushing by the slender cellular lysate of capture, while making the target released after cell cracking
The capture probe for the modified microsphere surface being arranged in molecule and chip array micropore 411 carries out hybridization reaction, realizes to target
Mark the capture of molecule；
It then carries out carrying out corresponding qualitative, quantitative detection to the target molecules of capture by signal amplification technique.
The modified microballoon being arranged in the chip array micropore 411：According to the bore dia of array micropore 411 in chip
Microballoon is chosen, microsphere diameter is slightly less than micro-pore diameter, ensures that single microballoon can only be accommodated in each micropore, microsphere surface is modified with
For the capture probe of specific objective object, each microballoon modifies a kind of capture probe, and the microballoon in each chip unit can be
The capture probe of same class microballoon, i.e. microsphere surface modification is identical, can also be different types of microballoon, i.e. in a unit
There are many capture probes for modification in numerous microballoons, but each microsphere surface only modifies a kind of capture probe.If it is a chip
Modification is chosen in unit, and there are many microballoons of capture probe, in order to effectively identify that it is glimmering that the microballoon type of different capture probes need to be chosen
Pumped FIR laser microballoon, convenient for the positioning addressing of microballoon in the chips.
Then the microspheres solution of surface modification capture probe is injected into chip, passes through standing, centrifugation or directional magnetic field
Microballoon is loaded into the array micropore 411 in chip by the mode of (being directed to magnetic microsphere), and extra microballoon is gone out chip, is protected
Demonstrate,prove in each micropore only that there are one microballoons, for use.
The signal amplification technique, optical signalling amplification and signal acquisition are to be continuously injected into or inject simultaneously branched chain DNA
Excess reagents are gone out chip, and carry out optical photographing, luminous microballoon by the related reagent of signal amplification after stable reaction
As capture the target molecules to match with microsphere surface capture probe.According to luminous to whether containing target molecules in cell
Carry out qualitative, the content progress quantitative analysis according to luminous intensity to target molecules.
The present invention is had the advantage that：
The present invention is combined branched chain DNA signal amplification techniques with micro-fluidic chip, can be realized in a chip slender
Born of the same parents sieve, cracking, target nucleic acid capture, the sequence of operations such as signal amplification detection, without to unicellular middle denier target core
Acid carries out PCR amplification and detection can be realized, and highest detection limit can reach monomolecular nucleic acid.The characteristics of chip is by unicellular sieve
It point is integrated on a micro-fluidic chip with the functions such as nucleic acid extraction, detection, the nucleic acid released after slender cellular lysate can be by
Microballoon carries out target acquistion on chip, then carries out the purpose that detection of nucleic acids is realized in the amplification of branched chain DNA signals；It will be to nucleic acid
Ultramicron detection is of great significance.
Description of the drawings
Fig. 1 is chip overall effect figure provided in an embodiment of the present invention；
Fig. 2 is chip portfolio component provided in an embodiment of the present invention；
Fig. 3 is cellular construction component provided in an embodiment of the present invention；
Fig. 4 is that cell sieve unit provided in an embodiment of the present invention just attempts；
Fig. 5 is cell sieve unit partial enlarged view provided in an embodiment of the present invention；
Fig. 6 is egative film partial enlarged view provided in an embodiment of the present invention.
Specific implementation mode
The present invention is combined branched chain DNA signal amplification techniques with micro-fluidic chip, can be realized in a chip slender
Born of the same parents sieve, cracking, target nucleic acid capture, the sequence of operations such as signal amplification detection, without to unicellular middle denier target core
Acid carries out PCR amplification and detection can be realized, and highest detection limit can reach monomolecular nucleic acid.The characteristics of chip is by unicellular sieve
It point is integrated on a micro-fluidic chip with the functions such as nucleic acid extraction, detection, the nucleic acid released after slender cellular lysate can be by
Microballoon carries out target acquistion on chip, then carries out the purpose that detection of nucleic acids is realized in the amplification of branched chain DNA signals.
Embodiment 1
By the visible micro-fluidic chips of Fig. 1-4, multiple cellular constructions are evenly equipped on chip 1, the chip 1 include from top to bottom according to
Secondary stacked upper cover 2, cell sieve unit 3 and egative film 4, the cellular construction 31 in cell sieve unit 3 include fluid passage 312
And it is set to the unicellular screening structure 311 in fluid passage 312, the cellular construction 21 in upper cover 2 includes inlet 211 and goes out
Liquid mouth 212, the inlet 211 and liquid outlet 212 are located at the top at 312 both ends of fluid passage, liquid by inlet 211 into
Enter, flowed out again by liquid outlet 212 after fluid passage 312；Unicellular screening structure 311 includes unicellular screening capture knot
Structure A and unicellular screening arresting structure B, the unicellular screening arresting structure A and unicellular screening arresting structure B are close to inlet
It is fixed single celled space between 211 one end, liquid is by unicellular screening arresting structure A and unicellular screening arresting structure
It is flowed between B, between unicellular screening arresting structure A and fluid passage 312 and between unicellular screening arresting structure B and 312
It crosses, any unicellular unicellular screening arresting structure A and unicellular screening arresting structure B of being fixed in liquid is close to feed liquor
Between one end of mouth 211；Cellular construction 41 on egative film 4 is along the uniformly distributed multiple micropores 411 of liquid flow direction, the micropore
Between unicellular screening structure 311 and liquid outlet 212.
The distance between the unicellular screening arresting structure A and unicellular screening arresting structure B are less than target cell
The diameter range of diameter, the specially 10-50% of target cell diameter, target cell is 1-50 microns.
The overall structure that the unicellular screening arresting structure A and unicellular screening arresting structure B is combined into can be ellipse
It is round, or splayed.The unicellular screening arresting structure A and unicellular screening arresting structure B can be as schemed institute
That shows is abnormally-structured, can also be simple triangular prism or quadrangular or other multiple knots that may be implemented to fix individual cells
Structure combination.
The specific preparation method of chip is：
Step 1：Select the silicon chip of a single-sided polishing；
Step 2：Silicon chip is cleaned；
Step 3：One layer of uniform photoresist of spin coating on silicon wafer polishing face is shifted egative film mask plate patterns by exposing
Mask is formed on to photoresist；
Step 4：The dry etching silicon slice under mask forms array micropore 411 in egative film, the diameter in hole on the surface of silicon chip
It it is 6 microns, the number in hole is 20, and depth is the sum of micropore target depth and cell sieve unit layer thickness, and micropore target is deep
Degree is 5 microns, and cell sieve unit layer thickness is 30 microns.
Step 5：Remove photoresist.
Step 6：Continue one layer of uniform photoresist of spin coating on the silicon chip after removing photoresist, by exposing cell sieve unit
Mask plate patterns, which are transferred on photoresist, forms mask；
Step 7：The dry etching silicon slice under mask forms unicellular screening knot in cell sieve unit on the surface of silicon chip
Structure 311 and fluid passage 312, depth are 30 microns of cell sieve unit layer target thickness.Form egative film 4 and cell sieve unit
3 combination；
Step 8：Upper cover plate 2 is chosen, material is quartz glass or PMMA, according to the channel two in unicellular sieve unit 3
End position gets through hole as inlet 211 and liquid outlet 212；
Step 9：Upper cover plate 2 is bonded with the silicon chip that step 7 obtains, completes the preparation of chip 1.Unicellular screening is caught
It is 20 microns to obtain the distance between structure A and unicellular screening arresting structure B d.
Embodiment 2
Unicellular capture is carried out using above-mentioned acquisition chip to test, and is as follows：
Step 1 microballoon is chosen：5 micron polystyrene microspheres, microballoon are chosen according to the bore dia of array micropore 411 in chip
Diameter is less than 1 micron of micro-pore diameter, ensures that single microballoon can only be accommodated in each micropore, microsphere surface is modified with for specific mesh
The capture probe of object is marked, each microballoon modifies a kind of capture probe, and the microballoon in each chip unit can be same class microballoon,
That is the capture probe of microsphere surface modification is identical, can also be different types of microballoon, i.e. in numerous microballoons in a unit
There are many capture probes for modification, but each microsphere surface only modifies a kind of capture probe.If it is being chosen in a chip unit
There are many microballoons of capture probe for modification, in order to effectively identify that it is fluorescence-encoded micro- that the microballoon type of different capture probes need to be chosen
Ball, convenient for the positioning addressing of microballoon in the chips.
Step 2 microballoon loads：The microspheres solution of surface modification capture probe is injected into chip, by standing, centrifuging
Or microballoon is loaded into the array micropore 411 in chip by the mode of directional magnetic field (be directed to magnetic microsphere), and by extra microballoon
Chip is gone out, ensures that only there are one microballoons in each micropore.
The unicellular capture of step 3：Cell solution to be detected is injected into chip, until individual cells be fixed on it is slender
Born of the same parents sieve at structure 311, replace caching liquid and unwanted cell is gone out chip.
The slender cellular lysate of step 4 and target molecules capture：Cell pyrolysis liquid is injected by slender cellular lysate, while making cell
The capture probe of the target molecules and microsphere surface that are released after cracking carries out hybridization reaction, realizes the capture to target molecules.
Step 5 optical signalling amplifies and signal acquisition：It is continuously injected into or injects simultaneously the correlation of branched chain DNA signals amplification
Excess reagents are gone out chip, and carry out optical photographing by reagent after stable reaction, luminous microballoon be capture with it is micro-
The target molecules that ball surface capture probe matches.It is recorded according to the prior art, can be to whether containing in cell according to shining
Target molecules progress is qualitative, and quantitative analysis can be carried out to the content of target molecules according to luminous intensity.
Another chip of the present invention can be applied to most usual cells for example：Microorganism, virus, zooblast
Deng.
Claims (4)
1. a kind of micro-fluidic chip detecting unicellular amplifying nucleic acid using branched chain DNA signal amplification techniques detects unicellular center
The method of acid, it is characterised in that：
Micro-fluidic chip, chip are evenly equipped with multiple cellular constructions on (1), the chip (1) include from top to bottom be sequentially stacked it is upper
(2), cell sieve unit (3) and egative film (4) are covered, the cellular construction (31) in cell sieve unit (3) includes fluid passage
(312) and the unicellular screening structure (311) that is set in fluid passage (312), the cellular construction (21) in upper cover (2) includes
Inlet (211) and liquid outlet (212), the inlet (211) and liquid outlet (212) are located at fluid passage (312) both ends
Top, liquid by inlet (211) enter, after fluid passage (312) again pass through liquid outlet (212) flow out；It is unicellular
Screening structure (311) includes unicellular screening arresting structure A and unicellular screening arresting structure B, the unicellular screening capture knot
Structure A and unicellular screening arresting structure B is fixed single celled space between one end of inlet (211), and liquid is by list
Cell sieves between arresting structure A and unicellular screening arresting structure B, unicellular screening arresting structure A and fluid passage (312)
Between and unicellular screening arresting structure B and fluid passage (312) between flow through, any in liquid unicellular is fixed in
Unicellular screening arresting structure A and unicellular screening arresting structure B are between one end of inlet (211)；On egative film (4)
Cellular construction (41) is along the uniformly distributed multiple micropores (411) of liquid flow direction, and the micropore is located at unicellular screening structure
(311) between liquid outlet (212)；
Cell solution to be detected is injected into the chip, liquid is caught by unicellular screening arresting structure A and unicellular screening
It obtains between structure B, between unicellular screening arresting structure A and fluid passage (312) and unicellular screening arresting structure B and liquid
Body channel is flowed through between (312), until individual cells are fixed at unicellular screening structure (311), i.e., unicellular screening is caught
Structure A and unicellular screening arresting structure B are obtained between one end of inlet (211), caching liquid is replaced and rushes unwanted cell
Go out chip；
Cell pyrolysis liquid is injected after flushing by the slender cellular lysate of capture, while making the target molecules released after cell cracking
Hybridization reaction is carried out with the capture probe for the modified microsphere surface being arranged in chip array micropore (411), is realized to target
The capture of molecule；
It then carries out carrying out corresponding qualitative, quantitative detection to the target molecules of capture by signal amplification technique.
2. the micro-fluidic chip as described in claim 1 for detecting unicellular amplifying nucleic acid using branched chain DNA signal amplification techniques
The method for detecting unicellular amplifying nucleic acid, it is characterised in that：The unicellular screening arresting structure A and unicellular screening arresting structure
The distance between B is the 10-50% of target cell diameter.
3. the micro-fluidic chip as described in claim 1 for detecting unicellular amplifying nucleic acid using branched chain DNA signal amplification techniques
The method for detecting unicellular amplifying nucleic acid, it is characterised in that：The micropore（ 411）Array arranges；Micropore（ 411）It is a diameter of
0.5-25 microns, depth is 0.7-30 microns.
4. the micro-fluidic chip as described in claim 1 for detecting unicellular amplifying nucleic acid using branched chain DNA signal amplification techniques
The method for detecting unicellular amplifying nucleic acid, it is characterised in that：The upper cover（ 2）For the transparent material of thickness 0.5-3mm；Cell sieve
Subdivision（ 3）For 10-100 microns of silicon, PDMS, SU8 photoresist, PMMA or PC plastic of thickness；Egative film（ 4）For thickness
Silicon, quartz glass, PMMA or the PC plastic of 0.5-3mm.
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| CN111514947B (en) * | 2020-04-20 | 2021-07-09 | 南京航空航天大学 | Micro-fluidic chip for cell electrical impedance spectroscopy measurement |
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