CN106047677A - Micro-fluidic chip and method for detecting nucleic acid in single cells - Google Patents
Micro-fluidic chip and method for detecting nucleic acid in single cells Download PDFInfo
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Abstract
The invention belongs to the technical field of nucleic acid detection and analysis, and in particularly relates to a micro-fluidic chip for detecting nucleic acid in single cells by virtue of a branched chain DNA signal amplification technology. A plurality of unit structures are uniformly distributed on the chip; the chip comprises an upper cover, a cell sieving unit and a bottom sheet which are sequentially superposed from top to bottom; unit structures on the cell sieving unit comprise liquid passages and single cell sieving structures which are arranged in the liquid passages; unit structures on the upper cover comprise liquid inlets and liquid outlets; and the liquid inlets and the liquid outlets are formed in the upper parts of two ends of the liquid passages. According to the micro-fluidic chip disclosed by the invention, the branched chain DNA signal amplification technology and the micro-fluidic chip are combined, and the chip has the characteristic that such functions as single cell sieving, nucleic acid extraction, nucleic acid detection and the like are integrated on one micro-fluidic chip; the nucleic acid, which is released from single cell lysis, can be captured by capturing probes on microspheres on the chip as a target; and then, branched chain DNA signal amplification is implemented, so that the purpose of nucleic acid detection is achieved.
Description
Technical field
The invention belongs to nucleic acid detection assay technical field, be specifically related to a kind of micro-fluidic chip utilizing branch's chain DNA signal amplification technique unicellular amplifying nucleic acid of detection and the method detecting unicellular amplifying nucleic acid thereof.
Background technology
Along with the arrival in accurate medical science (Precision Medicine) epoch, accurately detection, the ultramicron even demand of unimolecule accuracy detection technology for nucleic acid grow to even greater heights.It is still PCR-based know-why in the foranalysis of nucleic acids detection field widely used technology overwhelming majority at present, since round pcr is born, has 30 years.From classical PCR, real-time quantitative PCR digital pcr the most till now, this technology is in our visual field of the most never fading out of constantly changing in quality.Although round pcr is described as one of greatest invention of biological technical field by scientific circles, and obtain Nobel chemistry Prize in 1993, but in place of regular-PCR technology also has some shortcomings: 1. Taq enzyme or reverse transcription AMV enzyme does not all have 3 '～5 ' exonuclease activities, it is susceptible to base mispairing.The mispairing frequency of Taq enzyme is 1/9 000, and AMV reverse transcriptase, under conditions of high dNTP and Mg2+ exists, arises that the mispairing of a base every about 500 bases；2. the pairing of primer 3 ' end portion may result in non-specific amplification；3. exogenous pollution easily causes false positive；The most all factors affecting nucleic acid amplification all can affect it, and influence factor is too many, reacts poor repeatability；5. due in PCR amplification procedure influence factor too many, it is impossible to ensure that in course of reaction amplification efficiency keeps the amplification efficiency between constant and actual sample from standard sample and different sample to be identical.Even if amplification efficiency is identical, the mutant gene extremely low for content is easily covered by other gene signals a large amount of after amplification.For these reasons, utilize the effect of denier nucleic acid in round pcr detection great amount of samples unsatisfactory, even cannot detect.Therefore the present invention proposes to utilize branch's chain DNA signal amplification technique (branched DNA) to combine microflow control technique and carries out the technology of unicellular amplifying nucleic acid detection, target is the weak point evading round pcr, it is achieved the effective detection to unicellular middle denier even monomolecular nucleic acid.
Branch's chain DNA signal amplification technique is that Urdea etc. develops after synthesizing pectination DNA.This is a kind of nucleic acid hybridization signal amplification detection technology being independent of PCR amplification, this technology overcomes the defect in traditional PCR technique and uncertain factor, without sample is stripped purification, without reverse transcription, expand without PCR, as long as after mixing the sample with the cracking of specific cleavage liquid, gene quantification result can be rapidly be after probe hybridization is amplified with signal.Cardinal principle is to utilize the alkali phosphatase enzyme mark probe molecule connected on pectination DNA molecular and side chain to carry out signal amplification, and the signal of each DNA or RNA molecule about can amplify 2400 times.Owing to this technology specificity is high, it is not necessary to sample is carried out the purification step of complexity, have also been obtained the favor of research worker in terms of transcriptome analysis.Development now with detection technique of fluorescence, it is analyzed having become as reality to single biomolecule by single molecular fluorescence, what after the fluorescence signal of individual molecule is exaggerated thousands of times, identification can become is more prone to, and the detection carrying out mononucleotide molecule hence with bDNA technology is feasible.But the most not yet find to utilize bDNA technology to carry out the research work of nucleic acid coherent detection at unicellular single molecules level.
Summary of the invention
Present invention aim at providing a kind of and utilize the micro-fluidic chip that branch's chain DNA signal amplification technique detects unicellular amplifying nucleic acid and the method detecting unicellular amplifying nucleic acid thereof.
For achieving the above object, the technical solution used in the present invention is:
A kind of micro-fluidic chip utilizing branch's chain DNA signal amplification technique to detect unicellular amplifying nucleic acid, multiple cellular construction it is evenly equipped with on chip 1, this chip 1 includes the upper cover 2 being from top to bottom sequentially stacked, cell sieve unit 3 and egative film 4, cellular construction 31 in cell sieve unit 3 includes fluid passage 312 and the unicellular screening structure 311 being arranged in fluid passage 312, cellular construction 21 on upper cover 2 includes inlet 211 and liquid outlet 212, this inlet 211 and liquid outlet 212 lay respectively at the top at fluid passage 312 two ends, liquid is entered by inlet 211, flowed out by liquid outlet 212 again after fluid passage 312；Unicellular screening structure 311 includes unicellular screening arresting structure A and unicellular screening arresting structure B, this unicellular screening arresting structure A and unicellular screening arresting structure B is fixing single celled space between one end of inlet 211, liquid is by between unicellular screening arresting structure A and unicellular screening arresting structure B, flow through between unicellular screening arresting structure A and fluid passage 312 and between unicellular screening arresting structure B and fluid passage 312, arbitrary unicellular unicellular screening arresting structure A and the unicellular screening arresting structure B of being fixed in liquid is between one end of inlet 211；Cellular construction 41 on egative film 4 is that described micropore is between unicellular screening structure 311 and liquid outlet 212 along the uniform multiple micropores 411 of liquid flow direction.
Distance between described unicellular screening arresting structure A and unicellular screening arresting structure B is less than target cell diameter, the specially 10-50% of target cell diameter, and the diameter range of target cell is 1-50 micron.
The overall structure that described unicellular screening arresting structure A and unicellular screening arresting structure B is combined into can be oval, it is also possible to for splayed.
Described unicellular screening arresting structure A and unicellular screening arresting structure B can be the most abnormally-structured, it is also possible to is simple triangular prism or quadrangular, or other can realize multiple structure coalitions of fixing individual cells.
Described micropore 411 array arranges；Micropore 411 a diameter of 0.5-25 micron, the degree of depth is 0.7-30 micron.
Described upper cover 2 is the transparent material of thickness 0.5-3mm；Cell sieve unit 3 is the silicon of thickness 10-100 micron, PDMS, SU8 photoresist, PMMA or PC plastics；The silicon of thickness 0.5-3mm that egative film 4 is, quartz glass, PMMA or PC plastics.
A kind of method of the micro-fluidic chip utilizing branch's chain DNA signal amplification technique the to detect unicellular amplifying nucleic acid unicellular amplifying nucleic acid of detection:
Cell solution to be detected is injected in described chip, liquid is flow through by between unicellular screening arresting structure A and unicellular screening arresting structure B, between unicellular screening arresting structure A and fluid passage 312 and between unicellular screening arresting structure B and 312, until individual cells is fixed at unicellular screening structure 311, the most unicellular screening arresting structure A and unicellular screening arresting structure B, between one end of inlet 211, changes caching liquid and unwanted cell is gone out chip；
Injecting the cell pyrolysis liquid unicellular cracking by capture after flushing, the capture probe of the modified microsphere surface arranged in the target molecules discharged after simultaneously making cell cracking and chip array micropore 411 carries out hybridization, it is achieved the capture to target molecules；
Then carry out the target molecules of capture is carried out the most qualitative, quantitative detection by signal amplification technique.
The modified microsphere arranged in described chip array micropore 411: choose microsphere according to the bore dia of array micropore 411 in chip, microsphere diameter is slightly less than micro-pore diameter, ensure each micropore can only accommodate single microsphere, microsphere surface is modified with the capture probe for specific objective thing, a kind of capture probe modified by each microsphere, microsphere in each chip unit can be same class microsphere, the capture probe that i.e. microsphere surface is modified is identical, it can also be different types of microsphere, numerous microspheres in i.e. one unit are modified with multiple capture probe, but each microsphere surface only modifies a kind of capture probe.If a chip unit chooses the microsphere being modified with multiple capture probe, in order to effectively identify that the microsphere kind of different capture probe need to choose fluorescence-encoded micro-beads, it is simple to microsphere location addressing in the chips.
The microspheres solution that surface is then modified capture probe is injected in chip, in the array micropore 411 standing, being centrifuged or by the way of directional magnetic field (for magnetic microsphere), microsphere is loaded in chip, and unnecessary microsphere is gone out chip, ensure only one of which microsphere in each micropore, stand-by.
Described signal amplification technique, optical signalling amplifies and signals collecting is to be continuously injected into or be injected simultaneously into the related reagent that branch's chain DNA signal amplifies, after question response is stable, Excess reagents is gone out chip, and carrying out optical photographing, luminous microsphere is and captures the target molecules matched with microsphere surface capture probe.Carry out qualitative according to luminescence to whether cell contains target molecules, according to luminous intensity, the content of target molecules is carried out quantitative analysis.
The present invention is had an advantage in that
Branch's chain DNA signal amplification technique is combined by the present invention with micro-fluidic chip, unicellular screening can be realized at a chip, cracking, target nucleic acid captures, the sequence of operations such as signal amplification detection, can realize detection without unicellular middle denier target nucleic acid carries out PCR amplification, highest detection limit can reach monomolecular nucleic acid.The feature of this chip is unicellular screening and the function such as nucleic acid extraction, detection to be integrated on a micro-fluidic chip, the nucleic acid discharged after unicellular cracking can be carried out target acquistion by microsphere on chip, then carries out branch's chain DNA signal and amplifies the purpose realizing detection of nucleic acids；To the ultramicron of nucleic acid be detected significant.
Accompanying drawing explanation
The chip whole structure figure that Fig. 1 provides for the embodiment of the present invention；
The chip portfolio parts that Fig. 2 provides for the embodiment of the present invention；
The cellular construction parts that Fig. 3 provides for the embodiment of the present invention；
The cell sieve unit that Fig. 4 provides for the embodiment of the present invention just attempts；
The cell sieve unit partial enlarged drawing that Fig. 5 provides for the embodiment of the present invention；
The egative film partial enlarged drawing that Fig. 6 provides for the embodiment of the present invention.
Detailed description of the invention
Branch's chain DNA signal amplification technique is combined by the present invention with micro-fluidic chip, unicellular screening can be realized at a chip, cracking, target nucleic acid captures, the sequence of operations such as signal amplification detection, can realize detection without unicellular middle denier target nucleic acid carries out PCR amplification, highest detection limit can reach monomolecular nucleic acid.The feature of this chip is unicellular screening and the function such as nucleic acid extraction, detection to be integrated on a micro-fluidic chip, the nucleic acid discharged after unicellular cracking can be carried out target acquistion by microsphere on chip, then carries out branch's chain DNA signal and amplifies the purpose realizing detection of nucleic acids.
Embodiment 1
By micro-fluidic chip seen from Fig. 1-4, multiple cellular construction it is evenly equipped with on chip 1, this chip 1 includes upper cover 2, cell sieve unit 3 and the egative film 4 being from top to bottom sequentially stacked, cellular construction 31 in cell sieve unit 3 includes fluid passage 312 and the unicellular screening structure 311 being arranged in fluid passage 312, cellular construction 21 on upper cover 2 includes inlet 211 and liquid outlet 212, this inlet 211 and liquid outlet 212 lay respectively at the top at fluid passage 312 two ends, and liquid is entered by inlet 211, flowed out by liquid outlet 212 after fluid passage 312 again；Unicellular screening structure 311 includes unicellular screening arresting structure A and unicellular screening arresting structure B, this unicellular screening arresting structure A and unicellular screening arresting structure B is fixing single celled space between one end of inlet 211, liquid is by between unicellular screening arresting structure A and unicellular screening arresting structure B, flow through between unicellular screening arresting structure A and fluid passage 312 and between unicellular screening arresting structure B and 312, arbitrary unicellular unicellular screening arresting structure A and the unicellular screening arresting structure B of being fixed in liquid is between one end of inlet 211；Cellular construction 41 on egative film 4 is that described micropore is between unicellular screening structure 311 and liquid outlet 212 along the uniform multiple micropores 411 of liquid flow direction.
Distance between described unicellular screening arresting structure A and unicellular screening arresting structure B is less than target cell diameter, the specially 10-50% of target cell diameter, and the diameter range of target cell is 1-50 micron.
The overall structure that described unicellular screening arresting structure A and unicellular screening arresting structure B is combined into can be oval, it is also possible to for splayed.Described unicellular screening arresting structure A and unicellular screening arresting structure B can be the most abnormally-structured, it is also possible to is simple triangular prism or quadrangular, or other can realize multiple structure coalitions of fixing individual cells.
The concrete preparation method of chip is:
Step 1: select the silicon chip of a single-sided polishing；
Step 2: silicon chip is carried out；
Step 3: one layer of uniform photoresist of spin coating on silicon wafer polishing face, is transferred on photoresist form mask by egative film mask plate patterns by exposure；
Step 4: dry etching silicon slice under mask, array micropore 411 in the surface of silicon chip forms egative film, a diameter of 6 microns of hole, the number in hole is 20, the degree of depth is micropore target depth and cell sieve unit layer thickness sum, micropore target depth 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, is transferred on photoresist form mask by cell sieve unit mask plate patterns by exposure；
Step 7: dry etching silicon slice under mask, unicellular screening structure 311 and fluid passage 312 in the surface of silicon chip forms cell sieve unit, the degree of depth is cell sieve unit layer target thickness 30 microns.Form egative film 4 and the coalition of cell sieve unit 3；
Step 8: choose upper cover plate 2, material is quartz glass or PMMA, gets through hole as inlet 211 and liquid outlet 212 according to the passage end positions in unicellular sieve unit 3；
Step 9: be bonded by the silicon chip that upper cover plate 2 obtains with step 7, completes the preparation of chip 1.Distance d between unicellular screening arresting structure A and unicellular screening arresting structure B is 20 microns.
Embodiment 2
Utilize above-mentioned acquisition chip to carry out unicellular capture experiment, specifically comprise the following steps that
Step 1 microsphere is chosen: choose 5 micron polystyrene microspheres according to the bore dia of array micropore 411 in chip, microsphere diameter is less than micro-pore diameter 1 micron, ensure each micropore can only accommodate single microsphere, microsphere surface is modified with the capture probe for specific objective thing, a kind of capture probe modified by each microsphere, microsphere in each chip unit can be same class microsphere, the capture probe that i.e. microsphere surface is modified is identical, it can also be different types of microsphere, numerous microspheres in i.e. one unit are modified with multiple capture probe, but each microsphere surface only modifies a kind of capture probe.If a chip unit chooses the microsphere being modified with multiple capture probe, in order to effectively identify that the microsphere kind of different capture probe need to choose fluorescence-encoded micro-beads, it is simple to microsphere location addressing in the chips.
Step 2 microsphere loads: the microspheres solution that surface is modified capture probe is injected in chip, in the array micropore 411 standing, being centrifuged or by the way of directional magnetic field (for magnetic microsphere), microsphere is loaded in chip, and unnecessary microsphere is gone out chip, it is ensured that only one of which microsphere in each micropore.
The unicellular capture of step 3: be injected in chip by cell solution to be detected, until individual cells is fixed at unicellular screening structure 311, changes caching liquid and unwanted cell is gone out chip.
The unicellular cracking of step 4 and target molecules capture: inject cell pyrolysis liquid and unicellular cracking, the target molecules discharged after simultaneously making cell cracking are carried out hybridization with the capture probe of microsphere surface, it is achieved the capture to target molecules.
Step 5 optical signalling amplifies and signals collecting: be continuously injected into or be injected simultaneously into the related reagent that branch's chain DNA signal amplifies, after question response is stable, Excess reagents being gone out chip, and carries out optical photographing, luminous microsphere is and captures the target molecules matched with microsphere surface capture probe.Record according to prior art, can carry out qualitative to whether cell contains target molecules according to luminescence, the content of target molecules can be carried out quantitative analysis according to luminous intensity.
The chip that separately the present invention relates to can be applicable to overwhelming majority usual cell such as: microorganism, virus, zooblast etc..
Claims (5)
1. one kind utilizes the miniflow that branch's chain DNA signal amplification technique detects unicellular amplifying nucleic acid
Control chip, it is characterised in that: chip is evenly equipped with multiple cellular construction on (1), this chip (1)
Including the upper cover (2) being from top to bottom sequentially stacked, cell sieve unit (3) and egative film (4),
Cellular construction (31) in cell sieve unit (3) includes fluid passage (312) and arranges
Unicellular screening structure (311) in fluid passage (312), the unit on upper cover (2)
Structure (21) includes inlet (211) and liquid outlet (212), this inlet (211) and
Liquid outlet (212) lays respectively at the top at fluid passage (312) two ends, and liquid is by inlet
(211) enter, flowed out by liquid outlet (212) again after fluid passage (312)；
Unicellular screening structure (311) includes that unicellular screening arresting structure A and unicellular screening are caught
Obtaining structure B, this unicellular screening arresting structure A and unicellular screening arresting structure B is near entering
For fixing single celled space between one end of liquid mouth (211), liquid is caught by unicellular screening
Obtain between structure A and unicellular screening arresting structure B, unicellular screening arresting structure A and liquid
Between body passage (312) and unicellular screening arresting structure B and fluid passage (312) it
Between flow through, arbitrary unicellular in liquid is fixed in unicellular screening arresting structure A and slender
Born of the same parents sieve arresting structure B between one end of inlet (211)；List on egative film (4)
Meta structure (41) is along the uniform multiple micropores (411) of liquid flow direction, described micropore position
Between unicellular screening structure (311) and liquid outlet (212).
2. utilize the detection of branch chain DNA signal amplification technique unicellular as described in claim 1
The micro-fluidic chip of amplifying nucleic acid, it is characterised in that: described unicellular screening arresting structure A and list
The 10-50% that distance is target cell diameter between cell screening arresting structure B.
3. utilize the detection of branch chain DNA signal amplification technique slender as described in claim 1
The micro-fluidic chip of born of the same parents' amplifying nucleic acid, it is characterised in that: described micropore 411 array arranges；Micro-
Hole 411 a diameter of 0.5-25 micron, the degree of depth is 0.7-30 micron.
4. utilize the detection of branch chain DNA signal amplification technique slender as described in claim 1
The micro-fluidic chip of born of the same parents' amplifying nucleic acid, it is characterised in that: described upper cover 2 is thickness 0.5-3mm's
Transparent material；Cell sieve unit 3 is the silicon of thickness 10-100 micron, PDMS, SU8 light
Photoresist, PMMA or PC plastics；The silicon of thickness 0.5-3mm that egative film 4 is, quartz glass,
PMMA or PC plastics.
5. the branch's chain DNA signal amplification technique that utilizes described in a claim 1 detects list
The micro-fluidic chip of cell amplifying nucleic acid detects the method for unicellular amplifying nucleic acid, it is characterised in that:
Being injected in described chip by cell solution to be detected, liquid is by unicellular screening capture knot
Between structure A and unicellular screening arresting structure B, unicellular screening arresting structure A and liquid leads to
Flow between road (312) and between unicellular screening arresting structure B and fluid passage (312)
Cross, until individual cells is fixed on unicellular screening structure (311) place, the most unicellular sieve
Point arresting structure A and unicellular screening arresting structure B near one end of inlet (211) it
Between, change caching liquid and unwanted cell is gone out chip；
Inject the cell pyrolysis liquid unicellular cracking by capture after flushing, make cell crack simultaneously
After the modified microsphere that arranges in the target molecules that discharges and chip array micropore (411)
The capture probe on surface carries out hybridization, it is achieved the capture to target molecules；
Then carry out carrying out the target molecules of capture determining accordingly by signal amplification technique
Property, quantitative detection.
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| CN107012220A (en) * | 2017-04-10 | 2017-08-04 | 厦门大学 | It is a kind of to utilize the method for matching the unicellular inclusion of micro-fluidic chip high throughput analysis |
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