CN109852530A - A kind of micro-fluidic chip and its device and method integrating circulating tumor cell capture, cracking and detection of nucleic acids - Google Patents
A kind of micro-fluidic chip and its device and method integrating circulating tumor cell capture, cracking and detection of nucleic acids Download PDFInfo
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Abstract
The present invention provides a kind of micro-fluidic chip and its device and method for integrating circulating tumor cell capture, cracking and detection of nucleic acids.Micro-fluidic chip is bonded by glass substrate layers and polydimethylsiloxanechip chip layer, and polydimethylsiloxanechip chip layer includes: circulating tumor cell trapping region；Detection of nucleic acids area, comprising: the sample intake passage with elastic sidewall, microcavity array area and outlet；And valve control zone, it include: the T shape pipeline configuration being respectively arranged in sample intake passage two sides, the structure includes the first pipe that opposite sample intake passage extends in parallel, and the opposite vertically extending second pipe of sample intake passage, second pipe is connect with a gas supply mechanism, by controlling the opening and closing of gas supply mechanism, the closure and unlatching of sample intake passage are realized.According to the present invention, a kind of high throughput is provided, low cost is highly sensitive, easy to operate, the time-consuming short micro-fluidic chip and its device and method for integrating circulating tumor cell capture, cracking and detection of nucleic acids.
Description
Technical field
The present invention relates to micro-fluidic field, relate more specifically to a kind of collection circulating tumor cell capture, cracking is examined with nucleic acid
Survey the micro-fluidic chip and its device and method in one.
Background technique
Worldwide, cancer morbidity remains high for a long time, and is the main reason for tumor patient is dead.
It is domestic, no matter rural city, no matter men and women, cancer is still high one of the disease of the death rate, and it is public to become influence society
The significant problem of many health.It and is exactly early diagnosis and therapy to primary cancer to the key of anticancer.
Circulating tumor cell (CTCs) is the tumour cell that Peripheral Circulation is fallen off and invaded from tumour primary lesion, due to
The development transfer of CTCs and cancer is closely related, and is sending out in addition to containing the inhereditary material similar to primary tissue in CTC
Other additional genetic information can be also obtained in journey, thus, molecular level change can provide foundation for individuation disease treatment,
The molecule genetic analysis of CTC is expected to tumor genotype variation in monitoring treatment of diseases, to adjust for cancer patient drug
Foundation is provided.But the CTC content in peripheral blood is more rare, usually in about 100,000,000 leucocytes and 50,000,000,000 red blood cells only
Then several tumour cells are again analyzed its characterization of molecules therefore, it is necessary to first have to be enriched with CTCs.
In recent years, micro-fluidic chip relies on the advantages such as its channel size is suitable with cell size, integrated level is high, becomes CTCs
The reliable platform of detection.It can concentrate on the operations such as the capture, cracking, detection of nucleic acids of cell on one piece of small chip, use
To realize the various functions in standard biologic laboratory, has many advantages, such as that small in size, consumption reagent is few, is swift in response.Currently, existing
Circulating tumor cell is much detected based on micro-fluidic method, but is mostly the independent capture and cracking point realized respectively to cell
Analysis, it is less to realize the micro-fluidic chip that the capture, cracking and detection of nucleic acids of circulating tumor cell are integrated in one.
Patent document 1 (CN103732731A): micro-current controlled cell capture and analytical equipment for high throughput analysis, design
It is a kind of can to the micro-fluidic chip of quantitative unicellular carry out genetic analysis, but this chip cannot ensure to realize in design
Capture and analysis to circulating tumor cell rare in blood.Patent document 2 (CN102360010A): a kind of whole blood tumour is thin
Born of the same parents capture integrated microfluidic chip, devise a micro-fluidic chip that whole blood tumour cell capture and cracking may be implemented, but
It is that it only realizes the capture of cancer cell, analysis detection cannot be carried out to cancer cell nucleic acid.
In the technology having disclosed at present, it is related to capture, cracking and the detection of nucleic acids to CTCs in the documents and materials of one
It is fewer and fewer, it is necessary to which that the integrated microfluidic chip of a kind of capture that can be realized to CTCs, cracking and detection of nucleic acids is provided.
Summary of the invention
Integrate the micro-fluidic of circulating tumor cell capture, cracking and detection of nucleic acids the object of the present invention is to provide a kind of
Chip and its device and method, so that the capture, cracking and core of circulating tumor cell cannot be realized simultaneously by solving the prior art
The problem of acid detects, thus causes process cumbersome, the defect of higher cost.
In order to solve the above-mentioned technical problem, the invention adopts the following technical scheme:
According to the first aspect of the invention, provide it is a kind of integrate circulating tumor cell capture, cracking and detection of nucleic acids
Micro-fluidic chip, the micro-fluidic chip is bonded by glass substrate layers and polydimethylsiloxanechip chip layer, described poly-
Dimethyl siloxane chip layer includes: circulating tumor cell trapping region, comprising: cell capture injection port；It is formed by pillar array
The first agglomerate filtration zone；Target cell screening areas, several main pipelines and side ducts including extension spaced in parallel to each other,
It is connected between the main pipeline and side ducts by filtration channel, the main pipeline and side ducts are respectively provided with greater than circulating tumor
First height of cell size, the filtration channel have the second height less than circulating tumor cell size；And cell is caught
Obtain outlet；The polydimethylsiloxanechip chip layer further include: detection of nucleic acids area, comprising: with the cell capture outlet
The sample intake passage of connection, the sample intake passage have elastic sidewall；The microcavity array area being made of several microcavitys；And nucleic acid inspection
Survey outlet；And the valve control zone of connection the circulating tumor cell trapping region and the detection of nucleic acids area, comprising: respectively
It is arranged in the T shape pipeline configuration of the sample intake passage two sides, the T shape pipeline configuration includes that the relatively described sample intake passage prolongs in parallel
The first pipe stretched, and the relatively described vertically extending second pipe of sample intake passage, the second pipe and a gas supply mechanism
Connection realizes the closure and unlatching of sample intake passage by controlling the opening and closing of gas supply mechanism.
The micro-fluidic chip provided according to the present invention, circulating tumor cell trapping region and detection of nucleic acids area be not too many
It improves, and main improved inventive point is, a valve is provided between circulating tumor cell trapping region and detection of nucleic acids area
Control zone, the opening and closing of the sample intake passage for controlling connection circulating tumor cell trapping region and detection of nucleic acids area.Valve control
The working principle in area are as follows: the elastic sidewall of sample intake passage occurs by external gas supply mechanism pressurized control T shape pipeline configuration
Deflection, so that sample intake passage is closed；On the contrary, closing gas supply mechanism, T shape pipeline configuration does not apply any pressure to sample intake passage
Power, sample intake passage are opened.
Preferably, distance of the first pipe of T shape pipeline configuration apart from sample intake passage is 10~20 μm, the width of second pipe
Degree is 40~60 μm, and most preferably, distance of the first pipe of T shape pipeline configuration apart from sample intake passage is 15 μm, second pipe
Width be 50 μm, by gas supply mechanism, optionally, nitrogen is provided and applies certain pressure control sample intake passage, keeps sample introduction logical
The elastic sidewall in road deflects, and can close the sample intake passage, to prevent cell from by sample intake passage, valve control zone
Connect CTCs trapping region and detection of nucleic acids area.
The micro-fluidic chip provided according to the present invention, all structure features of circulating tumor cell trapping region substantially with
ZL201610398852X is similar, and difference is to eliminate the flow dividing structure of front end at cell capture injection port, only stays and cell
The identical branch of outlet is captured to reduce flow resistance.And, it is preferable that inlet and outlet structure is identical, there is hexagon column
Daughter cell group's filtration zone, that is, the target cell screening areas and cell capture outlet of the circulating tumor cell trapping region
Between be equipped with the second agglomerate filtration zone corresponding with the first agglomerate filtration zone so that the entire circulating tumor cell
Trapping region is configured to axially symmetric structure, so that subsequent cell nucleic acid and PCR reaction solution can be at identical conditions into two sides
Detection of nucleic acids area.
Wherein, it is connected between main pipeline and side ducts by filter pipe, the front end of main pipeline is opened wide to be entered for solution, side
The closing of pipeline front end, end is opened wide flows out for waste liquid, wherein main pipeline and side ducts are respectively provided with the first height, filtration channel
With the second height, the first height is greater than the size of circulating tumor cell, and the second height is less than the size of circulating tumor cell.
The quantity of the main pipeline of the circulating tumor cell trapping region provided according to the present invention, side ducts and filter pipe can
To change according to actual needs, height is optimized according to the size difference of target cell He other cells, can also
To change according to actual needs.The big circulating tumor cell of size by filter pipe height intercept, the small leucocyte of size and
Red blood cell can reach wing passage by filtration channel, to flow out from outlet.The present invention creatively uses height to sieve
Cell is selected, so that mould process is low to required precision, low manufacture cost, step is simple and easy.First height is 40~60 μ
M, most preferably 50 μm, the second height are 8~15 μm, most preferably 10 μm, and the circle that is used to support is equipped in filtration channel
Pillar.The first, second agglomerate filtration zone preferably includes the primary agglomerate mistake being arranged successively from cell capture injection port
Region and secondary agglomerate filtration zone are filtered, the minimum spacing between the hexagon pillar of primary agglomerate filtering area is 50 μm~60 μm,
Greater than 15 μm~25 μm of minimum spacing between the hexagon pillar of secondary agglomerate filtration zone, which only allows list
Cell passes through, and cell mass and impurity can be intercepted, so that chip CTCs trapping region be avoided to block.
The micro-fluidic chip provided according to the present invention, it is preferable that the polydimethylsiloxanechip chip layer includes two cores
Sour detection zone, described two detection of nucleic acids areas are symmetrically arranged in the two sides of circulating tumor cell trapping region, and catch respectively with cell
Injection port is obtained to connect with cell capture outlet.
Correspondingly, the polydimethylsiloxanechip chip layer includes two valve control zones.
As previously described, the micro-fluidic chip provided according to the present invention, detection of nucleic acids area substantially with it is public in the prior art
The structure opened is similar, but one of the improvement of amplifying nucleic acid detection zone of the present invention is, the microcavity array in the detection of nucleic acids area
Area includes several Rectangular Microchannels arranged in parallel, and several microcavitys communicated therewith in the two sides of the Rectangular Microchannel,
Right angle in 90 ° is different between microcavity and Rectangular Microchannel described in the prior art, excellent between the microcavity and Rectangular Microchannel
Selection of land is at 30 °~60 ° of acute angle.Under the angle design, make nucleic acid and PCR reaction solution straight compared to 90 ° by centrifugally operated
The mechanism design at angle is easier to enter microcavity.Most preferably, acute angle at 45 ° between the microcavity and Rectangular Microchannel.
According to a preferred embodiment of the present invention, what the detection of nucleic acids area was connect with sample intake passage, which is continuous one point, is
Two Rectangular Microchannel, it is therefore an objective to which the distance for enabling injection port reach each microchannel in array area is approximately equal.The array area
Straight line Rectangular Microchannel has 128, and equidistant arrangement forms, 80 μm wide, 50 μm high, and the both sides 45° angle of every Rectangular Microchannel is more
80 μm of cylindrical type microcavity diameters of narrow microchannel linking, 50 μm high, i.e. dPCR reacts microcavity, is used for store sample drop, horizontal
It is 40 μm to spacing between two neighboring microcavity.
According to a preferred embodiment of the present invention, the entire array area cylindrical type microcavity number in detection of nucleic acids area is 6400
It is a, but 6400 are not limited only to, it can change according to actual needs.Preferably, which includes two detection of nucleic acids
Area, that is to say, that respectively there is an array on chip both sides, therefore it is 12800 that the chip, which includes cylindrical type microcavity sum,.Nucleic acid inspection
It surveys outlet and is designed as 8 outlets, for chip preferably to be discharged in waste liquid, keep the sample in chip microcavity anti-in thermal cycle
Not disturbed and pollution in answering.
The array cylindrical microcavity structure be cellular nucleic acid and PCR mix are successfully imported cylindrical microcavities, and it is each
Microcavity can finally come independently of each other, then carry out augmentation detection, i.e., a kind of new nucleic acid to the cellular nucleic acid in each microcavity
Quantitative technique-digital pcr (dPCR), its basic principle are to can be summarized as " dividing and rule ": a sample equivalent is diluted to
Tens arrive tens of thousands of parts, are assigned to different reaction members, and each unit includes that the target molecule of zero or one copy is (initial
DNA profiling), the PCR amplification of single copy DNA is independently carried out in each reaction member.After amplification, to there is fluorescence signal
Positive unit (include target molecule) counted (fluorescence signal that negative unit does not become clear then), count results are just
The copy number of target template DNA in beginning sample.The quantity of dPCR reaction member is more, to the recognition capability of target gene or mutation
Stronger, i.e. sensitivity is higher, and accuracy is also higher, accordingly, with respect to regular-PCR and qPCR technology, dPCR with its independent of
External reference the characteristics of influence by PCR amplification efficiency, realizes reliable and stable absolute quantitation detection, has been used as grinds in recent years
Study carefully and is widely used with the analysis tool of clinical application, the inspection of detection, monocell expressing spectrum including allelic gene typing
It surveys, the detection of single nucleotide polymorphism (SNPs) detection and low-copy target gene.Micro-fluidic core based on microelectric technique
The development of piece processing technology provides more suitable platform for dPCR technology, the micromation of micro-fluidic chip, integrated, rapid height
Effect, required sample is few and so that dPCR technology has been obtained bigger development space suitable for the advantages that on-site test.The present invention uses
The method of dPCR carries out detection of nucleic acids to the circulating tumor cell captured.
According to the second aspect of the invention, providing a kind of includes capturing, splitting for circulating tumor cell as described above
The micro fluidic device of the micro-fluidic chip of solution and detection of nucleic acids.
The micro fluidic device includes: sequentially connected solution locker room, the micro-fluidic chip, waste collection syringe,
And sampling pump, it further include the gas supply mechanism being connect with the T shape pipeline configuration of the valve control zone of the micro-fluidic chip.
The gas supply mechanism includes the pulsometer and nitrogen gas tank by piping connection.
According to the third aspect of the invention we, it provides a kind of while carrying out circulating tumor cell capture, cracking and detection of nucleic acids
Method, the described method comprises the following steps: 1) a kind of micro fluidic device as described above is provided；2) gas supply mechanism is opened,
The closure of the sample intake passage is realized to sample intake passage pressure by the first pipe of the T shape pipeline configuration；3) negative pressure is utilized
Sample to be detected is pumped into the circulating tumor cell trapping region of micro-fluidic chip by cell capture injection port, size is smaller
Cell be rushed out micro-fluidic chip and enter in waste collection syringe, and larger-size circulating tumor cell is intercepted at
In micro-fluidic chip；4) circulation that cell pyrolysis liquid is pumped into micro-fluidic chip is swollen by cell capture injection port using negative pressure
In oncocyte trapping region, the circulating tumor cell intercepted in micro-fluidic chip is cracked；5) pass through cell using negative pressure
PCR reaction solution is pumped into the circulating tumor cell trapping region of micro-fluidic chip by capture injection port；6) gas supply mechanism, institute are closed
It states sample intake passage and restores normal circulation status, vacuumizing keeps the cell pyrolysis liquid of the circulating tumor cell trapping region and PCR anti-
The mixed liquor of liquid is answered to flow into detection of nucleic acids area, centrifugally operated carries out pcr amplification reaction；And 7) after pcr amplification reaction,
The micro-fluidic chip is placed in fluorescence microscopy under the microscope, and analyzes the fluorescence feelings of each microcavity in the detection of nucleic acids area
Condition, so that the nucleic acid situation to circulating tumor cell is analyzed.
The step 6) further include: before pcr amplification reaction, using mineral oil and aluminium-foil paper to the detection of nucleic acids area into
Row closing.
In the method for the CTCs detection of nucleic acids provided according to the present invention, into blood sample before need first by CTCs trapping region and
The sample intake passage of nucleic acid amplification area connection is closed, and cell is avoided to enter detection of nucleic acids area.The pulsometer used in detection can be with
8 valves are controlled simultaneously, and there are two parallel channels, examine respectively with detection of nucleic acids injection port and nucleic acid by venous transfusion needle tubing
Outlet connection is surveyed, blood sample and cell pyrolysis liquid are extracted into the circulating tumor cell trapping region on chip using negative pressure, it can
To detect two samples simultaneously, it can also according to actual needs, increase the number of pulsometer and be come using the sampling pump of more multichannel
Detection while realizing multiple samples.
In conclusion CTCs capture, cracking and detection of nucleic acids are creatively focused on same chip for the first time by the present invention
On, integration degree is high, substantially increases the detection speed and sensitivity of CTCs nucleic acid.CTCs trapping region and core in the device
Sour detection zone is similar with the corresponding construction of the prior art.The present invention is at injection port for the improvement of CTCs trapping region
The flow dividing structure of front end is eliminated, stays branch identical with outlet only to reduce flow resistance, and the complete phase of inlet and outlet structure
Together, there is hexagon pillar cell mass filtration zone, to achieve the effect that both sides symmetrical configuration, so that subsequent cell lysate can
Enter the detection of nucleic acids area on both sides at identical conditions.The present invention is also partly improved detection of nucleic acids area, wherein cylinder
By being that acute angle is connect with 90 ° of angle Innovation of Connection of Rectangular Microchannel, preferably 45° angle connects shape microcavity, i.e., tilts at microcavity
45° angle, by centrifugally operated, so that nucleic acid and amplification liquid phase are easier to enter microcavity than right-angle structure.The present invention is relatively existing
The core of technological improvement is to be to devise a kind of micro-valve, that is, passes through a sample intake passage for CTCs trapping region and detection of nucleic acids area
It links together, and respectively designs the T shape pipeline configuration that can be passed through gas, the T shape pipeline knot in the two sides of sample intake passage
Structure includes the first pipe that the relatively described sample intake passage extends in parallel, and vertically extending second pipe of the relatively described sample intake passage
Road, the second pipe are connect with a gas supply mechanism, by controlling the opening and closing of gas supply mechanism, are realized the closure of sample intake passage and are opened
It opens, to realize on a single die while carry out the whole process of the capture of CTCs, cracking and digital pcr augmentation detection.
Inventor passes through to air pressure size, and groping for the various conditions such as complexity of PDMS ratio and realization has just finally determined this
A kind of valve control structure of structure of sample, this structure easily manipulates, effect is good.
Compared with existing circulating tumor cell nucleic acid detection system, micro fluidic device provided by the invention and method are being followed
Ring tumour cell nucleic acid has very big advantage in terms of detecting speed, simplicity and cost.On the one hand, the microfluidic chip structure
Simply, easy to make, and silicon wafer mold carry out after can reuse；On the other hand, the microfluidic chip structure is small, needs
Amount of reagent it is few, only need the other amount of micro updating that can detect desired as a result, greatly reducing cost.In addition, of the invention
The micro fluidic device of offer is that a kind of capture of circulating tumor cell, cracking and the integrated device of detection of nucleic acids, circulating tumor are thin
Born of the same parents directly crack after being enriched in the chips and nucleic acid amplification and abrupt climatic change, do not need to be turned after cell enrichment
It moves, then its nucleic acid is detected, avoid the loss of target cell, to increase the sensitivity of cellular nucleic acid detection.And
The first technology of the application, patent of invention ZL201610398852X are " a kind of based on the micro-fluidic of size detection circulating tumor cell
Device and method " merely provides the method that circulating tumor cell capture counts, and feelings are expressed and be mutated for intracellular nucleic acid
Condition powerlessly detects.
In short, low cost is highly sensitive the present invention provides a kind of high throughput, easy to operate, time-consuming short collection circulation is swollen
Oncocyte capture, cracking and detection of nucleic acids are in the micro-fluidic chip and its device and method of one.
Detailed description of the invention
Shown in FIG. 1 is the structural schematic diagram of the micro-fluidic chip provided according to a preferred embodiment of the present invention；
Fig. 2 is the detail enlarged diagram of the circulating tumor cell trapping region of micro-fluidic chip as shown in Figure 1；
Fig. 3 is the structural schematic diagram of the valve control zone of micro-fluidic chip as shown in Figure 1；
Fig. 4 is the structural schematic diagram in the detection of nucleic acids area of micro-fluidic chip as shown in Figure 1；
Fig. 5 is the structural schematic diagram of the micro fluidic device of a preferred embodiment according to the present invention；
Fig. 6 is the CTCs capture rate curve graph in embodiment 4；
Fig. 7 is the solution distribution situation in the detection of nucleic acids area microcavity after vacuumizing and be centrifuged in embodiment 5；
Fig. 8 is the comparison diagram of solution evaporation situation in the microcavity of PDMS closing front and back when dPCR reacts in embodiment 6；
Fig. 9 is detection 21 exon L858 mutated gene of lung cancer circulating tumor cell H1975EGFR in embodiment 7
Fluorescence microscopy figure.
Specific embodiment
Below in conjunction with specific embodiment, the present invention will be further described.It should be understood that following embodiment is merely to illustrate this
The range of invention and is not intended to limit the present invention.
The preparation of 1 micro-fluidic chip of embodiment
1.1 firstly, be directed to micro-fluidic chip 100 structure division, draw out required knot with AutoCAD drawing software
Composition shape makes mask, carries out thermal oxide by substrate of four cun of monocrystalline silicon pieces, forms one layer 2 μm of oxide layer, then is applied
Glue photoetching, reactive ion etching, cleaning of removing photoresist, gluing photoetching, deep reaction ion etching etch the filtering of circulating tumor trapping region
The height in channel.Remove photoresist cleaning removal photoresist again, carries out deep reaction ion etching according to silicon dioxide pattern, etches core
The height in other channels of piece, and the depth of nucleic acid microcavity is etched, finally just obtain the silicon wafer mold with micro-structure.
1.2 are placed in a vacuum drying oven silicon wafer mold and open centrifuge tube equipped with 10 μ L silicon fluorides, are evacuated to negative
Pressure, vaporizes silicon fluoride, mold stands 5-6 hour in silicon fluoride steam.In ventilation, drying box is opened, ventilation 1
After a hour, by silicon chip extracting.The purpose of this step is to deposit one layer of organic matter in silicon chip surface, convenient for subsequent PDMS chip
Production.
1.3 weigh PDMS prepolymer and curing agent according to weight ratio 10:1 respectively, are then mixed and stirred for uniformly, being placed in true
It is vacuumized in empty drying box, stands 30min under negative pressure.After PDMS substantially without bubble after, be cast on silicon wafer mold, it is quiet
30min is set, then, puts it into 65 DEG C of baking oven heating 1h.Finally the PDMS layer being cured is stripped down from mold, according to
Inlet and outlet on figure is punched, and the redundance other than structure is cut away.Finally by 101 structure of PDMS chip layer
It is put into plasma cleaner with glass substrate layers 102 up and cleans 1min, fit together rapidly, that is, complete after taking-up
The encapsulation of micro-fluidic chip 100.
The specific structure of micro-fluidic chip 100 is described in detail as follows:
As shown in Figure 1, being a kind of micro-fluidic chip 100 provided according to a preferred embodiment of the present invention, the miniflow
Control chip 100 integrates circulating tumor cell capture, cracking and detection of nucleic acids, and micro-fluidic chip is by dimethyl silicone polymer core
Lamella 101 and glass substrate layers 102 are bonded, and polydimethylsiloxanechip chip layer 101 includes: circulating tumor cell trapping region
10, detection of nucleic acids area 20 and valve control zone 30.
Wherein, it as shown in Figure 1, Figure 2, circulating tumor cell trapping region 10 is centrally arranged on the chip, specifically includes that
Cell capture injection port 1；The the first agglomerate filtration zone 2 formed by pillar array；Target cell screening areas 3, including each other
Several main pipelines and side ducts that parallel interval extends, are connected between the main pipeline and side ducts by filtration channel, described
Main pipeline and side ducts are respectively provided with the first height greater than circulating tumor cell size, and the filtration channel, which has, is less than circulation
Second height of tumour cell size；The the second agglomerate filtration zone 4 formed by pillar array；And cell capture outlet 5.
According to the preferred embodiment, detection of nucleic acids area 20 is two, is symmetrically arranged in circulating tumor cell trapping region 10
Two sides are connect with cell capture injection port 1 and cell capture outlet 5 respectively, comprising: are connect with cell capture outlet 5
Sample intake passage 6, the sample intake passage 6 have elastic sidewall；The microcavity array area 7 being made of several microcavitys；And detection of nucleic acids goes out
Sample mouth 8.
As shown in figure 3, valve control zone 30 is used for connection ring tumour cell trapping region 10 and detection of nucleic acids area 20, comprising:
It is respectively arranged the T shape pipeline configuration 9 in 6 two sides of sample intake passage, which includes that the relatively described sample intake passage is parallel
The first pipe 91 of extension, and the opposite vertically extending second pipe 92 of sample intake passage 6, the second pipe 92 and a gas supply
The closure and unlatching of sample intake passage 6 can be realized by controlling the opening and closing of gas supply mechanism in mechanism connection.
According to the preferred embodiment, distance of the first pipe 91 of the T shape pipeline configuration apart from sample intake passage 6 is 15 μm,
The width of second pipe 92 is 50 μm.
As shown in figure 4, the microcavity array area 7 in detection of nucleic acids area 20 includes several Rectangular Microchannels 71 arranged in parallel,
And several microcavitys 72 communicated therewith in the two sides of Rectangular Microchannel 71, it is at 45 ° between microcavity 72 and Rectangular Microchannel 71
Angle.It should be understood that the angle is not limited in 45° angle, any acute angle between 30 °~60 ° can also be.
According to the preferred embodiment, polydimethylsiloxanechip chip layer 101 includes two valve control zones 30.
The assembling of 2 micro fluidic device of embodiment
As shown in figure 5, being the micro fluidic device that a preferred embodiment according to the present invention provides, the micro fluidic device
Include: sequentially connected solution locker room 200, micro-fluidic chip 100, waste collection syringe 300 and sampling pump 400, also wraps
Include the pulsometer 500 and nitrogen gas tank 600 connecting with the T shape pipeline configuration 9 of the valve control zone 30 of micro-fluidic chip 100.Wherein,
Needle tubing 700 is conveyed by solution between micro-fluidic chip 100 and solution locker room 200 and waste collection syringe 300.
It should be understood that admission gear is not limited in pulsometer 500 and nitrogen gas tank used in the present embodiment
600, it can also be any device that can be connect with T shape pipeline configuration 9 and supply to second pipe 92.
Embodiment 3 carries out capture, cracking and the nucleic acid amplification detection of circulating tumor cell using micro fluidic device
It is to handle blood sample first, is handled using Ficoll-Paque plus reagent, then with 4% poly
Cell is fixed in formaldehyde, and centrifugation goes supernatant 1mL PBS to be resuspended, and it is stand-by to be put in 4 DEG C of refrigerators.When detection, air pressure is first opened
500 offer air pressure of pump is closed sample intake passage 6, then sets 10mL/h for the flow velocity of sampling pump 300, diameter is set as
19mm, one end of waste collection syringe 300 are connected with the cell capture outlet 5 of micro-fluidic chip 100, utilize sampling pump 300
Sample is pumped into micro-fluidic chip 100 by the negative pressure of offer from the cell capture injection port 1 of micro-fluidic chip 100, size compared with
Small leucocyte and red blood cell is rushed out micro-fluidic chip 100 and enters in waste collection syringe 300, and larger-size circulation
Tumour cell is intercepted in micro-fluidic chip 100；Then chip is added in 10 μ L cell pyrolysis liquids with same method
Circulating tumor cell trapping region 10 cracks circulating tumor cell；Cell cracking terminates, 10 μ that will be prepared in the same way
L PCR reaction solution imports circulating tumor cell trapping region 10, mixes with cell pyrolysis liquid, it should be appreciated that according to target cell
Difference, different PCR reaction solutions can be replaced, to its nucleic acid carry out augmentation detection；It is then turned off pulsometer 500, makes sample introduction
Normal circulation status is restored in channel 6, then makes circulating tumor cell trapping region using the mode of centrifugation (revolving speed 3000rpm, 20min)
The mixed liquor of 10 cell pyrolysis liquid and PCR mix flow into detection of nucleic acids area 20 and expand to the nucleic acid of target cell, PCR journey
Sequence: 50 DEG C, 30min；94 DEG C, 30s；12 circulation (94 DEG C, 30s；62 DEG C, 40s)；33 circulation (94 DEG C, 30s；60 DEG C,
40s), it should be appreciated that according to target cell or nucleic acid difference, different PCR reaction conditions can be replaced；Reaction knot
Micro-fluidic chip 100 is placed in the fluorescence of each microcavity in observation analysis nucleic acid amplification area under fluorescence microscope by Shu Hou, thus
The nucleic acid situation of circulating tumor cell is analyzed.
Embodiment 4
The detection method that the micro fluidic device and embodiment 2 provided using embodiment 1 is provided, first to the lung of known concentration
Cancer cell line H1975 suspension is tested, and is calculated the capture rate of cell in the case where different cell quantities, is as a result seen Fig. 6.From figure
In as can be seen that the chip in circulating tumor cell trapping region 10 cell capture rate more than 80%.
Embodiment 5
Since the mixed liquor of cellular nucleic acid and PCR mix in circulating tumor cell trapping region 10 is successfully entered detection of nucleic acids
Area 20 be realize micro-fluidic chip function premise and basis, it would therefore be desirable to test in advance this vacuumize and be centrifuged
The feasibility of sample introduction.Since real sample reagent is colourless transparent solution, for convenience of test result is observed, we use blue
The dyestuff of color replaces authentic sample to be tested.We are first by 1 He of cell capture injection port of circulating tumor cell trapping region 10
Cell capture outlet 5 is closed with uv-curable glue, vacuumizes 30s, and solution is made slowly to flow into detection of nucleic acids area 20, then from
The heart (3000rpm, 20min) makes solution completely into each microcavity 72, as a result as shown in fig. 7, cellular nucleic acid is with PCR mix's
Mixed liquor (blue ink replacement) can be completely into each microcavity 72.
Embodiment 6
Since PDMS gas permeability is higher, under the high temperature conditions, the solution in microcavity easily evaporates, to solve this problem, this reality
It tests and is closed using mineral oil and aluminium-foil paper, mineral oil closure is good, and airtight, thermostabilization is good, not soluble in water, aluminium foil paper wood
Expect thin, good heat conductivity, and opaque, it is pollution-free, such as Fig. 8, the result after preceding and closing is closed for PDMS, it can be seen that closing
Preceding the solution in most of microcavity all evaporates after PCR reacts, and not volatilizing then after closing.
Embodiment 7
According to the condition that each step in front has been groped, 21 exon of EGFR is carried out to lung cancer cell line H1975
The detection of L858 gene mutation, dPCR after reaction, according to the fluorescent marker of probe institute band, are selected in general channels HEX
(excitation wavelength 533nm, launch wavelength 558nm), under mutant channel FAM (excitation wavelength 485nm, launch wavelength 536nm) fluorescence
By micro- sem observation fluorescence results, as a result as shown in Figure 9, it can be seen that part microcavity has fluorescence signal, that is, has jump signal.
Above-described, only presently preferred embodiments of the present invention, the range being not intended to limit the invention, of the invention is upper
Stating embodiment can also make a variety of changes.Letter made by all claims applied according to the present invention and description
Single, equivalent changes and modifications, fall within the claims of the invention patent.The not detailed description of the present invention is normal
Advise technology contents.
Claims (10)
1. a kind of micro-fluidic chip for integrating circulating tumor cell capture, cracking and detection of nucleic acids, the micro-fluidic chip
It is bonded by glass substrate layers and polydimethylsiloxanechip chip layer, the polydimethylsiloxanechip chip layer includes:
Circulating tumor cell trapping region, comprising: cell capture injection port；The the first agglomerate filtration zone formed by pillar array；
Target cell screening areas, several main pipelines and side ducts including extension spaced in parallel to each other, the main pipeline and side ducts
Between be connected to by filtration channel, the main pipeline and side ducts are respectively provided with first high greater than circulating tumor cell size
Degree, the filtration channel have the second height less than circulating tumor cell size；And cell capture outlet；
It is characterized in that, the polydimethylsiloxanechip chip layer further include:
Detection of nucleic acids area, comprising: the sample intake passage connecting with the cell capture outlet, the sample intake passage have elastic side
Wall；The microcavity array area being made of several microcavitys；And detection of nucleic acids outlet；And
Connect the valve control zone of the circulating tumor cell trapping region and the detection of nucleic acids area, comprising: be respectively arranged in institute
The T shape pipeline configuration of sample intake passage two sides is stated, the T shape pipeline configuration includes first that the relatively described sample intake passage extends in parallel
Pipeline, and the relatively described vertically extending second pipe of sample intake passage, the second pipe connect with a gas supply mechanism, pass through
The opening and closing for controlling gas supply mechanism, realizes the closure and unlatching of sample intake passage.
2. micro-fluidic chip according to claim 1, which is characterized in that the first pipe distance of the T shape pipeline configuration
The distance of sample intake passage is 10~20 μm, and the width of second pipe is 40~60 μm.
3. micro-fluidic chip according to claim 1, which is characterized in that the target of the circulating tumor cell trapping region is thin
The second agglomerate filtering corresponding with the first agglomerate filtration zone is additionally provided between born of the same parents' screening areas and cell capture outlet
Region, so that the entire circulating tumor cell trapping region is configured to axially symmetric structure.
4. micro-fluidic chip according to claim 1, which is characterized in that the microcavity array area packet in the detection of nucleic acids area
Several Rectangular Microchannels arranged in parallel, and several microcavitys communicated therewith in the two sides of the Rectangular Microchannel are included, it is described
At 30 °~60 ° of acute angle between microcavity and Rectangular Microchannel.
5. micro-fluidic chip according to claim 1, which is characterized in that the polydimethylsiloxanechip chip layer includes two
A detection of nucleic acids area and two valve control zones, described two detection of nucleic acids areas are symmetrically arranged in circulating tumor cell trapping region
Two sides, and connect respectively with cell capture injection port and cell capture outlet.
6. a kind of examine including the capture of collection circulating tumor cell described according to claim 1~any one of 5, cracking and nucleic acid
It surveys in the micro fluidic device of the micro-fluidic chip of one.
7. micro fluidic device according to claim 6, which is characterized in that the micro fluidic device includes: sequentially connected
Solution locker room, the micro-fluidic chip, waste collection syringe and sampling pump further include the valve with the micro-fluidic chip
The gas supply mechanism of the T shape pipeline configuration connection of door control zone.
8. micro fluidic device according to claim 7, which is characterized in that the gas supply mechanism includes by piping connection
Pulsometer and nitrogen gas tank.
9. a kind of method for carrying out circulating tumor cell capture, cracking and detection of nucleic acids simultaneously, which is characterized in that the method packet
Include following steps:
1) a kind of micro fluidic device according to any one of claim 6~8 is provided；
2) gas supply mechanism is opened, the sample introduction is logical to be realized to sample intake passage pressure by the first pipe of the T shape pipeline configuration
The closure in road；
3) circulating tumor cell that sample to be detected is pumped into micro-fluidic chip is caught by cell capture injection port using negative pressure
It obtains in area, the lesser cell of size is rushed out micro-fluidic chip and enters in waste collection syringe, and larger-size circulation is swollen
Oncocyte is intercepted in micro-fluidic chip；
4) circulating tumor cell that cell pyrolysis liquid is pumped into micro-fluidic chip is caught by cell capture injection port using negative pressure
It obtains in area, the circulating tumor cell intercepted in micro-fluidic chip is cracked；
5) circulating tumor cell that PCR reaction solution is pumped into micro-fluidic chip is captured by cell capture injection port using negative pressure
Qu Zhong；
6) gas supply mechanism is closed, the sample intake passage restores normal circulation status, and vacuumizing captures the circulating tumor cell
The cell pyrolysis liquid in area and the mixed liquor of PCR reaction solution flow into detection of nucleic acids area, and centrifugally operated carries out pcr amplification reaction；And
7) after pcr amplification reaction, the micro-fluidic chip is placed in fluorescence microscopy under the microscope, and analyzes the nucleic acid inspection
The fluorescence for surveying each microcavity in area, so that the nucleic acid situation to circulating tumor cell is analyzed.
10. micro fluidic device according to claim 9, which is characterized in that the step 6) further include: anti-in PCR amplification
Ying Qian closes detection of nucleic acids area using mineral oil and aluminium-foil paper.
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