CN105420086A - Single-cell positioning microporous membrane, application and single-cell automatic acquisition device - Google Patents
Single-cell positioning microporous membrane, application and single-cell automatic acquisition device Download PDFInfo
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- CN105420086A CN105420086A CN201510996170.4A CN201510996170A CN105420086A CN 105420086 A CN105420086 A CN 105420086A CN 201510996170 A CN201510996170 A CN 201510996170A CN 105420086 A CN105420086 A CN 105420086A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M47/00—Means for after-treatment of the produced biomass or of the fermentation or metabolic products, e.g. storage of biomass
- C12M47/04—Cell isolation or sorting
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D69/00—Semi-permeable membranes for separation processes or apparatus characterised by their form, structure or properties; Manufacturing processes specially adapted therefor
- B01D69/02—Semi-permeable membranes for separation processes or apparatus characterised by their form, structure or properties; Manufacturing processes specially adapted therefor characterised by their properties
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D71/00—Semi-permeable membranes for separation processes or apparatus characterised by the material; Manufacturing processes specially adapted therefor
- B01D71/02—Inorganic material
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D71/00—Semi-permeable membranes for separation processes or apparatus characterised by the material; Manufacturing processes specially adapted therefor
- B01D71/02—Inorganic material
- B01D71/024—Oxides
- B01D71/027—Silicium oxide
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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
- C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
- C12Q1/24—Methods of sampling, or inoculating or spreading a sample; Methods of physically isolating an intact microorganisms
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D2325/00—Details relating to properties of membranes
- B01D2325/02—Details relating to pores or porosity of the membranes
- B01D2325/028—Microfluidic pore structures
Abstract
The invention relates to a single-cell positioning microporous membrane, application and a single-cell automatic acquisition device. The single-cell positioning microporous membrane comprises a porous membrane equipped with a plurality of through holes and a filter membrane covering the through holes, and the filter membrane in the through holes is provided with at least one micropore. The single-cell automatic acquisition device consists of a bracket, a membrane breaking mechanism disposed on the bracket, a first platform below the membrane breaking mechanism and a second platform disposed between the membrane breaking mechanism and the first platform, and the first platform and the second platform do planar translation in the X direction and Y direction, and the membrane breaking mechanism does vertical motion in the Z direction. The first platform is provided with a container, and the second platform is equipped with the single-cell positioning microporous membrane. The technical scheme provided by the invention is accurate and reliable, can automatically complete acquisition of target single cells and process samples with a very small number of cells, realizes very low loss rate, and can realize automatic operation. At the same time, the cost of the device is significantly lower than other existing equipment.
Description
Technical field
The invention belongs to rare cell detection field, be specifically related to a kind of microporous membrane obtaining individual cells from cell suspension, and adopt this microporous membrane to obtain the method for individual cells, also relate to a kind of unicellular automatization acquisition device.
Background technology
Single cell analysis has become the hot fields of biomedical research, it can disclose the heterogeneous characterization of molecules compared with maxicell colony, particularly some quantity are extremely rare, but there is the cell of important biomolecule function or clinical detection meaning, as the circulating tumor cell in human peripheral, circulating endothelial cells, by the cell of virus infection, from the erythroblast of fetus in maternal blood, and the tumor stem cell etc. in a small amount of noumenal tumour sample, to its to catch and detect analysis significance just more great.But how reliably to obtain individual cells to analyze, especially for the rare cell that number is few, current technical bottleneck beyond doubt.
These rare cells often needed in addition enrichment before analyzing further, even if but after the enrichment, the number of these rare cells is still few and there is other interference cell a certain amount of, needs a kind of reliable and level of automation higher method the cell of these preciousnesses can be taken out one by one and analyze.
Be belong to the sample preparation processes before to single cell analysis to the manipulation of individual cells, require target cell to separate from background sample and be then transported to specified location or be in designated environment so that next step single cell analysis.A typical application scenarios is through in the circulating tumor cell sample of enrichment containing 80 circulating tumor cells, 500 white corpuscles, wherein circulating tumor cell and white corpuscle can be distinguished by the immunofluorescence dyeing for surface of cell membrane albumen, in order to study the characterization of molecules of circulating tumor cell from unicellular yardstick, need 80 circulating tumor cells to take out one by one and carry out the order-checking of lysis, genome amplification and target gene respectively.
The method of existing multiple Manipulation of single cells at present, commonly obtain target cell by micrurgy instrument, laser microprobe dating or light tweezer, can process a small amount of cell sample, but level of automation is low, the operating time is long, need well-trained operator and in operating process, cell exists certain rate of loss.Flow cytometer is another kind of common cell sorting equipment, cell can be assigned to one by one in each hole in such as 96 orifice plates, but the weak point of this technology is it cannot process the cell of fewer number of, be difficult to only carrying out reliable sorting containing the hundreds of even sample of tens cells.
Micro-fluidic chip is a kind of chip technology manipulating micro liquid flowing, is highly suitable for operation and the analysis of micro liquid biological specimen, on a small quantity cell, and is easy to the integration that automatization also can realize multiple function or module.The such as C1 of Fluidigm company
tMunicellular automatic preprocessing system (Single-CellAutoPrepSystem), is trapped in specified location by the geometry of deft design on micro-fluidic chip by individual cells thus realizes single celled catching.Every chip block has 96 capturing units, when carrying out the lysis on chip after individual cells is captured immediately, the steps such as nucleic acid amplification, complete whole Manipulation of single cells and treating processes, another equipment being transferred to the said firm from single celled nucleic acid obtained carries out analyzing to realize single celled genetic analysis.But, this system is still not too applicable to the manipulation of few number cell, and it is low that reason is that it handles accuracy rate, thus target cell rate of loss is higher, and the sample being mixed with other cells except target cell cannot be processed, the micro-fluidic chip simultaneously for sorting individual cells is expensive.The another kind of mode handled individual cells applies inhomogenous electric field outward at chip, thus by different signaling to specific position, the such as DEPArray of SiliconBiosystems company
tMsystem can be handled moveable dielectrophoretic trap and take out specific individual cells from cell colony, although handle accurately, loss cell rate is low, but its operating time is longer, flux is very low, and this system price is extremely expensive, be difficult to equip common laboratory, more cannot be applied to clinical, popularization is poor.
Described in summary, in prior art, be summarized in table 1 for the relative merits of Manipulation of single cells device.
Table 1
| Method | Rare cell sample | Be mixed with other cells | Rate of loss | Operating time | Level of automation | Price |
| Micrurgy instrument | Be | Passable | Medium | Long | Low | Medium |
| Laser microprobe dating | Be | Passable | Medium | Long | Low | High |
| Fluidigm C1 TM | Be | Cannot | Higher | Short | High | High |
| DEPArray TM | Be | Passable | Low | Long | Higher | Very high |
| Flow cytometer | No | Passable | High | Short | High | High |
Summary of the invention
The invention provides a kind of microporous membrane obtaining individual cells from cell suspension, and adopt this microporous membrane to obtain the method for individual cells, low in order to the unicellular extraction accuracy solved in the detection of traditional rare cell, rate of loss is high, the problem that the cycle is long.
Present invention also offers a kind of unicellular automatization acquisition device, in order to solve, current acquisition device level of automation is low or equipment cost is too high, is unfavorable for the problem popularized.
In order to solve the problems of the technologies described above, technical scheme of the present invention is: described unicellular location microporous membrane, comprise the porous-film offering some through holes and the filter membrane covering described through hole, in described through hole, filter membrane is provided with at least one micropore, described micro-pore diameter is 3-8 μm, preferably 5 μm, the thickness of described filter membrane is 0.5-2 μm, preferably 1 μm.The aperture of 5 microns is less than the rare cell of the overwhelming majority, as the circulating tumor cell in blood, circulating endothelial cells, fetal nucleated red blood in pregnant woman blood, and the Iisolated tumor cells in the body fluid such as hydrothorax, cerebrospinal fluid, therefore the micropore of 5 microns can these rare cells above in entrapped cell suspension, the filter membrane of 0.5-2 μm of thickness can ensure successfully to retain on the one hand in addition, can be easier to poke on the other hand when locating and reclaiming.
Preferably, the material of described porous-film is silicon, and the material of described filter membrane is silicon nitride.Silicon nitride material is more crisp, can ensure the carrying of cell, easily be broken by sharp weapon again.
Preferably, on described porous-film, through hole is array distribution, and quantity is 10000-100000.If the number of cell is much smaller than the number (more than 10000) of through hole on filter membrane in cell suspension, according to Poisson's distribution, substantially is individual cells on each micropore, the through hole of array distribution, coordinate is easily determined, thus the unicellular coordinate fallen in through hole is determined, realize unicellularly quick and precisely locating.
Preferably, the through-hole diameter on described porous-film is 20-100 μm, preferably 70 μm.The diameter of porous-film, wants on the one hand in unit surface, to hold as far as possible many holes, and another aspect will ensure follow-up when abolishing filter membrane, and the notice that mechanical devices can poke film can not encounter cell wherein.
Present invention also offers and adopt above-mentioned unicellular location microporous membrane to carry out the unicellular qualitative and method obtained, it comprises the steps:
1) imposed on by sample on the microporous membrane of described unicellular location, cells in sample falls in described through hole, and by described membrane retention, so far each cell coordinate is determined by lead to the hole site;
2) by the imaging of microscope light field or fluorescein-labelled cell and each through hole inner cell classification is determined in imaging, thus target cell position is determined;
3) container is placed in below the microporous membrane of described unicellular location, gets rid of the stop of filter membrane in through hole residing for target cell, make it fall in described container, complete the collection of target cell.
The rare cell retained in cell suspension, can be arrived by microscopical light field imaging, if also there is the cell of other kinds in cell suspension, can directly by different sorts being distinguished for the immunofluorescence dyeing of surface of cell membrane specific proteins on filter membrane.Such as, there is tumour cell and the white corpuscle of nonsmall-cell lung cancer in cell suspension simultaneously, these cells are after by membrane retention, the antibody of the fluorescein-labeled human leucocyte antigen CD45 of FITC (being green under microscope) can be used, and the antibody of the fluorescein-labeled epithelium mark of APC EpCAM and EGFR (for red under microscope) dyes to cell, then stronger red fluorescence will be shown under the microscope, the cell not showing green fluorescence regards as tumour cell, stronger green fluorescence will be shown, the cell not showing red fluorescence regards as white corpuscle.
Preferably, described target cell is Iisolated tumor cells in circulating tumor cell in blood, circulating endothelial cells, fetal nucleated red blood, body fluid and/or white corpuscle.
Preferably, lysis and the amplification of unicellular full-length genome is carried out after described container collection to target cell further wherein.After simple process, can be used for follow-up goal gene amplification, carry out a series of detection.
Present invention also offers a kind of unicellular automatization acquisition device, comprise support, the rupture of membranes mechanism be arranged on support, the first platform below described rupture of membranes mechanism and the second platform of being arranged between described rupture of membranes mechanism and described first platform, described first platform and the second platform at XY to plane translation, described rupture of membranes mechanism is at Z-direction vertical motion, described first platform arranges container, described second platform is arranged above-mentioned unicellular location microporous membrane.
Preferably, described container is microwell plate.Such as 96 orifice plates or 384 orifice plates.
Preferably, the kinematic accuracy of described rupture of membranes mechanism is 0.1-10 μm, preferably 5 μm, and the kinematic accuracy of described second platform is 0.1-5 μm, preferably 1 μm, and the kinematic accuracy of described first platform is 0.1-10 μm, preferably 5 μm.
Preferably, described unicellular automatization acquisition device also comprises microscope.
Preferably, described rupture of membranes mechanism comprises the 3rd platform and is fixed on membrane puncture needle on the 3rd platform, and described 3rd platform moves along Z-direction.
The unicellular each through hole falling into unicellular location microporous membrane in technical scheme provided by the invention in cell suspension, and intercepted and captured by filter membrane, differentiate unicellular in each through hole, wherein target cell completes location because of through hole addressable, then through hole residing for target cell is moved to above accommodating container, punctured by filter membrane, target cell falls in container.This microporous membrane, method and apparatus can complete the unicellular acquisition of target accurately, reliably, automatically, the sample of few number cell can be processed, can realize extremely low target cell rate of loss, can realize automated operation, the cost with timer is starkly lower than other existing installations.
Accompanying drawing explanation
Fig. 1 is the structural representation of unicellular automatization acquisition device one embodiment of the present invention;
Fig. 2 is the structural representation of unicellular location of the present invention microporous membrane one embodiment;
Fig. 3 is the structural representation of porous-film of the present invention and filter membrane one embodiment;
Fig. 4 a and Fig. 4 b is unicellular location microporous membrane working process microscope figure of the present invention;
Fig. 5 a and Fig. 5 b is the process schematic that unicellular location of the present invention microporous membrane obtains target cell.
Shown in figure: 1-support, 21-the 3rd platform, 22-membrane puncture needle, 31-first platform, 32-384 orifice plate, 41-second platform, 42-unicellular location microporous membrane, 421-porous-film, 422-filter membrane, 423-through hole, 424-micropore, 5-microscope, 6-target cell.
Embodiment
Below in conjunction with specific embodiment, set forth the present invention further.Should be understood that these embodiments are only not used in for illustration of the present invention to limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usually conveniently condition, or according to the condition that manufacturer advises.
The unicellular automatization acquisition device of embodiment 1
As shown in Figure 1, described unicellular automatization acquisition device comprises support 1, the rupture of membranes mechanism be arranged on support 1, the first platform 31 below described rupture of membranes mechanism 2 and the second platform 41 be arranged between described rupture of membranes mechanism, described first platform 31 and microscope 5, described rupture of membranes mechanism 2 comprises the 3rd platform 21 and is fixed on membrane puncture needle 22 on the 3rd platform 21, described first platform 31 arranges container-384 orifice plate 32, described second platform 41 arranges unicellular location microporous membrane 42.
Described first platform 31 can move along X to Y-direction under motor drives, stroke is 10cm, precision is 5 μm, described second platform 41 can move along X to Y-direction under motor drives, stroke is 10cm, and precision is 1 μm, and described 3rd platform 21 can move along Z-direction under motor drives, stroke is 5cm, and precision is 5 μm.
As shown in Figures 2 and 3, above-mentioned unicellular location microporous membrane 42 comprises two-layer, one deck is porous-film 421, another layer is filter membrane 422, and described pore membrane 421 offers about 10000 through holes 423 of array distribution, in described through hole 423, filter membrane 422 is provided with a micropore 424, described through hole 423 diameter is 70 μm, the degree of depth is 50 μm, and the thickness of described filter membrane 422 is 1 μm, and described micropore 424 diameter is 5 μm.
The material of described porous-film 421 is silicon, and the material of described filter membrane 422 is silicon nitride.
Embodiment 2 application test-unicellular recovery is tested with order-checking
To mix with 1000 undyed white corpuscles after 100 non-small cell lung cancer cell H1975 dyestuff VybrantDiI (LifeTehnologies) dyeing and be diluted to 1 milliliter with phosphate buffered saline buffer, make sample cell suspension, adopt the unicellular automatization acquisition device of embodiment 1 to catch wherein target cell 6 i.e. H1975 cell, set forth its principle of work and process with this sample.
Imposed on by sample cell suspension on described unicellular location microporous membrane 42, because gravity liquid flows out, cell is trapped within micropore 424.Because the number of cell in cell suspension is much smaller than the number of through hole 423, according to Poisson's distribution, as shown in fig. 4 a, is individual cells substantially in each through hole 423, cell is positioned on micropore 424, can identify and record the position of H1975 cell with fluorescent microscope.As shown in Figure 5 a, motor drives described second platform 41 to do tangential movement, through hole 423 residing for H1975 cell is placed in immediately below described membrane puncture needle 22, wherein membrane puncture needle 22 targeted by the position near through hole 423 edge, motor drives described first platform 31 simultaneously, hole hope being obtained target cell 6 moves to immediately below membrane puncture needle 22, aims at through hole 423 place at H1975 cell place on unicellular location microporous membrane 42.Then, as shown in Figure 5 b, motor drives described 3rd platform 21 to move downward along Z-direction, the membrane puncture needle 22 be fixed on the 3rd platform 21 impacts the filter membrane 422 with mechanical equivalent of light fragility bottom porous-film 421, make it cracked, filter membrane 422 falls into the hole of 384 orifice plates 32 of below together with the target cell 6 on it, thus realizes unicellular recovery, now as shown in Figure 4 b, through hole 423 place of unicellular location microporous membrane 42 is without filter membrane 422 and target cell 6.
The above step of cyclical operation, by all kinds that unicellular location microporous membrane 42 is intercepted and captured in unicellular each addressable hole of reclaiming people 384 orifice plate, then can carry out subsequent disposal determination and analysis respectively.
For confirming the feasibility of the method, following subsequent disposal is carried out to the target cell 6 reclaimed.
With the addition of 4 microlitre phosphoric acid buffers in advance in the hole of 384 orifice plates.Then REPLI-gSingleCellKit (Qiagen, USA) test kit is adopted target cell 6 cracking and full-length genome to be amplified.Concrete steps are, add 3 microliters of denatured liquid, 65 DEG C of high temperature incubation after 10 minutes in the hole containing target cell 6, add 3 micro litre stop-buffer and stop sex change.Then, add 29 microlitre reaction solutions, 2 microlitre phi29DNA polysaccharases successively, and moisturizing is to volume 50 microlitre, 30 degree of reactions 8 hours, last 65 DEG C of high temperature continue reaction makes enzyme deactivation in 3 minutes, stops whole amplification process.Phenol chloroform alcohol precipitation increases for follow-up goal gene after purifying.
We detect the L858R sudden change in EGFR gene 20 exon and the T790M sudden change in 21 exons, and the primer of use is:
EGFR20F(CTTTATCCAATGTGCTCCTC)
EGFR20R(TCTCCCTTCCCTGATTACCT)
EGFR21F(TTCGCCAGCCATAAGTCCT)
EGFR21R(TCATTCACTGTCCCAGCAAG)；
Selective annealing temperature 59 DEG C, 30 circulations.Experimental result shows, and target cell 6 Successful amplification reclaimed goes out 20,21 exons of EGFR gene, and detects that target cell 6 has L858R and T790M sudden change by Sanger order-checking, thus demonstrates the feasibility of this technology.
Claims (10)
1. a unicellular location microporous membrane, it is characterized in that, comprise the porous-film offering some through holes and the filter membrane covering described through hole, in described through hole, filter membrane is provided with at least one micropore, described micro-pore diameter is 3-8 μm, and the thickness of described filter membrane is 0.5-2 μm.
2. a kind of unicellular location microporous membrane according to claim 1, it is characterized in that, the material of described porous-film is silicon, and the material of described filter membrane is silicon nitride.
3. a kind of unicellular location microporous membrane according to claim 1, it is characterized in that, on described porous-film, through hole is array distribution, and quantity is 10000-100000.
4. claim 1-3 arbitrary described a kind of unicellular location microporous membrane carry out unicellular qualitative and obtain method, it is characterized in that, comprise the steps:
1) imposed on by sample on the microporous membrane of described unicellular location, cells in sample falls in described through hole, and by described membrane retention, so far each cell coordinate is determined by lead to the hole site;
2) by the imaging of microscope light field or fluorescein-labelled cell and each through hole inner cell classification is determined in imaging, thus target cell position is determined;
3) container is placed in below the microporous membrane of described unicellular location, gets rid of the stop of filter membrane in through hole residing for target cell, make it fall in described container, complete the collection of target cell.
5. the unicellular qualitative and method obtained according to claim 4, is characterized in that, described target cell is Iisolated tumor cells in circulating tumor cell in blood, circulating endothelial cells, fetal nucleated red blood, body fluid and/or white corpuscle.
6. the unicellular qualitative and method obtained according to claim 4, is characterized in that, carry out lysis and unicellular full-length genome further wherein and amplify after described container collection to target cell.
7. a unicellular automatization acquisition device, it is characterized in that, comprise support, the rupture of membranes mechanism be arranged on support, the first platform below described rupture of membranes mechanism and the second platform of being arranged between described rupture of membranes mechanism and described first platform, described first platform and the second platform at XY to plane translation, described rupture of membranes mechanism is at Z-direction vertical motion, described first platform arranges container, described second platform is arranged claim 1-3 arbitrary described unicellular location microporous membrane.
8. a kind of unicellular automatization acquisition device according to claim 7, it is characterized in that, described container is microwell plate, preferably 96 orifice plates or 384 orifice plates.
9. a kind of unicellular automatization acquisition device according to claim 7, it is characterized in that, the kinematic accuracy of described rupture of membranes mechanism is 0.1-10 μm, and the kinematic accuracy of described second platform is 0.1-5 μm, and the kinematic accuracy of described first platform is 0.1-10 μm.
10. a kind of unicellular automatization acquisition device according to claim 7, is characterized in that, described rupture of membranes mechanism comprises the 3rd platform and is fixed on membrane puncture needle on the 3rd platform, and described 3rd platform moves along Z-direction.
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| CN115253682B (en) * | 2022-07-29 | 2024-02-02 | 烟台至公生物医药科技有限公司 | Bladder cancer cell capturing device and capturing method |
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