CN107389642A - Unicellular efficient capture, the imaging of high intension and full transcriptome analysis apparatus and method - Google Patents
Unicellular efficient capture, the imaging of high intension and full transcriptome analysis apparatus and method Download PDFInfo
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Abstract
The invention belongs to unicellular capture, analysis technical field, and in particular to unicellular efficient capture, the imaging of high intension and full transcriptome analysis apparatus and method.Described device is unicellular for capturing, and described device includes the micro- well array chip of dimethyl silicone polymer and Tissue Culture Plate or Tissue Culture Dish substrate；Micro- well array chip is fitted in the Tissue Culture Plate substrate or the Tissue Culture Dish substrate.Unicellular high flux imaging analysis and the single celled full transcriptome analysis of target, the heterogeneous apparatus and method from the single celled complete horizontal slender intercellular of further investigation of transcript profile can be carried out simultaneously the invention provides a kind of.
Description
Technical field
The invention belongs to unicellular capture, analysis technical field, and in particular to unicellular efficient capture, high intension imaging and
Full transcriptome analysis apparatus and method.
Background technology
The unicellular response of high-throughout monitoring, for drug test, the various applications such as toxicology and elementary cell biology are
It is very important.Nearest research highlights the genome across cancer cell population, transcript profile and Proteomics it is notable different
Matter, this heterogeneity is characterized for understanding that tumour progression is most important.Single cell analysis is early diagnosed to major disease, controlled
Treatment, drug screening and cell physiological, the research of pathologic process are significant, turn into one of focus of research at present.
Capillary Electrophoresis (CE) separation method to grow up at the beginning of the 1980's is because its sampling volume is small, separative efficiency
High, the advantages that separating rate is fast, suitable for the measure to various ingredients and quantitative, unicellular multi-component detection is had been used for, is obtained
Some achievements.But limited by capillary one-dimentional structure, single-cell injection and molten membrane operations are complicated.With traditional fluidic cell
Cell count and screening instrument of the instrument for representative, though having realized integrated and automation, equally have equipment instrument big, internal
The shortcomings of complicated, fragile.
Micro-fluidic chip is the another important tool for promoting single cell analysis.In recent years based on the unicellular of microflow control technique
Analysis, due to its sensitivity and high-throughout advantage, a very powerful work is provided to study the heterogeneity of biosystem
Tool.Has the analysis that document report carries out cell imaging using the method for micro- well array at present（Huang,L.; Chen, Y.;
Chen, Y.; Wu,H. Anal. Chem. 2015, 87, 12169−12176）.But although this method can be analyzed slender
The fluorescence imaging signal of born of the same parents, but it is difficult to that follow-up molecular biological analysis, particularly transcript profile will be carried out after unicellular recovery
Analysis.In addition, also there are research paper and patent (Tang, Y.; Wang,Z.; Li, Z.; Kim, J.; Deng, Y.;
Li, Y.; Heath, J. R.; Wei, W.; Lu, S.; Shi, Q. Proc Natl Acad Sci USA. 114:
2544–2549;The patent No.:CN105954246 A) report captured based on micro- well array addressable it is swollen in rare body fluid
Oncocyte.But, this method scope of application is the rare tumour cell in body fluid, in addition, containing incessantly in each individually micro- well
There is a cell, this is that single celled graphical analysis brings difficulty, does not also show the possibility of RNA sequencing analysis.
Therefore, the method that single cell analysis is carried out using microflow control technique reported at present can not be realized single celled height
Flux imaging representation and full transcriptome analysis combine, thus exist in terms of research cell phenotype with the correlation of genotype
Defect.The heterogeneous apparatus and method of slender intercellular can be explored from full transcript profile level there is an urgent need to a kind of.
The content of the invention
For above-mentioned technical problem, the present invention provides a kind of unicellular efficient capture, the imaging of high intension and transcribes component entirely
Analysis apparatus and method.Unicellular high flux acquisition equipment of the present invention can quickly and efficiently capture cell, and the present invention also carries
For a kind of method of unicellular high intension imaging, this method utilizes unicellular high flux acquisition equipment, and high flux obtains unicellular
Image information, the present invention also provides a kind of positioning single celled method of picking, and this method utilizes unicellular high flux capture dress
Put, picking is unicellular can be used in single celled full transcriptome analysis for positioning.
The present invention is achieved by the following technical solutions：
A kind of device of unicellular high flux capture, described device is unicellular for capturing, and described device includes poly dimethyl silicon
The micro- well array chip of oxygen alkane and Tissue Culture Plate or Tissue Culture Dish substrate；Micro- well array chip fits in the cell training
Support in plate substrate or the Tissue Culture Dish substrate, to the surface of the Tissue Culture Plate substrate or the Tissue Culture Dish substrate
Do not process.
Further, it is distributed with micro- well on micro- well array chip, a diameter of 23 μm ± 5 μm of micro- well, depth
For 25 ± 10 μm, the distance of center circle of the adjacent micro- well of any two is 46 μm ± 10 μm.
Further, the preparation method of micro- well array chip is：Produced using optical etching technology comprising microtrabeculae battle array
The mould of row, then dimethyl silicone polymer is directly poured and is filled on the mould, mould is separated after solidification, obtain described poly- two
The micro- well array chip of methylsiloxane.
Further, the Tissue Culture Plate substrate or the Tissue Culture Dish substrate are glass, silicon chip, metal or high score
Sub- material.
A kind of method of unicellular high flux capture, comprises the following steps：
Step 1: using the dust of the micro- well array chip bottom surface of adhesive tape sticky removing dimethyl silicone polymer, it is suitable to be cut to
After shapes and sizes, it is close to be positioned in Tissue Culture Plate substrate or Tissue Culture Dish substrate, obtains unicellular high flux capture
Device；
Step 2: the micro- well array chip of the dimethyl silicone polymer is subjected to infiltration processing to improve its surface hydrophilicity；
Step 3: preparing the aaerosol solution of cell and injecting the top of the micro- well array chip of the dimethyl silicone polymer, stand
Or centrifugation, by unicellular capture well in a subtle way；
Step 4: the cell of the micro- well array chip excess surface of the dimethyl silicone polymer is washed away with PBS solution.
Further, if the micro- well array chip of the dimethyl silicone polymer is placed in into the Tissue Culture Plate substrate
On, detailed process is：The card punch for being less than or equal to the orifice plate internal diameter of the Tissue Culture Plate using internal diameter cuts out circle
Micro- well array chip, the blank space of micro- well array chip of the circle is then pierced into needle point, vertically by micro- well battle array of the circle
Row chip is put into the orifice plate bottom of Tissue Culture Plate；
If the micro- well array chip of the dimethyl silicone polymer is placed in the Tissue Culture Dish substrate, detailed process is：
After the dust of the micro- well array chip bottom surface of dimethyl silicone polymer described in sticky removing, micro- well array of target sizes is cut out with cutting knife
Chip, directly micro- well array chip after cutting is close in Tissue Culture Dish with hand or tweezers, then in micro- well array chip
Upper surface fitting place dimethyl silicone polymer material cofferdam surround micro- well array chip.
Further, the micro- well array chip of the dimethyl silicone polymer is subjected to infiltration processing to improve its surface hydrophilic
Property, it is specially：The Pluronic that the priority ethanol that use quality percentage is 75% successively, pure water, mass percent are 2%
F127 or F108 is injected into the micro- well array chip surface of the dimethyl silicone polymer as size, will be whole after injection
Unicellular high flux acquisition equipment is positioned in vavuum pump, enters liquid to extract the gas in micro- well, is incubated infiltrating time
It respectively is 10 min, 10 min, 30 min.
Further, in the step 3, lung carcinoma cell, the fibrosarcoma cells of the aaerosol solution behaviour of the cell
With the suspension of any one cell in breast cancer cell, cell solution is blown and beaten repeatedly with liquid-transfering gun when preparing suspension, so that
Cell is single celled state in suspension, in the aaerosol solution of the cell, cell concentration 106Individual/ml.
Further, in the step 3, after injecting the aaerosol solution of cell, according to the mode of standing, static conditions
For：Time of repose is 30 min-40min, and temperature is room temperature；
According to the mode of centrifugation, centrifugal condition is：Centrifugation time is 5-7min, and centrifuging temperature is 4 DEG C, rotating speed 1000
rpm.Set low temperature effectively cell can be prevented to unite during cell settlement, keep single celled state.
Further, in step 4, the micro- well array chip excess surface of the dimethyl silicone polymer is washed away with PBS solution
Cell when, it is impossible to chip surface liquid is all siphoned away, it is ensured that micro- well array chip surface has a small amount of liquid to coat；Fluid injection
When with liquid-transfering gun instill drop from micro- well array chip upper vertical, speed is about 1 drop per second, and apart from chip upper surface about 2
cm；Ensure that chip level is placed, and repeats imbibition fluid injection about 4 ~ 5 times during imbibition fluid injection, can be by the micro- well of the dimethyl silicone polymer
The cell clearance of array chip excess surface.
A kind of method of unicellular high flux imaging analysis, comprises the following steps：
It is placed under microscope Step 1: single celled unicellular high flux acquisition equipment will have been captured, is then looked under low power lens
To unicellular capture region；
Step 2: being converted into high power lens, unicellular capture region is imaged successively according to micro- well array module numbering, every piece
Region is intended to shoot its corresponding light field figure and fluorescence field figure；
Step 3: after the completion of shooting, single celled image information is obtained, then using image processing software to the glimmering of every piece of region
Light field figure carries out fluorescence intensity quantitative analysis, obtains each single celled fluorescence intensity information；
Step 4: utilize the single celled fluorescence intensity distribution situation of data processing software high throughput analysis.
One kind positioning single celled method of picking, comprises the following steps：
Step 1: prepare the capillary needle that needle point internal diameter is about 30 ~ 50 μm；
Step 2: imaging of being taken pictures to unicellular region, obtains some region of light field figure and fluorescence field figure, selected with two width figures and
It is unicellular to position target；
Step 3: observe tip position in eyepiece, when it is located at target unicellular top, it is vertically goed deep into rapidly the mesh
Mark in unicellular corresponding micro- well, cell is inhaled into capillary needle under capillary force effect, is then taken out capillary needle, is inserted
In the one end for entering rubber tube, unicellular lysate ready in advance is blown into by unicellular from the rubber tube other end with mouth
In, carry out the single celled full transcriptome analysis of target.
Further, the needle point grinder buffing of the capillary needle is gone out it is suitably sized, to guarantee and can only cover
One micro- well.
Further, the capillary needle is bent into rectangular-shaped, it is allowed to human hand held one end, and another needle tip extends vertically into
Chip, to avoid hand from sheltering from the observation visual field of eyepiece.
The advantageous effects of the present invention：
The apparatus structure of unicellular high flux capture of the present invention is simple, it is unicellular quickly and efficiently to capture, and uses
It can be reused in the mould for making the micro- well array chip of dimethyl silicone polymer, only with the micro- well array chip for preparing individual layer
It can efficiently capture unicellular, production process is simpler；Micro-pillar array template obtained by photoetching can be repeated several times use, micro- well
Mm -1 cm of chip thickness about 0.2, can greatly reduce the consumption of dimethyl silicone polymer, relatively other single celled methods of capture
It is more convenient, it is effectively and economical；
The positioning single celled method of picking provided by the invention utilizes unicellular high flux acquisition equipment, positions the unicellular of picking
It can be used in single celled full transcriptome analysis.
The invention provides one kind can carry out unicellular high flux imaging analysis and the single celled full transcript profile of target simultaneously
Analysis, the heterogeneous apparatus and method from the single celled complete horizontal slender intercellular of further investigation of transcript profile.
And chip is used in combination with Tissue Culture Plate, can prepare multiple capturing units simultaneously, for example, with 96 orifice plate knots
Close and use, 96 capturing units can be prepared simultaneously, and the capturing unit is permanently effective, the potentiality for possessing batch production.
Brief description of the drawings
Fig. 1 is to carry out single cell analysis schematic flow sheet using micro- well array chip；
Fig. 2 is the unicellular schematic diagram of picking target；
Fig. 3 is that the micro- well array chip of dimethyl silicone polymer is placed in into 96 orifice plate substrate pictorial diagrams；
Fig. 4 is that the micro- well array chip of dimethyl silicone polymer is placed in into Tissue Culture Dish substrate pictorial diagram；
After the completion of Fig. 5 A are unicellular capture, the light field figure that is imaged using inverted microscope to micro- well array chip；
After the completion of Fig. 5 B are unicellular capture, the fluorescence field figure that is imaged using inverted microscope to micro- well array chip；
Fig. 6 A are after medicine acts on 1 h, by high flux imaging analysis, a large amount of single celled fluorescence intensity profile of gained；
Fig. 6 B are after medicine acts on 2 h, by high flux imaging analysis, a large amount of single celled fluorescence intensity profile of gained；
When Fig. 7 A are that picking target is unicellular, the design sketch of tip position is observed in eyepiece.
Fig. 7 B be picking target it is unicellular before, the micro- well array chip light field figure of dimethyl silicone polymer under eyepiece；
Fig. 7 C be picking target it is unicellular after, the micro- well array chip light field figure of dimethyl silicone polymer under eyepiece.
Embodiment
In order to make the purpose , technical scheme and advantage of the present invention be clearer, it is right below in conjunction with drawings and Examples
The present invention is explained in further detail.It should be appreciated that specific embodiment described herein is used only for explaining the present invention, and
It is not used in the restriction present invention.
On the contrary, the present invention covers any replacement done in the spirit and scope of the present invention being defined by the claims, repaiied
Change, equivalent method and scheme.Further, in order that the public has a better understanding to the present invention, below to the thin of the present invention
It is detailed to describe some specific detail sections in section description.Part without these details for a person skilled in the art
Description can also understand the present invention completely.
Embodiment 1
As shown in Figure 1-2, a kind of device of unicellular high flux capture is present embodiments provided, described device is used to capture list
Cell, described device include micro- well array chip and Tissue Culture Plate or Tissue Culture Dish substrate；Micro- well array chip patch
Together in the Tissue Culture Plate substrate or the Tissue Culture Dish substrate, the Tissue Culture Plate substrate or the cell are trained
The surface for supporting ware substrate does not process.
Micro- well is distributed with micro- well array chip, a diameter of 23 μm ± 5 μm of micro- well, depth is 25 ± 10
μm, the distance of center circle of the adjacent micro- well of any two is 46 μm ± 10 μm, and the specific size of micro- well can ensure to meet that capture is slender
The specific demand of born of the same parents.
The preparation method of micro- well array chip is：The mould comprising micro-pillar array is produced using optical etching technology,
Then dimethyl silicone polymer is directly poured and be filled on the mould, mould is separated after solidification, obtain the polydimethylsiloxanes
The micro- well array chip of alkane.
The Tissue Culture Plate substrate or the Tissue Culture Dish substrate are glass, silicon chip, metal or high polymer material, side
Just draw materials, cost is cheap.
In addition, the present embodiment also provides a kind of method of unicellular high flux capture, it is slender for high flux imaging analysis
Born of the same parents are heterogeneous, using said apparatus, comprise the following steps：
Step 1: using 3M adhesive tapes or the dust of any other micro- well array chip bottom surface of adhesive tape sticky removing dimethyl silicone polymer,
After being cut to suitable shapes and sizes using card punch or cutter, as shown in Figure 3-4, it is close to be positioned over Tissue Culture Plate
In substrate or Tissue Culture Dish substrate, unicellular high flux acquisition equipment is obtained；
If the micro- well array chip of the dimethyl silicone polymer is placed in the Tissue Culture Plate substrate, detailed process is：
The card punch for being less than or equal to the orifice plate internal diameter of the Tissue Culture Plate using internal diameter cuts out micro- well array chip of circle, so
The blank space of micro- well array chip of the circle is pierced into needle point afterwards, micro- well array chip of the circle is vertically put into cell training
Support the orifice plate bottom of plate；
The micro- well array chip of dimethyl silicone polymer is placed in the Tissue Culture Dish substrate, and detailed process is：Sticky removing institute
After the dust for stating the micro- well array chip bottom surface of dimethyl silicone polymer, micro- well array chip of target sizes is cut out with cutting knife,
Directly micro- well array chip after cutting is close in Tissue Culture Dish with hand or tweezers, then in the upper table of micro- well array chip
The cofferdam that dimethyl silicone polymer material is placed in face fitting surrounds micro- well array chip.
Step 2: the micro- well array chip of the dimethyl silicone polymer is subjected to infiltration processing to improve its surface hydrophilic
Property；Specially：The Pluronic that the priority ethanol that use quality percentage is 75% successively, pure water, mass percent are 2%
F127 or F108 is injected into the micro- well array chip surface of the dimethyl silicone polymer as size, will be whole after injection
Unicellular high flux acquisition equipment is positioned in vavuum pump, enters liquid to extract the gas in micro- well, is incubated infiltrating time
It respectively is 10 min, 10 min, 30 min.
Step 3: cell induction apoptosis and dyeing：Same apoptosis is carried out using adriamycin to two kinds of breast cancer cells to lure
Lead.Five concentration gradients and four time gradients are respectively provided with the present embodiment：Concentration gradient is 0,10,20,40,80 μM,
Time gradient is 2,4,6,8 h；By two kinds of cancer cell kinds in normal growth state in 24 orifice plates, treat that its is adherent and covers with
About bottom area 80% when, apply the medicine of various concentrations, after 2,4,6,8 h, medicine removed, and with PBS one time
Afterwards, immunofluorescent reagent CellEvent Caspase-3/7 Green are added and immunofluorescence dyeing, duration 30 is carried out to cell
Min, the fluorometric reagent can be to the cell dyeing of apoptosis, and the apoptosis degree of fluorescence intensity and cell is into positive correlation.After 30 min,
Remove fluorometric reagent.
Step 4: cell dissociation is got off using pancreatin, the culture medium of same volume is added, then moves into cell solution
Centrifuged in 1.5 ml centrifuge tube, after outwelling supernatant, add PBS（Phosphate buffer solution）, cell is entered with liquid-transfering gun
Row is blown and beaten repeatedly, in order to is allowed the cell of adhesion to be separated into unicellular one by one, is easy to ensuing unicellular capture.Will
Ready cell solution is added to bottom and is placed with 96 orifice plates of chip, then using horizontal freezing centrifuge carry out from
The heart, temperature be 4 DEG C, the rpm of rotating speed 1000, the min of time 5, after the completion of the cell of excess surface is washed off with PBS, capture complete.
Specifically, be directed to specific cell, two kinds of breast cancer cells in step 3 be respectively wild-type cell system MCF-7 and
Drug-resistant type cell line MCF/Adr.
Specifically, unicellular capture also can realize efficient capture, the min of time of repose about 30 by standing in step 4.
Embodiment 2
The present embodiment provides a kind of method of unicellular high flux capture, for the unicellular heterogeneity of high flux imaging analysis, adopts
With said apparatus, comprise the following steps：
Step 1: cell induction apoptosis and dyeing：Apoptosis induction is carried out to human lung carcinoma cell using Reversine.The present embodiment
In be respectively provided with four concentration gradients and two time gradients：Concentration gradient is 0,10,20,30 μM, time gradient 1,2
h；By the human lung carcinoma cell kind in normal growth state in 24 orifice plates, when adherent when its and covering with about bottom area 80%,
Apply the medicine of various concentrations, after 1 h or 2 h, medicine is removed, and with after PBS one time, add immunofluorescent reagent
CellEvent Caspase-3/7 Red carry out immunofluorescence dyeing to cell, and the min of duration 30, the fluorometric reagent can be to apoptosis
Cell dyeing, and the apoptosis degree of fluorescence intensity and cell is into positive correlation.After 30 min, fluorometric reagent is removed.
Step 2: cell dissociation is got off using pancreatin, the culture medium of same volume is added, then moves into cell solution
Centrifuged in 1.5 ml centrifuge tube, after outwelling supernatant, add PBS（Phosphate buffer solution）, cell is entered with liquid-transfering gun
Row is blown and beaten repeatedly, in order to is allowed the cell of adhesion to be separated into unicellular one by one, is easy to ensuing unicellular capture.Will
Ready cell solution is added in the chip cofferdam for being positioned over Tissue Culture Dish substrate, then stands 30 min, after the completion of
The cell of excess surface is washed off with PBS, capture is completed.
Step 3: the device for having captured single celled unicellular high flux capture is placed under microscope, then in 4 times of mirrors
Under find unicellular capture region.
Step 4: being converted into 10 times or 20 times of mirrors, unicellular capture region is entered successively according to micro- well array module numbering
Row imaging, every piece of region are intended to shoot its corresponding light field figure and fluorescence field figure, as shown in Figure 5 A and 5B.
Step 5: after the completion of shooting, a large amount of single celled image informations are obtained, then using image processing software to every piece
The fluorescence field figure in region carries out fluorescence intensity quantitative analysis, obtains each single celled fluorescence intensity signals.
Step 6: utilize the single celled fluorescence intensity distribution situation of data processing software high throughput analysis.Such as Fig. 6 A and 6B
Shown, every curve includes about 2000 single celled fluorescence intensity information in figure, and each data point represents strong in the fluorescence
Single cells population in the range of degree ± 50 accounts for the ratio of total statistics single cells population.Specifically, specific cell is directed to, in step 1
Human lung carcinoma cell be A549 cell lines.
Embodiment 3
The present embodiment provides a kind of method that positioning picking is unicellular, carries out full transcriptome analysis, for high flux imaging analysis
Unicellular heterogeneity, using said apparatus, comprise the following steps：
Step 1: prepare the capillary needle that needle point internal diameter is about 30 ~ 50 μm；
Step 2: imaging of being taken pictures to unicellular region, obtains some region of light field figure and fluorescence field figure, selected with two width figures and
It is unicellular to position target；
Step 3: extending vertically into capillary needle, tip position is observed in eyepiece, as shown in Figure 7 A-7C, it is slender to treat that it is located at target
When above born of the same parents, it is rapid deeply, cell is inhaled into capillary needle under capillary force effect, is then taken out capillary needle, is inserted
Enter in long rubber tube, be blown into mouth from the rubber tube other end by unicellular in unicellular lysate ready in advance, carried out
The real-time quantitative PCR of the single celled full transcriptome analysis of target or specific gene.
Specifically, in step 1 capillary needle point can be gone out with grinder buffing it is suitably sized, to guarantee and can only cover one
Individual micro- well.
Specifically, in step 1 capillary needle be bent into it is rectangular-shaped, it is allowed to human hand held one end, and needle tip extends vertically into core
Piece, avoid the observation visual field that hand shelters from eyepiece.
Specifically, the difference of the slender intercellular fluorescence intensity of fluorescence field figure reflects slender intercellular apoptosis degree in step 2
Difference, thus can picking specific fluorescent intensity it is unicellular, in the present embodiment extremely strong unicellular of picking fluorescence signal and
Extremely faint unicellular of fluorescence signal.
Device and method provided by the invention, provide first one kind can carry out simultaneously unicellular high flux imaging analysis and
The single celled full transcriptome analysis of target, the heterogeneous device from the single celled complete horizontal slender intercellular of further investigation of transcript profile
And method.Only need to prepare individual layer micro- well array chip can high flux capture it is unicellular, manufacture craft is simple.It is micro- obtained by photoetching
Post array mould plate can be repeated several times use, micro- mm -1 cm of well chip thickness about 0.2, can greatly reduce dimethyl silicone polymer
Use, relatively other single celled methods of capture are more convenient, effectively and economical.And chip is combined with Tissue Culture Plate made
With, multiple identical capturing units can be prepared simultaneously, such as be used in combination with 96 orifice plates, 96 capturing units can be prepared simultaneously,
And the capturing unit is permanently effective, the potentiality for possessing batch production.
The micro- well array chip of device dimethyl silicone polymer provided by the present invention, the chip allow using single celled heavy
Power acts on（Under nature or under apocarpy）Carry out single celled efficient capture.The present invention also provides 1）The preparation of the chip
Method, this method utilize photoetching technique, it is allowed to produce the multiple mould of repeatable utilization；2）Single celled device is captured to prepare
And operating method；3）The flow of unicellular high flux imaging analysis；With 4）The method of unicellular picking, the method allow unicellular
Full transcriptome analysis.Present invention also offers described device and technology in unicellular high flux imaging analysis and unicellular full transcription
Group analysis is studying the application example of unicellular apoptosis and resistance heterogeneity, and Single-cell imaging can only be carried out by breaching original technology
Analysis or the gene expression analysis of unicellular specific several genes.Described above is the preferred embodiment of the present invention, should be referred to
Go out, for those skilled in the art, on the premise of principle of the present invention is not departed from, can also make
Some improvements and modifications, these improvements and modifications also should be regarded as protection scope of the present invention.
Claims (14)
1. a kind of device of unicellular high flux capture, it is characterised in that described device is used to capture unicellular, described device bag
Include the micro- well array chip of dimethyl silicone polymer and Tissue Culture Plate or Tissue Culture Dish substrate；Micro- well array chip fitting
In in the Tissue Culture Plate substrate or the Tissue Culture Dish substrate.
A kind of 2. device of unicellular high flux capture according to claim 1, it is characterised in that micro- well array chip
On be distributed with micro- well, a diameter of 23 μm ± 5 μm of micro- well, depth is 25 ± 10 μm, the circle of the adjacent micro- well of any two
The heart is away from for 46 μm ± 10 μm.
A kind of 3. device of unicellular high flux capture according to claim 1, it is characterised in that micro- well array chip
Preparation method be：The mould comprising micro-pillar array is produced using optical etching technology, it is then that dimethyl silicone polymer is direct
Pour and be filled on the mould, mould is separated after solidification, obtain the micro- well array chip of the dimethyl silicone polymer.
A kind of 4. device of unicellular high flux capture according to claim 1, it is characterised in that the Tissue Culture Plate base
Bottom or the Tissue Culture Dish substrate are glass, silicon chip, metal or high polymer material.
A kind of 5. method of unicellular high flux capture, it is characterised in that comprise the following steps：
Step 1: using the dust of the micro- well array chip bottom surface of adhesive tape sticky removing dimethyl silicone polymer, it is suitable to be cut to
After shapes and sizes, it is close to be positioned in Tissue Culture Plate substrate or Tissue Culture Dish substrate, obtains unicellular high flux capture
Device；
Step 2: the micro- well array chip of the dimethyl silicone polymer is subjected to infiltration processing to improve its surface hydrophilicity；
Step 3: preparing the aaerosol solution of cell and injecting the top of the micro- well array chip of the dimethyl silicone polymer, stand
Or centrifugation, by unicellular capture well in a subtle way；
Step 4: the cell of the micro- well array chip excess surface of the dimethyl silicone polymer is washed away with PBS solution.
6. the method for unicellular high flux capture according to claim 5, it is characterised in that if by the poly dimethyl
The micro- well array chip of siloxanes is placed in the Tissue Culture Plate substrate, and detailed process is：It is less than or equal to using internal diameter described
The card punch of the orifice plate internal diameter of Tissue Culture Plate cuts out micro- well array chip of circle, is then pierced into the micro- of the circle with needle point
The blank space of well array chip, micro- well array chip of the circle is vertically put into the orifice plate bottom of Tissue Culture Plate；
If the micro- well array chip of the dimethyl silicone polymer is placed in the Tissue Culture Dish substrate, detailed process is：
After the dust of the micro- well array chip bottom surface of dimethyl silicone polymer described in sticky removing, micro- well array of target sizes is cut out with cutting knife
Chip, directly micro- well array chip after cutting is close in Tissue Culture Dish with hand or tweezers, then in micro- well array chip
Upper surface fitting place dimethyl silicone polymer material cofferdam surround micro- well array chip.
7. the method for unicellular high flux capture according to claim 5, it is characterised in that by the polydimethylsiloxanes
The micro- well array chip of alkane carries out infiltration processing to improve its surface hydrophilicity, is specially：Successively use quality percentage is successively
75% ethanol, pure water, the Pluronic F127 or F108 that mass percent is 2% are injected into described gather as size
The micro- well array chip surface of dimethyl siloxane, whole unicellular high flux acquisition equipment is positioned in vavuum pump after injection,
Enter liquid to extract the gas in micro- well, be incubated infiltrating time and respectively be 10 min, 10 min, 30 min.
8. the method for unicellular high flux capture according to claim 5, it is characterised in that described in the step 3
The suspension of any one cell in lung carcinoma cell, fibrosarcoma cells and breast cancer cell that the aaerosol solution of cell is behaved
Liquid, cell solution is blown and beaten repeatedly with liquid-transfering gun when preparing suspension, so that cell is single celled state in suspension, the institute
In the aaerosol solution for stating cell, cell concentration 106Individual/ml.
9. the method for unicellular high flux capture according to claim 5, it is characterised in that in the step 3, injection
After the aaerosol solution of cell, according to the mode of standing, static conditions are：Time of repose is 30 min-40min, and temperature is room
Temperature；
According to the mode of centrifugation, centrifugal condition is：Centrifugation time is 5-7min, and centrifuging temperature is 4 DEG C, rotating speed 1000
rpm。
10. the method for unicellular high flux capture according to claim 5, it is characterised in that molten with PBS in step 4
When liquid washes away the cell of the micro- well array chip excess surface of the dimethyl silicone polymer, it is impossible to all inhale chip surface liquid
Walk, it is ensured that micro- well array chip surface has a small amount of liquid to coat；With liquid-transfering gun from micro- well array chip upper vertical during fluid injection
Drop is instilled, speed is about 1 drop per second, and apart from the cm of chip upper surface about 2；Ensure that chip level is placed during imbibition fluid injection, weight
Liquid fluid injection about 4 ~ 5 times is relapsed, can be by the cell clearance of the micro- well array chip excess surface of the dimethyl silicone polymer.
A kind of 11. method of unicellular high flux imaging analysis, it is characterised in that comprise the following steps：
It is placed under microscope Step 1: single celled unicellular high flux acquisition equipment will have been captured, is then looked under low power lens
To unicellular capture region；
Step 2: being converted into high power lens, unicellular capture region is imaged successively according to micro- well array module numbering, every piece
Region is intended to shoot its corresponding light field figure and fluorescence field figure；
Step 3: after the completion of shooting, single celled image information is obtained, then using image processing software to the glimmering of every piece of region
Light field figure carries out fluorescence intensity quantitative analysis, obtains each single celled fluorescence intensity information；
Step 4: utilize the single celled fluorescence intensity distribution situation of data processing software high throughput analysis.
12. one kind positioning single celled method of picking, it is characterised in that comprise the following steps：
Step 1: prepare the capillary needle that needle point internal diameter is about 30 ~ 50 μm；
Step 2: imaging of being taken pictures to unicellular region, obtains some region of light field figure and fluorescence field figure, selected with two width figures and
It is unicellular to position target；
Step 3: observe tip position in eyepiece, when it is located at target unicellular top, it is vertically goed deep into rapidly the mesh
Mark in unicellular corresponding micro- well, cell is inhaled into capillary needle under capillary force effect, is then taken out capillary needle, is inserted
In the one end for entering rubber tube, unicellular lysate ready in advance is blown into by unicellular from the rubber tube other end with mouth
In, carry out the single celled full transcriptome analysis of target.
13. the positioning single celled method of picking according to claim 11, it is characterised in that by the needle point of the capillary needle
Gone out with grinder buffing it is suitably sized, to guarantee and can only cover a micro- well.
14. the positioning single celled method of picking according to claim 11, it is characterised in that be bent into the capillary needle
It is rectangular-shaped, it is allowed to human hand held one end, and another needle tip extends vertically into chip, to avoid hand from sheltering from the observation visual field of eyepiece.
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| CN116855366A (en) * | 2023-08-19 | 2023-10-10 | 北京航空航天大学 | Cell chip for metabolic fingerprint spectrum visualization and application method |
| CN116855366B (en) * | 2023-08-19 | 2024-10-18 | 北京航空航天大学 | Cell chip for metabolic fingerprint spectrum visualization and application method |
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