CN109706053B - Raman activated liquid drop sorting system and method - Google Patents
Raman activated liquid drop sorting system and method Download PDFInfo
- Publication number
- CN109706053B CN109706053B CN201711014803.2A CN201711014803A CN109706053B CN 109706053 B CN109706053 B CN 109706053B CN 201711014803 A CN201711014803 A CN 201711014803A CN 109706053 B CN109706053 B CN 109706053B
- Authority
- CN
- China
- Prior art keywords
- raman
- sorting
- unit
- flow
- droplet
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Images
Classifications
-
- 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
- C12M1/00—Apparatus for enzymology or microbiology
-
- 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
- C12M1/00—Apparatus for enzymology or microbiology
- C12M1/34—Measuring or testing with condition measuring or sensing means, e.g. colony counters
-
- 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
- C12M1/00—Apparatus for enzymology or microbiology
- C12M1/36—Apparatus for enzymology or microbiology including condition or time responsive control, e.g. automatically controlled fermentors
-
- 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
- C12M1/00—Apparatus for enzymology or microbiology
- C12M1/42—Apparatus for the treatment of microorganisms or enzymes with electrical or wave energy, e.g. magnetism, sonic waves
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
Abstract
The invention provides a Raman activated liquid drop sorting system and a sorting method, which can realize high-speed, accurate and lossless liquid drop sorting based on Raman spectrum. The Raman activated liquid drop sorting system sequentially comprises the following units from first to last according to the liquid flow direction: the liquid flow pinching unit, the signal detection unit, the liquid droplet generation unit, the sorting control unit, the liquid flow driving unit, the liquid droplet recovery unit and the liquid flow channel. The invention solves the problem that high-flux separation is difficult to carry out originally, and has the advantages of simplicity, feasibility, wide application range, strong expandability and the like.
Description
Technical Field
The invention belongs to the field of biotechnology and instrument science, in particular to a system and a method for Raman-activated liquid drop sorting, which can realize high-speed, accurate and lossless liquid drop sorting and are particularly suitable for single cell sorting.
Background
The living single cell is the basic unit of life activity and the basic unit of evolution, the research on the cell biological process from the single cell level has important significance, and the rapid separation and acquisition of the single cell becomes the key technology of single cell research and analysis. The ideal single cell analysis tool would like to process the cells in their native state in a non-invasive, label-free and high throughput manner. Based on the principle that Raman spectrum is the inherent biochemical curve and 'chemical image' of single cells, the Raman activated cell sorting has the single cell sorting capability of analyzing a plurality of characteristics or phenotypes simultaneously without marking and non-invasiveness, so that the Raman activated cell sorting method becomes an ideal method for single cell research.
In the prior art, a series of technologies related to raman-activated cell sorting, such as optical tweezers, laser jet coupling and the like, belong to a static version of the raman-activated cell sorting technology. Although these systems are simple and practical, their throughput is too low to prevent the use of raman spectroscopy techniques in high-throughput sorting. In order to improve the single cell sorting throughput, raman-activated sorting flow cytometry developed based on microfluidic technology is also appeared, in such technical schemes, cells are fixed by optical tweezers for measurement and dragged to collect the cells, but the finally realized throughput of such schemes is still not high (about 3 min/cell), and the actual demand cannot be completely met.
Disclosure of Invention
As described above, there is currently no raman-activated droplet sorting system and method that can meet the following requirements:
a) the requirement on the sorting throughput is high;
b) the sorting accuracy is high;
c) the sorted droplets are not damaged or only slightly damaged by the contents, such as single cells.
In addition, the liquid drop sorting system and the sorting method based on the raman spectrum have the following two technical problems: firstly, the lens effect generated by the convex/concave shape of the surface of the liquid drop can deform the focus and reduce the spatial resolution; secondly, the background of the Raman spectrum of the oil phase used for wrapping the liquid drops is too strong, thereby causing remarkable interference. Both of these problems can lead to difficulties in accurately obtaining raman signals when detecting droplets and/or their contents.
The present invention is intended to provide a raman-activated droplet sorting system and a raman-activated droplet sorting method that can solve the above-mentioned problems, thereby realizing high-speed, accurate, and lossless droplet sorting based on raman spectroscopy.
In a first aspect of the invention, a raman-activated droplet sorting system is provided, which is characterized in that the sorting system comprises the following units in sequence from first to last in the direction of liquid flow:
1) the liquid flow pinching unit is used for adjusting the flow rates of the cell/particle suspension and the buffer solution, and converging the cells/particles to be sorted in the middle of the liquid flow channel to form a stable single cell/particle flow;
2) the signal detection unit is used for generating Raman scattering photons, carrying out Raman laser signal detection on the single cell/particle flow formed by converging the liquid flow pinching unit, collecting detection signals and transmitting the detection signals to the sorting control unit;
3) the droplet generation unit is used for introducing an oil phase into the single cell/particle flow detected by the signal detection unit to form water-in-oil type droplets;
4) a sorting control unit for analyzing the detection signal from the signal detection unit and sorting the droplets generated by the droplet generation unit according to the signal;
the sorting system further comprises a liquid flow driving unit and a liquid flow channel, wherein the liquid flow driving unit is used for generating the driving force required by the liquid flow in the sorting system; the liquid flow channel is a carrier for liquid flowing in the system and a liquid inlet and a liquid outlet.
In a preferred form of the invention, the flow entrapment unit comprises at least one flow channel for the inflow of the cell/particle suspension and at least two flow channels for the inflow of the buffer.
In a preferred form of the invention, the flow entrapment unit is adapted to regulate the flow rates of the cell/particle suspension and the buffer to form a single cell/particle flow suitable for detection by the signal detection unit.
In a preferred form of the present invention, the signal detection unit includes a microscope, a stage, a raman laser light source, a signal collection element, and a high-speed CCD.
In a preferred form of the present invention, the raman laser signal alignment method used in the signal detection unit is selected from a combination of one or more of the following: unimodal comparison, multimodal comparison, full spectral comparison.
In a more preferred form of the invention, the signal collection element is a charge coupled device.
In a more preferred form of the present invention, the signal detection unit includes a dielectric capture unit, which is used to capture cells/particles by dielectric means to prolong the time for collecting raman laser signals and increase the intensity of collected signals.
In a preferred form of the invention, the droplet-generating unit comprises at least one flow channel for flowing the cell/particle suspension after detection by the signal-detecting unit, and at least one side-stream inlet for introducing the oil phase.
In a more preferred form of the invention, the droplet generation unit generates water-in-oil droplets containing on average no more than 1 single cell per particle per droplet.
In a more preferred form of the invention, the droplet generation unit generates water-in-oil droplets containing an average of 0.3 single cells/particle per droplet.
In one embodiment of the invention, the droplet generating unit generates droplets having a diameter of 30-80 μm.
In one embodiment of the invention, the oil phase used in the droplet generation unit is a mineral oil containing 2.5% Span 80.
In one embodiment of the invention, the oil phase flow rate used in the droplet generation unit is 120 μ L/h.
In one embodiment of the invention, the droplet generation unit generates water-in-oil droplets having a diameter of 50 μm.
As a preferred form of the present invention, the sorting control unit performs droplet sorting by any one of the following methods: dielectrophoresis, ultrasound, electric field, magnetic field, pressure squeezing or pressure sucking.
As a more preferred form of the invention, the sorting control unit comprises at least two dielectric electrodes for sorting the droplets by dielectrophoresis.
In a preferred form of the invention, the sorting system further comprises at least one droplet collection unit for collecting and recovering sorted droplets.
In a preferred form of the invention, the flow channel is a microfluidic chip.
In a more preferred form of the invention, the width of the flow channel is 25 to 100 μm and the depth is 40 to 60 μm.
In a second aspect of the invention, there is provided a method of raman-activated droplet sorting, comprising the steps of:
1) liquid flow convergence: converging cells/particles to be sorted with a buffer solution to form a stable single cell/particle flow;
2) signal detection: performing Raman spectrum detection on the single cells/particle flow one by one and collecting signals;
3) droplet generation: after the collection is finished, introducing an oil phase into a side stream, and forming water-in-oil micro-droplets containing single cells/particles through shearing;
4) liquid drop sorting: and acquiring the collected signals in the signal detection step, carrying out sorting operation on the correspondingly encapsulated water-in-oil droplets according to the signals, and collecting the droplets of the required part.
In a preferred aspect of the present invention, the sorting operation in the droplet sorting step in the above sorting method is performed by a dielectrophoresis method.
In a preferred form of the present invention, in the droplet sorting step of the above sorting method, there is a delay time between the acquisition of the acquisition signal and the execution of the sorting operation, and the delay time is calculated and determined by: the time interval is the length of the liquid flow/flow velocity, wherein the length of the liquid flow refers to the actual distance from the signal detection point to the sorting operation point.
The invention has the following advantages and beneficial effects:
1. the invention creatively solves the problem of signal interference caused by Raman detection by the bending interface of the liquid drop and the oil phase component in a mode of leading the Raman detection and then encapsulating the liquid drop,
2. the invention realizes the single cell high-throughput sorting based on the Raman spectrum, the throughput can reach 500 cells/minute, and the single cell sorting is improved by more than two orders of magnitude compared with other single cell sorting technologies based on the Raman spectrum.
3. The invention improves the flux and simultaneously ensures the sorting precision (more than 98%).
4. The sorting system and the method adopt a droplet microfluidic system, greatly reduce the damage to cells and facilitate downstream processing after sorting.
In a word, the invention provides a set of liquid drop sorting system based on Raman spectrum and a corresponding sorting method, solves the problem that high-throughput sorting is difficult to carry out originally, and has the advantages of simplicity, feasibility, wide application range, strong expandability and the like.
Drawings
Fig. 1 is a schematic diagram of the system components of a raman-activated droplet sorting system according to the present invention.
Fig. 2 is a schematic structural diagram of one implementation of a raman-activated droplet sorting system of the present invention.
FIG. 3 is a schematic diagram of another implementation of a Raman-activated droplet sorting system according to the present invention
Fig. 4 is a flow chart of the sorting control operation of the raman-activated droplet sorting system of the present invention.
FIG. 5 shows the result of the sorting efficiency verification in the sorting model of the microalgae with high astaxanthin yield by using the method.
Detailed Description
The present invention will be described in further detail with reference to the accompanying drawings and examples.
The prior art has disclosed the general framework design of a cell flow detection system based on raman signals, such as: CN102019277A, CN104877898A, etc., which are incorporated herein in their entirety. On the basis of the basic structure disclosed in the above-mentioned document, the raman-activated droplet sorting system of the present invention is basically composed as shown in fig. 1, and mainly includes a liquid stream pinching unit, a signal detecting unit, a droplet generating unit, and a sorting control unit.
Fig. 2 is an implementation of the raman-activated droplet sorting system of the present invention, specifically:
the flow cell 1 has at least one flow channel 5 for the inflow of the cell/particle suspension and at least two flow channels 6 and 7 for the inflow of buffer for adjusting the flow rates of the cell/particle suspension and the buffer. The flow rate is generally determined according to the size of the liquid drop expected to be generated, and meanwhile, the cells/particles to be sorted need to be gathered in the middle of the liquid flow channel to form a stable single cell/particle liquid flow suitable for being detected by the signal detection unit. To reduce interference with raman signal detection, aqueous based buffers with low raman signal are used for suspensions and buffers.
The signal detection unit 2 is used for generating Raman laser, detecting Raman laser signals of the single cell/particle flow formed by converging the liquid flow pinching unit, collecting detection signals and transmitting the detection signals to the sorting control unit. The device comprises a microscope, an object stage, Raman laser scattered photons, a signal collecting element, a high-speed CCD and other components. The signal comparison mode used for detection can be one or a combination of unimodal comparison, multimodal comparison or full spectrum comparison. In addition, the signal detection unit can be additionally provided with a dielectric capture unit, and cells/particles can be captured in a dielectric mode to prolong the acquisition time of the Raman laser signals and further improve the intensity of the acquired signals.
The droplet generating unit 3 comprises at least one flow channel for flowing the cell/particle suspension detected by the signal detecting unit, and at least one side flow inlet 8 for introducing an oil phase, and the oil phase is introduced into the single cell/particle flow detected by the signal detecting unit to form water-in-oil type droplets. Due to the requirements of subsequent cell sorting, on average no more than 1 single cell/particle per water-in-oil droplet. In practice it can be reduced to 0.3 single cells/particle or even lower per droplet.
A sorting control unit 4 for analyzing the detection signal from the signal detection unit and sorting the droplets generated by the droplet generation unit according to the signal. The actual sorting method can be realized by any one of dielectrophoresis, ultrasound, an electric field, a magnetic field, pressure extrusion or pressure suction and the like according to different requirements on flux. Among them, the separation based on dielectrophoresis is a better way to realize the high-throughput separation requirement, that is, based on the detection signal, the liquid drops flowing through one pair by at least two dielectric electrodes 9 and 10 are applied with different acting forces, and the liquid drop flow direction is controlled to realize the high-speed and convenient separation effect. There is a delay time between acquiring the acquisition signal and performing the sorting operation during sorting, the delay time being calculated and determined by: the time interval is the length of the liquid flow/flow velocity, wherein the length of the liquid flow refers to the actual distance from the signal detection point to the sorting operation point.
The collecting channel is connected to a collecting container (not shown in the figure), and collects the target droplets obtained by sorting as a droplet collecting unit.
Fig. 3 shows another form of implementing the raman-activated droplet sorting system of the present invention, and specifically, based on the structure shown in fig. 2 and the above description of the structure shown in fig. 2, a capture electrode 11, i.e., a dielectric capture unit, is added in the signal detection unit for capturing cells/particles by dielectric means to prolong the raman laser signal collection time, thereby increasing the collected signal intensity.
One possible sort control is shown in figure 4.
In addition to the above units, the raman-activated droplet sorting system of the present invention further comprises: a fluid flow driving unit for generating a driving force required for fluid flow in the sorting system; the liquid drop collecting unit is used for collecting and recovering the sorted liquid drops; and fluid flow channels as liquid flow carriers in the system and into and out of the ports.
As a conventional technical means in the field, the width of a liquid flow channel in the system is 25-100 μm, and the depth is 40-60 μm.
The invention is further illustrated below with reference to specific examples.
Examples
Design and manufacture of microfluidic sorting chip
We designed a single layer microfluidic chip with PDMS. Wherein the width for the flow channel is designed to be 50 μm. The microfluidic chip was fabricated by soft lithography and rapid prototyping techniques. Briefly, a 50 μm high SU-8TMThe die was added to a 3 inch silicon wafer. A PDMS layer was prepared by pouring a mixture of PDMS and a curing agent on a SU-8TM mold in a mass ratio of 10: 1. The PDMS sheets (3 mm thick) with channels were cut and peeled from the mold after curing in an oven at 70 ℃ for 2 hours. Inlet and outlet holes were punched using a Harris Uni-Core biopsy punch (Electron Microcopy Sciences) 0.75mm in diameter. In oxygen, etcAfter PLASMA (PLASMA-PREEN II-862, PLASMA Systems, Inc., United States), PDMS sheets were bonded with PDMS-coated glass substrates (75 mm. times.25 mm. times.1 mm). The sealed PDMS chip was then placed in an oven at 70 ℃ for at least 12 hours to recover its hydrophobicity.
The device was then heated to 100 ℃ on a hot plate and a low melting point In-Sn solder was filled into the electrode channels. And inserting small copper wires to be electrically connected with the solder electrodes and further protected by AB glue.
High throughput sorting system build
PEEK tubes (OD 0.03 inch, ID 0.012 inch; Cole-Parmer, USA) were used for connecting the microfluidic device, Pump-on syringes (LSP01-2A, ringer Pump, China) and tubes for cell collection. The cell loaded tubing and syringe was treated with hydrophilic agent "5% PF 127" for 10 minutes and then washed in sterile deionized water for 1 minute. Mineral oil containing 2.5% (w/w) Span 80 surfactant was used to generate the droplets. The DEP is generated to trigger the sorting of the target droplets by using a high-voltage amplifier (PC 2000, tianjin eastern high-voltage power supply limited, china) controlled by a digital I/O unit (DIO-1616LX-USB, CONTEC) connected to a computer.
Raman microscopy was performed on a custom-made LabRam HR micro-raman setup equipped with Nd: YAG 532nm Laser emitter (Ventus, Laser Quantum ltd., United Kingdom) as an excitation light source to generate scattered photons, a charge coupled device (EMCCD) for collecting raman signals (Newton DU970N-BV), a high speed CCD camera (Pike F-032, available Vision Technologies, China) for monitoring cell and droplet flow, and a 60 x water mirror (NA 1.0, Olympus, United Kingdom) to focus the Laser beam on the sample. Measurements were made using a 600 line/mm grating using a 660nm LED array as the light source for monitoring the sorting process.
The RADS device including the microfluid device, the Raman system and the DEP system are integrated together by controlling the electronic equipment (EMCCD, high-pressure amplifier and the like) and adjusting the system parameters (such as acquisition time and DEP duration) through self-designed and written software so as to automatically operate.
Sorting efficiency validation
Ethanol, isopropanol, Span 80, C2H3O 2. Na, MgCl 2. 6H2O, CaCl2, FeSO 4.7H 2O, NaNO3, K2HPO 4.3H 2O, KH2PO4, NaCl, MgSO 4.7H 2O, FeCl 3.6H 2O, MnCl 2.4H 2O, ZnSO 4.7H 2O, CoCl 2.6H 2O and Na2MO 4.2H 2O are chemical reagents from Chinese medicine (Shanghai, China). Mineral oil, L-asparagine, yeast extract, Propidium Iodide (PI) PF127 and Na2 EDTA.2H2O were purchased from Sigma-Aldrich (St. Louis, MO, USA). SU-8TM (GM 3025) was purchased from MicroChem (Massachusetts, USA). Polydimethylsiloxane (PDMS Sylgard) and curing agent (Sylgard 184) were purchased from Dow Corning (Midland MI USA). All reagents used in the experiment were analytically pure. All solutions were prepared with deionized water and filtered through 0.22 μm microporous membranes or sterilized in an autoclave at 121 ℃ for 30 minutes prior to use.
Microalgae H.pluvialis (NIES-144) was purchased from the microorganism culture Collection (NIES, Japan) and cultured in basal medium (1.197g/L C2H3O 2. Na, 0.357g/L L-asparagine, 2g/L yeast extract, 0.2g/L MgCl 2.6H 2O, 0.015g/L CaCl2, 0.01g/L FeCl 3.6H 2O and 20g/L agar) under continuous low light conditions (22 ℃, inoculated into liquid basal medium at 20. mu. mol photon m-2 s-1) and shaken manually once a day. To induce Astaxanthin (AXT) accumulation, exponentially growing cells were resuspended in triplicate modified BBM medium (no nitrogen source and containing 10mM NaAc) and exposed to 150 μmol photons m-2s-1 with continuous irradiation (creating an AXT content gradient). Cells with various average AXT contents were collected, filtered through a 40 μm microporous membrane to remove debris and cell clusters, and then washed 3 times with deionized water at 3000rpm for 3 minutes each. Cell density was measured using a cell counting plate and adjusted to about 8.02 × 106 cells/mL with a final cell density of 4.58 × 106 cells/mL after pinch loading). Finally, the mixture was encapsulated in 2.5% Span 80 mineral oil at a flow rate of 120. mu.L/h into droplets having a diameter of about 50 μm and containing an average of 0.3 cells per droplet.
Because of the need for centrifugation or demulsifying agents such as Pico-BreakTM in cell recovery based on other sorting methods, these methods are both damaging to the cells and inefficient. We used ultra-high porosity Parylene C membranes with a pore size of 12 μm to recover the sorted cells. In this way, targeted cell isolation yields can be as high as 96%.
In order to verify the sorting efficiency, the cells are mixed at a specific ratio and then subjected to Raman signal detection after being induced for 0 days and 3 days respectively. As shown in FIGS. 5a and 5b, more than 60 cells were randomly selected and their intracellular astaxanthin content was verified, of which only one did not meet the predetermined sorting criterion. The proportion of the astaxanthin high-yield cells is increased by 98 percent on average. These results indicate that the raman-activated droplet sorting of the present invention is very accurate and efficient.
It should be understood that after reading the above description of the present invention, various changes or modifications can be made by those skilled in the art to the relevant conditions of the present invention, and these equivalents also fall within the scope of the appended claims of the present application.
Claims (10)
1. A raman-activated droplet sorting device, comprising the following units in order from first to last in the direction of flow:
1) the liquid flow pinching unit is used for adjusting the flow rates of the cell/particle suspension and the buffer solution, and converging the cells/particles to be sorted in the middle of the liquid flow channel to form a stable single cell/particle flow;
2) the signal detection unit is used for generating Raman scattering photons, carrying out Raman laser signal detection on the single cell/particle flow formed by converging the liquid flow pinching unit, collecting detection signals and transmitting the detection signals to the sorting control unit;
3) the droplet generation unit is used for introducing an oil phase into the single cell/particle flow detected by the signal detection unit to form water-in-oil type droplets; and the droplet generation unit comprises at least one side flow inlet for introducing an oil phase, and also comprises at least one liquid flow channel for flowing in the cell/particle suspension detected by the signal detection unit, and the water-in-oil droplets generated by the droplet generation unit contain 0.3 single cells/particles on average in each droplet; and
4) a sorting control unit for analyzing the detection signal from the signal detection unit and sorting the droplets generated by the droplet generation unit in accordance with the signal;
the sorting device also comprises a liquid flow driving unit and a liquid flow channel, wherein the liquid flow driving unit is used for generating the driving force required by the liquid flow in the sorting device; the liquid flow channel is a carrier for liquid flowing in the device and a liquid inlet and a liquid outlet.
2. The raman-activated droplet sorting device of claim 1, wherein the fluid flow pinching unit comprises at least one fluid flow channel for flowing a cell/particle suspension and at least two fluid flow channels for flowing a buffer.
3. The raman-activated droplet sorting device of claim 1, wherein the fluid flow pinching unit is configured to adjust the cell/particle suspension and buffer flow rates to form a single cell/particle fluid flow suitable for detection by the signal detection unit.
4. The raman-activated droplet sorting device of claim 1, wherein the signal detection unit comprises a microscope, a stage, a raman laser light source, a signal collection element, and a high-speed CCD.
5. The raman-activated droplet sorting device of claim 1, wherein the raman laser signal alignment used in the signal detection unit is selected from the group consisting of one or more of: unimodal comparison, multimodal comparison, full spectral comparison.
6. The raman-activated droplet sorting device of claim 1, wherein the signal detection unit comprises a dielectric capture unit for dielectrically capturing cells/particles to prolong the time for collecting raman laser signals and increase the intensity of collected signals.
7. The raman-activated droplet sorting device of claim 1, wherein the sorting control unit performs droplet sorting by any one of the following methods: dielectrophoresis, ultrasound, electric field, magnetic field, pressure squeezing or pressure sucking.
8. The raman-activated droplet sorting device of claim 1, wherein the sorting system further comprises at least one droplet collection unit for collecting recovered sorted droplets.
9. A method of raman-activated droplet sorting using the raman-activated droplet sorting device of claim 1, comprising the steps of:
1) liquid flow convergence: converging cells/particles to be sorted with a buffer solution to form a stable single cell/particle flow;
2) signal detection: performing Raman spectrum detection on the single cells/particle flow one by one and collecting signals;
3) droplet generation: after the collection is finished, introducing an oil phase into a side stream, and forming water-in-oil micro-droplets containing single cells/particles through shearing; and
4) liquid drop sorting: and acquiring the acquisition signal in the signal detection step, carrying out sorting operation on the correspondingly encapsulated water-in-oil micro-droplets according to the signal, and collecting the required part of droplets.
10. The raman-activated droplet sorting method of claim 9, wherein in the droplet sorting step, there is a delay time between acquiring the acquisition signal and performing the sorting operation, the delay time being calculated by: the time interval is the length of the liquid flow/flow velocity, wherein the length of the liquid flow refers to the actual distance from the signal detection point to the sorting operation point.
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201711014803.2A CN109706053B (en) | 2017-10-25 | 2017-10-25 | Raman activated liquid drop sorting system and method |
| PCT/CN2018/111938 WO2019080905A1 (en) | 2017-10-25 | 2018-10-25 | Raman activated droplet sorting system and method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201711014803.2A CN109706053B (en) | 2017-10-25 | 2017-10-25 | Raman activated liquid drop sorting system and method |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN109706053A CN109706053A (en) | 2019-05-03 |
| CN109706053B true CN109706053B (en) | 2021-04-23 |
Family
ID=66247760
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201711014803.2A Active CN109706053B (en) | 2017-10-25 | 2017-10-25 | Raman activated liquid drop sorting system and method |
Country Status (2)
| Country | Link |
|---|---|
| CN (1) | CN109706053B (en) |
| WO (1) | WO2019080905A1 (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113189040A (en) * | 2021-04-28 | 2021-07-30 | 北京大学第三医院（北京大学第三临床医学院） | Method and system for efficiently and nondestructively detecting number and activity of tumor cells in sample |
| CN115025826B (en) * | 2022-06-24 | 2024-01-19 | 武汉大学 | Label-free high-flux rare cell enrichment microfluidic system and method |
Family Cites Families (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US8414755B2 (en) * | 2009-05-29 | 2013-04-09 | Ondavia, Inc. | Microfluidic separation device |
| CN103897985B (en) * | 2012-12-25 | 2016-08-10 | 中国科学院青岛生物能源与过程研究所 | living single cell sorting electronic control system |
| CN104677877B (en) * | 2013-11-26 | 2017-11-28 | 中国科学院青岛生物能源与过程研究所 | A kind of micro-fluidic chip and method for capturing collection cell/particle Raman spectrum |
| CN104677808A (en) * | 2013-11-26 | 2015-06-03 | 中国科学院青岛生物能源与过程研究所 | Pressure sucking-based cell/particle sorting system and method |
| CN104877898B (en) * | 2014-02-27 | 2017-12-15 | 中国科学院青岛生物能源与过程研究所 | A kind of low cost, efficiently separate the single celled system and method for acquisition |
-
2017
- 2017-10-25 CN CN201711014803.2A patent/CN109706053B/en active Active
-
2018
- 2018-10-25 WO PCT/CN2018/111938 patent/WO2019080905A1/en active Application Filing
Also Published As
| Publication number | Publication date |
|---|---|
| WO2019080905A1 (en) | 2019-05-02 |
| CN109706053A (en) | 2019-05-03 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| JP7071519B2 (en) | Formation of droplets enclosing a single particle with a microfluidic chip and methods for deriving each | |
| JP7283798B2 (en) | Single particle analysis method and system for the analysis | |
| Hunt et al. | Dielectrophoresis tweezers for single cell manipulation | |
| Wang et al. | Enhanced cell sorting and manipulation with combined optical tweezer and microfluidic chip technologies | |
| US8357530B2 (en) | Microfluidic device for trapping single cell | |
| WO2010113994A1 (en) | Device for concentrating and separating cells | |
| CN102174369B (en) | Micro-slit-structure-based full PDMS (polydimethylsiloxane) micro-fluidic cell capturing chip and manufacturing method thereof | |
| CN106497786B (en) | A kind of micro-fluidic chip for unicellular capture and culture | |
| CN109706053B (en) | Raman activated liquid drop sorting system and method | |
| CN104736718A (en) | Devices and methods for manipulating components in a fluid sample | |
| EP2964360A1 (en) | Devices, systems, and methods for acoustically -enhanced magnetophoresis | |
| CN111315892A (en) | Intracellular delivery and methods thereof | |
| WO2021084814A1 (en) | Minute particle collection method, microchip for aliquoting minute particles, minute particle collection device, production method for emulsion, and emulsion | |
| Lu et al. | A microfluidic approach towards hybridoma generation for cancer immunotherapy | |
| CN104974997B (en) | A kind of cell novel method for separating using parallel electric field formula photoelectric chip | |
| CN1886684A (en) | System and method for manipulating and processing materials using holographic optical trapping | |
| CN113874488A (en) | Erythrocyte removing device, monocyte recovering device, cell culture system, cell culture method, and monocyte recovering method | |
| US20170045430A1 (en) | Collecting apparatus for extracellular vesicles and method for using the same | |
| JP2009136789A (en) | Method for recovering minute particle and apparatus therefor | |
| JP2021506263A (en) | Inertial cell focusing and sorting | |
| CN113322181A (en) | Cell lysis device and cell purification and/or cell lysis method based on cell lysis device | |
| US9939353B2 (en) | Apparatus for cell observation and method for cell collection using the same | |
| Deivasigamani et al. | A correlation of conductivity medium and bioparticle viability on dielectrophoresis‐based biomedical applications | |
| CN112840015A (en) | Method and apparatus for controlling intracellular delivery of substances | |
| WO2022062897A1 (en) | Microfluidic chip for particle screening and separation |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |