CN104877898A - System and method for low-cost and efficient separation and obtaining of single cell - Google Patents
System and method for low-cost and efficient separation and obtaining of single cell Download PDFInfo
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- CN104877898A CN104877898A CN201410069484.5A CN201410069484A CN104877898A CN 104877898 A CN104877898 A CN 104877898A CN 201410069484 A CN201410069484 A CN 201410069484A CN 104877898 A CN104877898 A CN 104877898A
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- 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
- C12M23/00—Constructional details, e.g. recesses, hinges
- C12M23/02—Form or structure of the vessel
- C12M23/16—Microfluidic devices; Capillary tubes
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- 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
- C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
- C12M41/48—Automatic or computerized control
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- 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
Abstract
According to the present invention, based on a liquid droplet microfluidic chip technology, a system and a method for low-cost and efficient separation and obtaining of single cell is designed and constructed. The system comprises a microfluidic chip, a liquid droplet signal detector, a signal analysis and sorting control unit, an electromagnetic valve, a conduct pipe, and a liquid droplet collection micro-pipe. The single cell separation process comprises that: liquid droplets wrapping cells are subjected to online continuous generation and directional flow, signal acquisition and analysis and sorting control are performed when passing through a signal acquisition zone, and when the sorting condition (for example: the liquid droplet only warps single cell) is met, the work of the electromagnetic valve is controlled to make the force applying state and the flow direction of the liquid droplet be changed so as to separate and collect the single liquid droplet, such that the obtaining of the single cell can be achieved through the de-emulsification, the extraction and other operations. According to the technical method of the present invention, a set of the simple and feasible system and the simple and feasible method suitable for the liquid droplet sorting, cell separation and single cell obtaining of different detection methods are provided, and the method has advantages of high efficiency, short time consumption, low cost, no pollution, wide application range, strong extensibility and the like compared with the traditional single cell obtaining method.
Description
Technical field
The invention belongs to biotechnology and instrumental science field, specifically based on drop parcel on micro-fluidic chip with the method for drop sorting realizes low cost, high efficiency separation obtains single celled system and method.
Background technology
Living single cell is the elementary cell of vital movement and the fundamental unit of evolution, have great significance from individual cells layer viewpoint cell biological processes, can inherently disclose pathogenesis of cancer mechanism, understand cytodifferentiation and tissue development principle, identify gene expression characteristics and cell characteristic.
Sharp separation, obtain the unicellular gordian technique having become unicellular research and analysis.The important instrument platform of flow cell sorter in unicellular sorting and analysis is the effective means of isolated cell.But because the fluorescent effect of most active somatic cell own is more weak or do not have fluorescence, additional fluorescent mark is not suitable for active somatic cell screening.The integrated micro-fluidic chip single cell analysis instrument of commercialization can realize single celled certain or some object analyzed, but its integrated level is higher, extensibility is not strong, expensive, not easily realize single celled physical sepn to obtain, limit the autonomy-oriented analysis of single celled downstream, hinder its application in laboratory study.Unicellular separation method conventional in current laboratory study also comprises laser optical tweezer, micro-separation and infinite dilution method.First two technology platform is expensive, is separated flux lower; Infinite dilution method is simple and convenient, but cannot controlled separation obtain unicellular.
Micro-fluidic chip has that size is little, reagent consumption is few, high-throughput, the feature such as easy of integration, is applicable to the research and analysis work of microbody system; Microlayer model is as independently research and analysis unit, and making the microlayer model technology based on micro-fluidic chip be applied to unicellular research and analysis has huge advantage and prospect.
Summary of the invention
For above shortcomings part in prior art, the technical problem to be solved in the present invention is to provide a kind of low cost, high efficiency separation obtains single celled system and method.
The technical scheme that the present invention is adopted for achieving the above object is: a kind of low cost, high efficiency separation obtain single celled system, it is characterized in that, comprising: micro-fluidic chip, drop signal detector, signal analysis and selection control unit, magnetic valve, conduit, drop collect microtubule;
Described micro-fluidic chip inside comprises microchannel network, for making fluid in the flowing of its fluid channel interior orientation under external force drives, the generation of drop and sorting, and waste liquid, is sorted the derivation of drop;
Described drop signal detector is used for the optical signalling of acquisition stream through the drop of the detection zone of described micro-fluidic chip, and is sent to signal analysis and selection control unit;
Described signal analysis and selection control unit are for analyzing the signal from described drop signal detector, and the work of Controlling solenoid valve;
The selection control instruction that described magnetic valve sends for receiving described signal analysis and selection control unit, and perform corresponding drop in the direction of motion of micro-fluidic chip divide selection operation by changing drop;
Micro-fluidic chip and magnetic valve described in described tubes connection, for deriving the drop of sorting;
Described drop collects one end that microtubule connects described conduit, for collecting the dropping liquid that described micro-fluidic chip sub-elects.
Described micro-fluidic chip is by the upper and lower substrate key and form, and material all can be plastics, glass or quartz.
The upper strata of described micro-fluidic chip comprises microchannel network, and the gangway of described microchannel network comprises oil-continuous phase entrance, cell suspending liquid entrance, effluent oil phase entrance, magnetic valve Link Port, waste liquid outlet and is sorted drop outlets;
After microchannel that the microchannel that described oil-continuous phase entrance is communicated with is communicated with cell suspending liquid entrance is communicated with, then with described effluent oil phase entrance, magnetic valve Link Port, waste liquid outlet be sorted drop outlets and be communicated with.
Described microchannel is that 25-100 micron is wide, and 40-60 micron is dark, and the droplet dia of formation is 30-80 micron.
The described drop outlets that is sorted is the microchannel that 200-250 micron is wide, 200-250 micron is dark.
Described drop signal detector is any one in high speed camera, fluorimetric detector and Raman spectroscopic detector.
Described signal analysis and selection control unit are coupled by the signal analysis software corresponding with described drop signal detector and solenoid control software and hardware and are formed.
Described magnetic valve has suction, pushes away the controlled operating mode of two kinds of difference.
A kind of low cost, high efficiency separation obtain single celled method, comprise the following steps:
The formation of liquid injection and drop: utilize pressure-driven oil phase to enter a microchannel of micro-fluidic chip by oil-continuous phase entrance, drive cell suspending liquid aqueous phase to be entered another microchannel of micro-fluidic chip by cell suspending liquid entrance, by oil phase, water-in-oil microlayer model is formed to the shearing action of aqueous phase in the junction of above-mentioned two microchannels;
Stream of liquid droplets to control: from effluent oil phase entrance introduce oil phase, make water-in-oil microlayer model fluid channel interior orientation flowing, and acquiescence flow into waste liquid outlet;
Drop sampling and analysis of signal: when water-in-oil microlayer model flows through drop signal detector, the drop signal of water-in-oil microlayer model is by collected and be sent to signal analysis and selection control unit, by the sort regular of received signal and setting being compared, judge whether the drop signal gathered meets the condition of described sort regular;
Drop sorting: when the drop signal gathered meets separation condition, signal analysis and selection control unit triggers electromagnetic valve work, make water-in-oil microlayer model be subject to the power of magnetic valve applying, and then change its direction of motion, flow to and be sorted drop outlets;
The collection of drop and single celled acquisition: the conduit being sorted drop outlets place flows into drop and collects in microtubule, realize single drop and collect and obtain unicellular by follow-up deemulsification, extraction operation.
Described sort regular is be enclosed with in a drop and only have individual cells.
The present invention has the following advantages and beneficial effect:
1. the present invention effectively utilizes the microlayer model technique for packing of micro-fluidic chip, and by the method for drop parcel cell, improve the degree to unicellular manipulation and tolerance range, especially less to size cell, the advantage of this method is more outstanding.
2. the present invention effectively utilizes the microlayer model sorting technology of micro-fluidic chip, by the drop sorting to parcel cell, achieves the object to single celled separation and acquisition.
3. the present invention is by the time of electromagnetic valve for adjusting work, effectively controls the size that drop is stressed, expands the scope of application of this method to the different drop of size.
4. present invention optimizes micro-fluidic chip and the extraneous interface structure be connected, solve the problem that different scale on micro-mobility protocols interface etc. causes, achieve drop fluent, efficiently derive.
5. the technological method that the present invention proposes provides a set of simple possible, is applicable to the drop sorting of different detection method, cellular segregation and the single celled system and method for acquisition, and the method obtains single celled method compared to tradition and has high, consuming time short, the advantage such as cost is low, pollution-free, applied widely, extensibility is strong of efficiency.
Accompanying drawing explanation
Fig. 1 obtains single celled micro-fluidic chip schematic diagram for being separated:
Wherein, 1, oil-continuous phase entrance; 2, cell suspending liquid entrance; 3, effluent oil phase entrance; 4, magnetic valve Link Port; 5, waste liquid outlet; 6, drop outlets is sorted.
Fig. 2 obtains single celled system architecture schematic diagram for being separated:
Wherein, 7, micro-fluidic chip; 8, drop signal detector; 9, signal analysis and selection control unit; 10, magnetic valve; 11, conduit; 12, detection zone; 13, drop collects microtubule.
Embodiment
Below in conjunction with drawings and Examples, the present invention is described in further detail.
Embodiment 1: obtain unicellular based on laboratory conventional imaging technique low cost, high efficiency separation
As shown in Figure 1, 2, obtain single celled system constructed by this example based on laboratory conventional imaging technique low cost, high efficiency separation, the structure of micro-fluidic chip 7 is shown in Fig. 1, and it comprises: 1, oil-continuous phase entrance; 2, cell suspending liquid entrance; 3, effluent oil phase entrance; 4, magnetic valve Link Port; 5, waste liquid outlet; 6, drop outlets is sorted.Micro-fluidic chip 7 is by upper strata PDMS and lower floor's glass substrate key and form, it has 25-150 micron wide at the middle and upper levels, the dark microchannel network of 40-60 micron adopts wet etching and obtains, lower floor's glass substrate adheres to the PDMS of one deck about 1 mm thick, its effect is easy to produce water-in-oil drop and bilevel bonding sealing-in.Drop is formed the shearing of water by oil, makes it all even stable by adding tensio-active agent; The outlet of micro-fluidic chip 7 is connected with the external world with microtubule by fixed interface with entrance, realizes the importing of oil phase and aqueous phase, is sorted the derivation of drop and waste liquid.Specific embodiments is as follows: brewing yeast cell (Saccharomyces cerevisiae) is 10 through PBS damping fluid (phosphate buffered saline buffer) dilution
6-10
7individual/milliliter, with micro-injection pump with the flow of 0.2 μ l/min from cell suspending liquid entrance 2 sample introduction, oil phase from oil-continuous phase entrance 1 with the flow sample introduction of 1.5 μ l/min, formed water-in-oil (W/O) drop of about about 50 μm by the shearing action of oil phase, effluent oil phase flows to waste liquid outlet 5 for guiding drop from effluent oil phase entrance 3 with the flow sample introduction of 1.2 μ l/min simultaneously; By the number of the drop parcel cell of microscopic examination sorting place, when wrapping up individual cells, touch electromagnetic valve work, and then change the stressed and kinestate of this drop, thus make object droplet flow to single drop collection microtubule from being sorted drop outlets 6, so far, realize the object that single drop is collected, can individual cells be obtained by operations such as follow-up deemulsification, extractions.
Embodiment 2: obtain unicellular based on high speed camera and image processing techniques high efficiency separation
As shown in Figure 1, 2, obtain single celled system constructed by this example based on high speed camera and image processing method high efficiency separation, the structure of micro-fluidic chip 7 is shown in Fig. 1, and it comprises: 1, oil-continuous phase entrance; 2, cell suspending liquid entrance; 3, effluent oil phase entrance; 4, magnetic valve Link Port; 5, waste liquid outlet; 6, drop outlets is sorted.Micro-fluidic chip 7 is by upper strata PDMS and lower floor's glass substrate key and form, it has 25-150 micron wide at the middle and upper levels, the dark microchannel network of 40-60 micron adopts wet etching and obtains, lower floor's glass substrate adheres to the PDMS of one deck about 1 mm thick, its effect is easy to produce water-in-oil drop and bilevel bonding sealing-in.Drop is formed the shearing of water by oil, makes it all even stable by adding tensio-active agent; Chip outlet is connected with the external world with microtubule by fixed interface with entrance, realizes the importing of oil phase and aqueous phase, is sorted the derivation of drop and waste liquid.Specific embodiments is as follows: brewing yeast cell (Saccharomycescerevisiae) is 10 through PBS damping fluid (phosphate buffered saline buffer) dilution
6-10
7individual/milliliter, with micro-injection pump with the flow of 0.5 μ l/min from cell suspending liquid entrance 2 sample introduction, oil phase from oil-continuous phase entrance 1 with the flow sample introduction of 3.5 μ l/min, formed water-in-oil (W/O) drop of about about 50 μm by the shearing action of oil phase, effluent oil phase flows to waste liquid outlet 5 for guiding drop from flow measurement oil phase entrance 3 with the flow sample introduction of 2.9 μ l/min simultaneously; By high speed camera, imaging is carried out to drop, image processing software uses cell detecting algorithms to carry out Analysis And Evaluation to imaging, if judge this drop parcel individual cells, touch electromagnetic valve work, and then change the stressed and kinestate of this drop, thus make object droplet flow to single drop collect microtubule from being sorted drop outlets 6, so far, realize the object that single drop is collected, can individual cells be obtained by operations such as follow-up deemulsification, extractions.
Embodiment 3: obtain unicellular based on detection technique of fluorescence low cost, high efficiency separation
As shown in Figure 1, 2, obtain single celled system constructed by this example based on detection technique of fluorescence low cost, high efficiency separation, the structure of micro-fluidic chip 7 is shown in Fig. 1, and it comprises: 1, oil-continuous phase entrance; 2, cell suspending liquid entrance; 3, effluent oil phase entrance; 4, magnetic valve Link Port; 5, waste liquid outlet; 6, drop outlets is sorted.Micro-fluidic chip 7 is by upper strata PDMS and lower floor's glass substrate key and form, it has 25-150 micron wide at the middle and upper levels, the dark microchannel network of 40-60 micron adopts wet etching and obtains, lower floor's glass substrate adheres to the PDMS of one deck about 1 mm thick, its effect is easy to produce water-in-oil drop and bilevel bonding sealing-in.Drop is formed the shearing of water by oil, makes it all even stable by adding tensio-active agent; Chip outlet is connected with the external world with microtubule by fixed interface with entrance, realizes the importing of oil phase and aqueous phase, is sorted the derivation of drop and waste liquid.Specific embodiments is as follows: cell (expression have fluorescin or by fluorescent dye) is 10 through PBS damping fluid (phosphate buffered saline buffer) dilution
6-10
7individual/milliliter, with micro-injection pump with the flow of 0.2 μ l/min from cell suspending liquid entrance 2 sample introduction, oil phase from oil-continuous phase entrance 1 with the flow sample introduction of 1.5 μ l/min, formed water-in-oil (W/O) drop of about about 50 μm by the shearing action of oil phase, effluent oil phase flows to waste liquid outlet 5 for guiding drop from effluent oil phase entrance 3 with the flow sample introduction of 1.2 μ l/min simultaneously; Stream of liquid droplets is to a point constituency, its signal is caught by fluorimetric detector, and with decision rule comparison, if judge this drop parcel individual cells, touch electromagnetic valve work, and then change the stressed and kinestate of this drop, thus make object droplet flow to single drop collection microtubule from being sorted drop outlets 6, so far, realize the object that single drop is collected, can individual cells be obtained by operations such as follow-up deemulsification, extractions.
The present invention is based on drop microfluidic chip technology, design construction a kind of low cost, high efficiency separation obtain single celled system and method.System of the present invention comprises micro-fluidic chip, drop signal picker, signal analysis and selection control unit, magnetic valve, conduit, drop and collects microtubule.Unicellular sepn process is as follows: the drop on-line continuous of parcel cell generates and directed flow, when flowing through signals collecting district, signals collecting, analysis and selection control are carried out to drop, when meeting separation condition (as: be enclosed with in drop and only have individual cells), Controlling solenoid valve work makes drop strained condition and flows to change, thus be separated and collect single drop, and then realize by the operation such as deemulsification, extraction the object obtaining individual cells.The technological method that the present invention proposes provides a set of simple possible, is applicable to the drop sorting of different detection method, cellular segregation and the single celled system and method for acquisition, obtains single celled method have high, consuming time short, the advantage such as cost is low, pollution-free, applied widely, extensibility is strong of efficiency compared to tradition.
Claims (10)
1. a low cost, high efficiency separation obtain single celled system, it is characterized in that, comprising: micro-fluidic chip (7), drop signal detector (8), signal analysis and selection control unit (9), magnetic valve (10), conduit (11), drop collect microtubule (13);
Described micro-fluidic chip (7) inside comprises microchannel network, for making fluid in the flowing of its fluid channel interior orientation under external force drives, the generation of drop and sorting, and waste liquid, is sorted the derivation of drop;
Described drop signal detector (8) for the optical signalling of acquisition stream through the drop of the detection zone (12) of described micro-fluidic chip (7), and is sent to signal analysis and selection control unit (9);
Described signal analysis and selection control unit (9) are for analyzing the signal from described drop signal detector (8), and the work of Controlling solenoid valve (10);
The selection control instruction that described magnetic valve (10) sends for receiving described signal analysis and selection control unit (9), and perform corresponding drop in the direction of motion of micro-fluidic chip (7) divide selection operation by changing drop;
Described conduit (11) connects described micro-fluidic chip (7) and magnetic valve (10), for deriving the drop of sorting;
Described drop collects one end that microtubule (13) connects described conduit (11), for collecting the dropping liquid that described micro-fluidic chip (7) sub-elects.
2. a kind of low cost according to claim 1, high efficiency separation obtain single celled system, it is characterized in that, described micro-fluidic chip (7) is by the upper and lower substrate key and form, and material all can be plastics, glass or quartz.
3. a kind of low cost according to claim 1 and 2, high efficiency separation obtain single celled system, it is characterized in that, the upper strata of described micro-fluidic chip (7) comprises microchannel network, and the gangway of described microchannel network comprises oil-continuous phase entrance (1), cell suspending liquid entrance (2), effluent oil phase entrance (3), magnetic valve Link Port (4), waste liquid outlet (5) and is sorted drop outlets (6);
After microchannel that the microchannel that described oil-continuous phase entrance (1) is communicated with is communicated with cell suspending liquid entrance (2) is communicated with, then with described effluent oil phase entrance (3), magnetic valve Link Port (4), waste liquid outlet (5) be sorted drop outlets (6) and be communicated with.
4. a kind of low cost according to claim 3, high efficiency separation obtain single celled system, it is characterized in that, described microchannel is that 25-100 micron is wide, and 40-60 micron is dark, and the droplet dia of formation is 30-80 micron.
5. a kind of low cost according to claim 3, high efficiency separation obtain single celled system, it is characterized in that, described in be sorted drop outlets (6) for the microchannel that 200-250 micron is wide, 200-250 micron is dark.
6. a kind of low cost according to claim 1, high efficiency separation obtain single celled system, it is characterized in that, described drop signal detector (8) is any one in high speed camera, fluorimetric detector and Raman spectroscopic detector.
7. a kind of low cost according to claim 1, high efficiency separation obtain single celled system, it is characterized in that, described signal analysis and selection control unit (9) are coupled by the signal analysis software corresponding with described drop signal detector (8) and solenoid control software and hardware and are formed.
8. a kind of low cost according to claim 1, high efficiency separation obtain single celled system, it is characterized in that, described magnetic valve (10) has suction, pushes away the controlled operating mode of two kinds of difference.
9. low cost, high efficiency separation obtain a single celled method, it is characterized in that, comprise the following steps:
The formation of liquid injection and drop: utilize pressure-driven oil phase to enter a microchannel of micro-fluidic chip (7) by oil-continuous phase entrance (1), drive cell suspending liquid aqueous phase to enter another microchannel of micro-fluidic chip (7) by cell suspending liquid entrance (2), by oil phase, water-in-oil microlayer model is formed to the shearing action of aqueous phase in the junction of above-mentioned two microchannels;
Stream of liquid droplets to control: the oil phase introduced from effluent oil phase entrance (3), make water-in-oil microlayer model fluid channel interior orientation flowing, and acquiescence flow into waste liquid outlet (5);
Drop sampling and analysis of signal: when water-in-oil microlayer model flows through drop signal detector (8), the drop signal of water-in-oil microlayer model is by collected and be sent to signal analysis and selection control unit (9), by the sort regular of received signal and setting being compared, judge whether the drop signal gathered meets the condition of described sort regular;
Drop sorting: when the drop signal gathered meets separation condition, signal analysis and selection control unit (9) trigger magnetic valve (10) and work, the power making water-in-oil microlayer model be subject to magnetic valve (10) to apply, and then change its direction of motion, flow to and be sorted drop outlets (6);
The collection of drop and single celled acquisition: the conduit being sorted drop outlets (6) place flows into drop and collects in microtubule (13), realize single drop and collect and obtain unicellular by follow-up deemulsification, extraction operation.
10. a kind of low cost according to claim 9, high efficiency separation obtain single celled method, it is characterized in that, described sort regular is be enclosed with in a drop and only have individual cells.
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