CN104677808A - Pressure sucking-based cell/particle sorting system and method - Google Patents
Pressure sucking-based cell/particle sorting system and method Download PDFInfo
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- CN104677808A CN104677808A CN201310612205.0A CN201310612205A CN104677808A CN 104677808 A CN104677808 A CN 104677808A CN 201310612205 A CN201310612205 A CN 201310612205A CN 104677808 A CN104677808 A CN 104677808A
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Abstract
According to a solenoid valve switch principle comprising that at the solenoid valve opening moment, liquid in a liquid channel is instantly sucked, a pressure sucking-based flowing cell/particle sorting system and method are designed and constructed by effective utilization of sucking effects. The system and method effectively solve the problems of the pressure driving-based cell/particle sorting system and improve sorting accuracy and efficiency. The sorting system comprises a cell signal acquisition and analysis unit, a micro-fluidic chip and a pressure control unit. The continuously-flowing cell/particle flows flow in a micro-fluidic chip micro-channel under the power action, when the cells flow through a detection point of the cell signal acquisition and analysis unit, a cell signal is acquired and determined, and when the specific cells satisfying sorting conditions are detected, the determined signal activates the pressure control unit, and the pressure control unit sucks the specific cells/particles so that the specific cells/particles enter into the cell/particle sorting channel.
Description
Technical field
The invention belongs to biotechnology and instrumental science field, the principle that utilizes pressure to suck specifically builds the system and method realizing specific cells/particle under flow state and be separated with colony.
Background technology
Cell is the base unit of organism structure and function, to the research of unicellular individual difference mechanism, can disclose pathogenesis of cancer mechanism, understands Cell Differentiation and tissue development principle, identifies gene expression characteristics and cell characteristic.
Quick identification, separation obtain the unicellular gordian technique becoming single cell analysis of specific function phenotype.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 fluorescence labeling is not suitable for active somatic cell screening.Realize the physical separation method relative complex of its cell in addition.Single cell Raman spectrum can provide the bulk informations such as intracellular nucleic acid, protein, lipid content, the change of cellular elements structure can be monitored real-time dynamicly under cold condition, also can obtain " molecular fingerprint " of cell, there is high, the real-time detection of susceptibility, sample alive does not need numerous features such as fixing or dyeing, not damaging cells.When it is combined with microflow control technique, Raman spectrum can be realized and activate Living single cell sorting.
The method of the driving cell sorting/separation realized on micro-fluidic chip platform at present comprises: electric field force, dielectric power, light tweezer, pressure-driven etc.Pressure-driven comprises pulse laser and produces bubble promotion, the promotion of electric heating generation bubble, and these are all depart from flow pattern based on pressure promotion cell/particle to enter sort channel.Also has the switch by solenoid control ambient pressure in addition, realization treats that sorting cells/particle deflection flow pattern enters sort channel, this method is relatively simple, be easy to realize, but adopt this utilize solenoid valve to open there is a defect that cannot overcome in method that rear UNICOM ambient pressure realizes the deflection of cell/particle, namely when solenoid valve open in a flash can the flow pattern of disturbance fluid in microchannel to the effect of sucking of liquid, especially accurately control folder stream thus realize the unicellular application flowed through in the cell/particle sorting system of check point to be restricted at needs.
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 cell/particle sorting system of sucking based on pressure and method.
The technical scheme that the present invention is adopted for achieving the above object is: a kind of cell/particle sorting system sucked based on pressure, it is characterized in that, comprising: selection control device, pressure control unit, solenoid valve, liquid conduits, cell/particle signal detecting device, micro-fluidic chip;
Described cell/particle signal detecting device, connects the sorting cells/particle outlet of micro-fluidic chip, for the cell/particle signal of acquisition stream through this outlet;
Described selection control device, connect cell/particle signal detecting device, for the sort regular of gathered cell/particle signal and setting is compared, judge whether the cell/particle signal gathered meets separation condition, and result of determination is sent to pressure control unit;
Described pressure control unit, for the unlatching according to judged result Controlling solenoid valve;
Described solenoid valve, connects the sorting cells/particle outlet of micro-fluidic chip by liquid conduits;
Micro-fluidic chip, flows at microchannel interior orientation under external force drives for cell/particle suspension.
Described cell/particle signal collection analysis unit is that wherein one or several detecting devices and corresponding control software design are formed by high speed camera, fluorescence detector, Raman spectroscopic detector and impedance detector etc.
Described micro-fluidic chip is by upper and lower two-layer key and form, and forms the microchannel being used for fluid and passing through; Described microchannel interface comprises folder stream buffer inlet, cell suspension entrance, waste liquid outlet, sorting cells/particle outlet; Lower floor is provided with electrode pair, and distance is 15-30 micron.
Described microchannel is that 20-150 micron is wide, the microchannel network that 10-50 micron is dark.
The material of described micro-fluidic chip is plastics, glass or quartz.
Described pressure control unit comprises: pressure source, micro liquid solenoid valve, pressure control unit, liquid connect microtubular.
Described pressure control unit is used for the switch of Controlling solenoid valve, described pressure source connected electromagnetic valve entrance, and electromagnetic valve outlet is connected with the connector of described micro-fluidic chip by liquid connecting duct.
Described pressure source is static pressure or low pressure gas cylinder.
Based on cell/particle sorting method that pressure is sucked, comprise the following steps:
Liquid injects: pressure-driven cell suspension and folder stream damping fluid flow in microchannel through cell suspension entrance and folder stream buffer inlet respectively, regulate the flow velocity of cell and damping fluid, realizes stable unicellular folder stream;
Cell/particle signal collection and differentiation: when unicellular/grain flow is through cell/particle signal detecting device, the sort regular of gathered cell/particle signal and setting is compared, judge whether the cell/particle signal gathered meets separation condition, and result of determination is sent to pressure control unit;
The pressure of cell/particle sucks sorting: when the result of determination received in pressure control unit is "Yes", pressure control unit produces high level opens solenoid valve, produce one in the moment that solenoid valve is opened and suck effect, cell/particle is sucked sorting cells/particle outlet.
Described solenoid valve opens within rear 20-30 millisecond and automatically cuts out.
The present invention has the following advantages and beneficial effect:
1. the present invention effectively utilizes the principle of electromagnetic valve switch, the system and method that design utilizes pressure to suck to realize specific cells/particle to be separated with colony.
2. technical method provided by the invention can effectively solve because pressure-driven sorting is to the problem of flow pattern interference in microfluidic channel, provides a set of simple possible, is applicable to the pressure-driven cell/particle sorting system of different detection method and method.
Accompanying drawing explanation
Fig. 1 is the cell/grain sorting micro-fluidic chip schematic diagram sucked based on pressure;
Wherein, 1, folder stream buffer inlet; 2, cell suspension entrance; 3, waste liquid outlet; 4, sorting cells/particle outlet, for being connected with solenoid valve 9 through liquid conduits 10; 5,6, electrode pair, exports with high frequency ac signal generator and is connected;
Fig. 2 is system architecture schematic diagram of the present invention,
Wherein, 7, selection control device; 8, pressure control unit; 9, solenoid valve; 10, liquid conduits; 11, cell/particle signal detecting device; 12, micro-fluidic chip.
Embodiment
Below in conjunction with drawings and Examples, the present invention is described in further detail.
Embodiment 1: suck Raman based on pressure and activate unicellular sorting
As shown in Figure 1, 2, the quartzy micro-fluidic chip constructed by this example is used for sucking Raman based on pressure under flow state and activates unicellular sorting, and its chip structure is shown in Fig. 1, and comprising 1, accurate Micropump or static pressure drive folder stream buffer inlet; 2, accurate Micropump or static pressure drive cell suspension entrance; 3, waste liquid outlet; 4, sorting cells/particle outlet, for being connected with solenoid valve 9 through liquid conduits 10; 5,6, electrode pair, exports with high frequency ac signal generator and is connected.Chip is made up of upper and lower two-layer quartz substrate, cover plate comprises 20-150 micron wide, and the dark microchannel network of 10-50 micron adopts wet etching and obtain, and on lower floor's quartz substrate, microelectrode structure adopts photoetching and electrode etch liquid corrosion quarter and obtains.Upper and lower quartz substrate, through cleaning, is accurately aimed under the microscope, then heating in vacuum pressurization bonding.Electrode pair 5,6 is connected with signal generator by wire.Chip outlet is connected with the external world with microtubule by fixed interface with entrance, the importing realizing cell/particle be separated derivation.Chip described in this example and correlation technique are sucked Raman based on pressure under realizing flow state and are activated unicellular sorting.Yeast cells is 106-107/milliliter through the dilution of PBS damping fluid, promote to flow with 0.1-100 mm/second speed through static pressure or micro-injection pump, enabling signal generator periodically produces 5-12 volt, frequency is the high-frequency voltage signal of 0.5 million to 10 megahertz, when yeast cells flows through electrode pair 5,6, under dielectric effect, catch cell instantaneously, start selection control device 7 and cell/particle signal detecting device 11 start to gather yeast cells raman spectral signal and with decision rule comparison.When cell meets separation condition, activation pressure control module 10 makes solenoid valve 9 complete rapidly opening/closing action, treat the sucked cell collection passage entering sorting cells/particle outlet 4 place of sorting cells, the cell do not satisfied condition directly is flowed in waste liquid pool by waste liquid outlet 3.
Embodiment 2: the fluorecyte sorting of sucking based on pressure
Micro-fluidic chip constructed by this example is used for the fluorecyte sorting of sucking based on pressure under flow state, and it is identical that its chip structure comprises cell suspension entrance 2, folder stream buffer inlet 1, waste liquid outlet 3, sorting cells/particle outlet 4 and pressure deflection control mouth.Micro-fluidic chip 12 is made up of upper and lower two-layer matrix, and it is wide that upper substrate comprises 20-150 micron, the microchannel network that 10-50 micron is dark, and lower floor is cover plate.Upper subtegulum, through cleaning, is accurately aimed at, then bonding under the microscope.Chip outlet is connected with the external world with microtubule by fixed interface with entrance, the importing realizing cell/particle be separated derivation.Micro-fluidic chip 12 described in this example and correlation technique are sucked Raman based on pressure under realizing flow state and are activated unicellular sorting.Cell (expression has fluorescin) is 106-107/milliliter through the dilution of PBS damping fluid, promote through static pressure or micro-injection pump with 0.1-100 mm/second speed extraining sampling, its signal is caught by fluorescence detector, and with decision rule comparison, when cell meets separation condition, activation pressure control module 8, solenoid valve 9 is made to complete rapidly opening/closing action, treat the sucked cell collection passage entering sorting cells/particle outlet 4 place of sorting cells, the cell do not satisfied condition directly is flowed in waste liquid pool by waste liquid outlet 3.
Embodiment 3: the microlayer model sorting of sucking based on pressure
Micro-fluidic chip 12 constructed by this example and method suck the sorting of fluorescence microlayer model based on pressure under being used for flow state, and its chip structure comprises, and 1, oil phase entrance (folder stream buffer inlet); 2, droplet inlet (cell suspension entrance); 3, waste liquid outlet; 4, sorting drop outlets (sorting cells/particle outlet), it is identical that drop acquisition port and pressure deflection control mouth.Chip is made up of upper and lower two-layer matrix, and it is wide that upper substrate comprises 20-150 micron, the microchannel network that 10-50 micron is dark, and lower floor is cover plate.Upper subtegulum, through cleaning, is accurately aimed at, then bonding under the microscope.Chip outlet is connected with the external world with microtubule by fixed interface with entrance, the importing realizing cell/particle be separated derivation.Chip described in this example and correlation technique suck the sorting of fluorescence microlayer model based on pressure under realizing flow state.Cell is 106-107/milliliter through the dilution of PBS damping fluid, promote to flow with 0.1-100 mm/second speed through static pressure or micro-injection pump, the mineral oil being added with surfactant promotes admission passage through static pressure or micro-injection pump, form the microlayer model of cell encapsulation, enabling signal detects (detects fluorescence, light field cell growth state), and with decision rule comparison, when drop meets separation condition, activation pressure control module, solenoid valve completes rapidly opening/closing action, treat that sorting drop is sucked and enter drop acquisition channel, the drop do not satisfied condition flows directly in waste liquid pool.
The present invention utilizes electromagnetic valve switch principle, what namely the liquid of solenoid valve in the moment of opening in flow path had a moment sucks effect, and in traditional separation system promoted based on pressure, this effect of sucking can disturb flow pattern, affect the screening accuracy of cell/particle, and effectively utilize in the present invention and suck effect, cell/particle sorting system and method under a kind of flow state of sucking based on pressure of design construction, efficiently solve the problem in pressure-driven sorting cells/particle system, and improve sorting accuracy and the efficiency of separation.Described separation system comprises cell signal collection analysis unit, micro-fluidic chip and pressure control unit; Cell/the grain flow of continuous flow carries under power drive in micro-fluidic chip microchannel and flows, when stream of cells is through cell signal collection analysis unit inspection point, the collected judgement of cell signal, when the specific cells meeting separation condition is detected, specific cells/particle is sucked and is entered cell/grain sorting passage by judgment signal activation pressure control module.
Claims (9)
1. cell/the particle sorting system sucked based on pressure, it is characterized in that, comprising: selection control device (7), pressure control unit (8), solenoid valve (9), liquid conduits (10), cell/particle signal detecting device (11), micro-fluidic chip (12);
Described cell/particle signal detecting device (11), connects the sorting cells/particle outlet (4) of micro-fluidic chip (12), for the cell/particle signal of acquisition stream through this outlet;
Described selection control device (7), connect cell/particle signal detecting device (11), for the sort regular of gathered cell/particle signal and setting is compared, judge whether the cell/particle signal gathered meets separation condition, and result of determination is sent to pressure control unit (8);
Described pressure control unit (8), for the unlatching according to judged result Controlling solenoid valve (9);
Described solenoid valve (9), connects the sorting cells/particle outlet (4) of micro-fluidic chip (12) by liquid conduits (10);
Micro-fluidic chip (12), flows at microchannel interior orientation under external force drives for cell/particle suspension.
2. a kind of cell/particle sorting system sucked based on pressure according to claim 1, it is characterized in that, described cell/particle signal collection analysis unit is that wherein one or several detecting devices and corresponding control software design are formed by high speed camera, fluorescence detector, Raman spectroscopic detector and impedance detector etc.
3. a kind of cell/particle sorting system sucked based on pressure according to claim 1, is characterized in that, described micro-fluidic chip is by upper and lower two-layer key and form, and forms the microchannel being used for fluid and passing through; Described microchannel interface comprises folder stream buffer inlet (1), cell suspension entrance (2), waste liquid outlet (3), sorting cells/particle outlet (4); Lower floor is provided with electrode pair (5,6), and distance is 15-30 micron.
4. a kind of cell/particle sorting system sucked based on pressure according to claim 3, it is characterized in that, described microchannel is that 20-150 micron is wide, the microchannel network that 10-50 micron is dark.
5. a kind of cell/particle sorting system sucked based on pressure according to claim 1 or 3, is characterized in that, the material of described micro-fluidic chip is plastics, glass or quartz.
6. a kind of cell/particle sorting system sucked based on pressure according to claim 1, it is characterized in that, described pressure control unit comprises: pressure source, micro liquid solenoid valve, pressure control unit, liquid connect microtubular.
Described pressure control unit is used for the switch of Controlling solenoid valve, described pressure source connected electromagnetic valve entrance, and electromagnetic valve outlet is connected with the connector of described micro-fluidic chip by liquid connecting duct.
7. a kind of cell/particle sorting system sucked based on pressure according to claim 6, it is characterized in that, described pressure source is static pressure or low pressure gas cylinder.
8., based on cell/particle sorting method that pressure is sucked, it is characterized in that, comprise the following steps:
Liquid injects: pressure-driven cell suspension and folder stream damping fluid flow in microchannel through cell suspension entrance (2) and folder stream buffer inlet (1) respectively, regulate the flow velocity of cell and damping fluid, realizes stable unicellular folder stream;
Cell/particle signal collection and differentiation: when unicellular/grain flow is through cell/particle signal detecting device (11), the sort regular of gathered cell/particle signal and setting is compared, judge whether the cell/particle signal gathered meets separation condition, and result of determination is sent to pressure control unit (8);
The pressure of cell/particle sucks sorting: when the result of determination received in pressure control unit (8) is "Yes", pressure control unit (8) produces high level opens solenoid valve (9), produce one in the moment that solenoid valve (9) is opened and suck effect, cell/particle is sucked sorting cells/particle outlet (4).
9. a kind of cell/particle sorting method sucked based on pressure according to claim 8, is characterized in that, described solenoid valve (9) opens within rear 20-30 millisecond and automatically cuts out.
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| CN105238676A (en) * | 2015-10-15 | 2016-01-13 | 清华大学深圳研究生院 | Microfluidic chip for cell printing |
| CN106644900A (en) * | 2017-02-27 | 2017-05-10 | 大连海事大学 | Pulse impedance particle counting device based on non-uniform electric field and particle counting method |
| CN108369224A (en) * | 2015-10-02 | 2018-08-03 | 小利兰·斯坦福大学托管委员会 | It is sorted using the biology and abiological constitution of magnetic suspension |
| WO2019080905A1 (en) * | 2017-10-25 | 2019-05-02 | 中国科学院青岛生物能源与过程研究所 | Raman activated droplet sorting system and method |
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| CN111266140A (en) * | 2020-03-09 | 2020-06-12 | 厦门大学 | Oil-free sorting-direct injection-ICPMS single cell analysis system |
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| CN114149893A (en) * | 2021-11-23 | 2022-03-08 | 中国科学院青岛生物能源与过程研究所 | Particle self-clamping flow type micro-fluidic chip, manufacturing method thereof and particle self-dispersion method |
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