CN104677877A - Micro-fluidic chip and method for capturing and collecting Raman spectra of cells/particles - Google Patents
Micro-fluidic chip and method for capturing and collecting Raman spectra of cells/particles Download PDFInfo
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- CN104677877A CN104677877A CN201310613921.0A CN201310613921A CN104677877A CN 104677877 A CN104677877 A CN 104677877A CN 201310613921 A CN201310613921 A CN 201310613921A CN 104677877 A CN104677877 A CN 104677877A
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Abstract
The invention discloses a novel quartz micro-fluidic chip and method for capturing/releasing and continuously collecting the Raman spectra of the cells/particles through dielectric single cells under the flow condition. On the basis of the principle of dielectric capturing of the cells/particles, an electrode couple is integrated on a micro channel of the micro-fluidic chip, and high-frequency voltage signals are exerted periodically, so that the single cells/particles are captured/released; in the capturing period, Raman spectrum signals of the cells/particles are collected. Dielectric single cells can be captured and operated conveniently, the response is rapid, the cells cannot be damaged, and the micro-fluidic chip and the method are suitable for collecting the Raman spectra of the cells and the particles with the size scale from nano to micron under the flow condition and particularly suitable for Raman cell/particle flow sorting.
Description
Technical field
The invention belongs to biotechnology and instrumental science field, make use of the principle that cell/particle can be captured in non-uniform electric field specifically and propose a kind of micro-fluidic chip and method, the raman spectral signal of cell or particle can be measured under current system.
Background technology
Cell is the base unit of organism structure and function, and research method new since being found continues to bring out.But traditional information based on the measurement of cell colony levelness truly can not reflect bioprocess and the mechanism of cell interior.To the research of unicellular individual difference mechanism, pathogenesis of cancer mechanism can be disclosed, understand Cell Differentiation and tissue development principle, identify gene expression characteristics and cell characteristic.
Existing unicellular investigative technique is mainly based on grand genome Manganic pyrophosphate complex initiation and Fluorescence Activated Cell sorting.The former is the Main Means of research gene expression and uncultured microorganisms, but significantly relies on DNA sequencing analysis, not only directly can not disclose Gene Expression Mechanism, and can not be separated the microorganism with specific function gene.The latter 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.Therefore, only having by realizing proterties identification and sorting in Living single cell level, just can really understand and improve cell function.
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.But by the relatively low impact of Raman spectrum sensitivity, the acquisition time of single cell Raman spectrum usually second to minute the order of magnitude.Researchist adopts the technology of laser raman surface enhanced in the sensitivity improving Raman spectrum, but due to cell relative complex, find the SERS technology of energy stable application to cell Raman detection so far yet, and collection and the sorting that cell realizes Raman spectrum under flow state will be realized.
It is a kind of that to catch/discharge the unicellular and technology gathering Raman spectrum under current system be realize prerequisite based on cell/particle Raman spectrum flow sort and gordian technique.The technology the most often adopted is at present that Raman light tweezer is caught unicellular, but the method often needs complicated light path system, is not easy to realize; And long-time illumination can affect cell physiological state, especially will cause diverse measurement result to the cell of photosynthesis sensitivity.
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 micro-fluidic chip and method of continuous acquisition cell/particle Raman spectrum under flow state.
The technical scheme that the present invention is adopted for achieving the above object is: a kind of micro-fluidic chip of catching collection cell/particle Raman spectrum, comprises two-layer up and down, forms by aiming at sealing-in the microchannel being used for fluid and passing through; Described upper strata is the cover plate that surface has microchannel, and described lower floor is the substrate with electrode;
Described electrode is an electrode pair, is connected with high frequency signal generator by wire;
Described microchannel, is connected with buffer container with the sample in the external world by interface.
Described microchannel is that 20-150 micron is wide, the microchannel network that 10-50 micron is dark.
Described microchannel interface comprises folder stream buffer inlet, cell suspension entrance, waste liquid outlet.
The spacing of described electrode pair is 15-30 micron.
The material of described cover plate and substrate is quartz.
Catch a method for collection cell/particle Raman spectrum, comprise the following steps:
Liquid injects: miniflow syringe pump drives cell suspension and folder stream damping fluid to flow in microchannel through cell suspension entrance and folder stream buffer inlet respectively, regulates the flow velocity of cell and damping fluid, realizes stable unicellular folder stream;
Cell/particle dielectric is caught/is discharged: on electrode pair, apply high-frequency voltage signal, makes cell/particle capture to electrode zone;
Raman spectrum gathers: aim at the electrode zone capturing cell/particle with raman laser, and gather the Raman spectrum of cell/particle, cell/particle capture time and Raman acquisition time are by executing alive time controling
Described high-frequency voltage signal is 5-12 volt, and frequency is the voltage signal of 0.5 million to 10 megahertz.
The present invention has the following advantages and beneficial effect:
1. utilize the principle that cell/particle dielectric is caught, Integrated electrode pair on micro-fluidic chip microchannel, and periodically apply high-frequency voltage signal, catch/discharge unicellular/particle, between trapping period, gather its raman spectral signal.
2. dielectric is unicellular catches easy to operate, and response is rapidly, harmless to cell, is applicable to the collection of cell from nanometer to micro-meter scale and the Raman spectrum of particle under flow state, especially Raman cell/particle airflow classification.
Accompanying drawing explanation
Fig. 1 is the micro-fluidic chip schematic diagram of continuous acquisition cell/particle Raman spectrum under state;
Wherein, 1, folder stream buffer inlet; 2, cell suspension entrance; 3, waste liquid outlet; 4,5 electrode pairs, export with high frequency ac signal generator and are connected;
Fig. 2 is that schematic diagram is caught/discharged to unicellular dielectric;
Fig. 3 is that yeast single cell dielectric catches collection Raman spectrogram.
Embodiment
Below in conjunction with drawings and Examples, the present invention is described in further detail.
Concrete summary of the invention of the present invention is as follows: 1, and in order to reduce the Raman background of chip to the impact of cell/particle sizing, chip material involved in the present invention is quartzy material; 2, this chip is made up of two-layer up and down, and upper strata is wide for being processed with 20-150 micron, the quartzy cover plate of the microchannel network that 10-50 micron is dark, and lower floor is for being processed with electrode structure, and its electrode material is the quartz substrate of metal or other conductive materials; 3, upper and lower two-layer quartz substrate is accurately aimed at sealing-in and is become one piece of micro-fluidic chip, and electrode structure is exposed in microchannel; Chip is connected with buffer container with the sample in the external world by interface; 4, the electrode pair on chip is connected with signal generator by wire.5, continuous acquisition cell/particle Raman optical spectrum method under flow state according to claim 1, it is characterized in that: when electrode structure on chip of cell or grain flow, operation signal generator produces 5-24 volt manually or automatically, frequency is the voltage signal of 0.5 million to 10 megahertz, cell is accurately captured in electrode zone, raman laser hot spot is accurately aimed at capture electrode, start the Raman spectrum gathering cell/particle, cell capture time and Raman acquisition time are by executing alive time controling.Cell can gather enough Raman signals and differentiate for cell between trapping period, and provides foundation for sorting.
The following examples will be further described the present invention, but not thereby limiting the invention.
Embodiment 1: the quartzy micro-fluidic chip of continuous acquisition cell/particle Raman spectrum under flow state
Quartzy micro-fluidic chip constructed by this example is for continuous acquisition cell/particle Raman spectrum under flow state, and its chip structure is shown in Fig. 1.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.Chip electrode is connected with signal generator by wire.
Embodiment 2: under flow state, yeast cells Raman spectrum gathers
This example adopts chip described in example 1 and correlation technique to realize the unicellular collection of catching Raman spectrum of yeast cells under flow state.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, under dielectric effect, catch cell instantaneously, start Raman spectrum capture program and start to gather yeast cells raman spectral signal 1 second, acquisition time can require to adjust by basis signal.After completing the collection of cell Raman spectrum, stop voltage applying, cell discharges.Enter next cell capture Raman spectrum collection period again.Raman spectrogram is caught and gathered to yeast cells as shown in Figures 2 and 3.
Claims (7)
1. catching a micro-fluidic chip for collection cell/particle Raman spectrum, it is characterized in that, comprise two-layer up and down, forming by aiming at sealing-in the microchannel being used for fluid and passing through; Described upper strata is the cover plate that surface has microchannel, and described lower floor is the substrate with electrode;
Described electrode is an electrode pair (4,5), is connected with high frequency signal generator by wire;
Described microchannel, is connected with buffer container with the sample in the external world by interface.
2. a kind of micro-fluidic chip of catching collection cell/particle Raman spectrum according to claim 1, it is characterized in that, described microchannel is that 20-150 micron is wide, the microchannel network that 10-50 micron is dark.
3. a kind of micro-fluidic chip of catching collection cell/particle Raman spectrum according to claim 1 and 2, is characterized in that, described microchannel interface comprises folder stream buffer inlet (1), cell suspension entrance (2), waste liquid outlet (3).
4. a kind of micro-fluidic chip of catching collection cell/particle Raman spectrum according to claim 1, is characterized in that, the spacing of described electrode pair is 15-30 micron.
5. a kind of micro-fluidic chip of catching collection cell/particle Raman spectrum according to claim 1, is characterized in that, the material of described cover plate and substrate is quartz.
6. catch a method for collection cell/particle Raman spectrum, it is characterized in that, comprise the following steps:
Liquid injects: miniflow syringe pump drives cell suspension and folder stream damping fluid to flow in microchannel through cell suspension entrance (2) and folder stream buffer inlet (1) respectively, regulates the flow velocity of cell and damping fluid, realizes stable unicellular folder stream;
Cell/particle dielectric is caught/is discharged: on electrode pair (4,5), apply high-frequency voltage signal, make cell/particle capture to electrode zone;
Raman spectrum gathers: aim at the electrode zone capturing cell/particle with raman laser, and gather the Raman spectrum of cell/particle, cell/particle capture time and Raman acquisition time are by executing alive time controling.
7. a kind of method of catching collection cell/particle Raman spectrum according to claim 6, it is characterized in that, described high-frequency voltage signal is 5-12 volt, and frequency is the voltage signal of 0.5 million to 10 megahertz.
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| CN105647799A (en) * | 2016-02-24 | 2016-06-08 | 西安交通大学 | Circulating tumor cell separation device adopting combined field flow separation |
| CN108291859A (en) * | 2015-08-18 | 2018-07-17 | 财团法人卫生研究院 | Microfluid dynamical type for high-throughput most unicellular captures transports chip |
| CN108728328A (en) * | 2018-05-30 | 2018-11-02 | 中北大学 | The micro-current controlled cell for integrating unicellular capture sorts chip |
| 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 |
| WO2021208872A1 (en) * | 2020-04-13 | 2021-10-21 | 中国科学院青岛生物能源与过程研究所 | Electrohydrodynamics-based microfluidic chip, micro sample application device and method |
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| WO2021208872A1 (en) * | 2020-04-13 | 2021-10-21 | 中国科学院青岛生物能源与过程研究所 | Electrohydrodynamics-based microfluidic chip, micro sample application device and method |
| CN113522378A (en) * | 2020-04-13 | 2021-10-22 | 中国科学院青岛生物能源与过程研究所 | Microfluidic chip based on electrohydrodynamics, micro sample application device and method |
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