CN104677877B - A kind of micro-fluidic chip and method for capturing collection cell/particle Raman spectrum - Google Patents
A kind of micro-fluidic chip and method for capturing collection cell/particle Raman spectrum Download PDFInfo
- Publication number
- CN104677877B CN104677877B CN201310613921.0A CN201310613921A CN104677877B CN 104677877 B CN104677877 B CN 104677877B CN 201310613921 A CN201310613921 A CN 201310613921A CN 104677877 B CN104677877 B CN 104677877B
- Authority
- CN
- China
- Prior art keywords
- cell
- raman spectrum
- microchannel
- micro
- particle
- 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
Abstract
The present invention constructs the quartzy micro-fluidic chip and method for devising and the unicellular capture of dielectric/release continuous acquisition cell/particle Raman spectrum being used under a kind of new flow regime.It is the Integrated electrode pair on micro-fluidic chip microchannel using the principle of cell/particle dielectric capture, and periodically applies high-frequency voltage signal, capture/release is unicellular/particle, and its raman spectral signal is gathered during capture.The unicellular capture of dielectric is easy to operate, and response is rapid, harmless to cell, suitable for the collection of the Raman spectrum of the cell from nanometer to micro-meter scale and particle under flow regime, especially Raman cell/particle airflow classification.
Description
Technical field
The invention belongs to biotechnology and instrumental science field, specifically make use of cell/particle in uneven electricity
The principle that can be captured in proposes a kind of micro-fluidic chip and method, and the drawing of cell or particle can be determined under current system
Graceful spectral signal.
Background technology
Cell is organism structure and the base unit of function, and new research method continues to bring out since being found.But
It is the bioprocess and mechanism that traditional information based on the measurement of cell colony levelness can not truly reflect cell interior.
Research to unicellular individual difference mechanism, can reveal that pathogenesis of cancer mechanism, understand cell differentiation and tissue development principle,
Identify gene expression characteristics and cell characteristic.
Existing unicellular investigative technique is based primarily upon grand genome pyrosequencing and fluorescence activated cell sorts.The former is
The Main Means of gene expression and uncultured microorganisms are studied, but significantly rely on DNA sequencing analysis, not only can not directly be disclosed
Gene Expression Mechanism, and the microorganism with specific function gene can not be separated.The latter is a point cellifugal effective means, so
And because most active somatic cells fluorescent effects itself is weaker or is not suitable for active somatic cell without fluorescence, additional fluorescence labeling
Screening.Therefore, only it just can really understand by realizing character identification and sorting in Living single cell level and improve cell
Function.
Single cell Raman spectrum can provide the bulk informations such as intracellular nucleic acid, protein, lipid content, can be in non-marked
Under the conditions of real-time dynamicly monitor cellular elements structure change, can also obtain " molecular fingerprint " of cell, have sensitiveness high, real
When detection, sample living be not required to fixed or dye, not numerous features such as damaging cells.It is but relatively low by Raman spectral sensitivity
Influence, the acquisition time of single cell Raman spectrum is generally in the order of magnitude of second to minute.Researcher uses LR laser raman table
The technology of face enhancing is improving the sensitivity of Raman spectrum, but because cell is relative complex, not finding yet so far can be stable
The SERS technologies of cell Raman detection are applied to, and to realize cell and the collection of Raman spectrum is realized under flow regime and is divided
Choosing.
It is a kind of under current system can capture/release it is unicellular and gather the technology of Raman spectrum be realize based on cell/
The premise and key technology of particle Raman spectrum flow sort.Commonly used technology is that the capture of Raman optical tweezer is unicellular at present,
But this method generally requires the light path system of complexity, is not easy to realize；And long-time illumination can influence cell physiological state, especially
Its cell sensitive to photosynthesis will cause entirely different measurement result.
The content of the invention
For in place of above shortcomings in the prior art, the technical problem to be solved in the present invention is to provide one kind to flow
The micro-fluidic chip and method of continuous acquisition cell/particle Raman spectrum under dynamic state.
The used to achieve the above object technical scheme of the present invention is：One kind capture collection cell/particle Raman spectrum
Micro-fluidic chip, the microchannel passed through for fluid is formed by being directed at sealing-in including upper and lower two layers；The upper strata is surface
Cover plate with microchannel, the lower floor are the substrate with electrode；
The electrode is an electrode pair, is connected by wire with HF signal generator；
The microchannel, it is connected by interface with the sample and buffer container in the external world.
The microchannel is that 20-150 microns are wide, the deep microchannel network of 10-50 microns.
The microchannel interface includes folder stream buffer inlet, cell suspension entrance, waste liquid outlet.
Distance is 15-30 microns between the electrode pair.
The material of the cover plate and substrate is quartz.
A kind of method for capturing collection cell/particle Raman spectrum, comprises the following steps：
Liquid injects：Miniflow syringe pump drives cell suspension and folder stream buffer solution slow through cell suspension entrance and folder stream respectively
Fliud flushing entrance flows in microchannel, adjusts the flow velocity of cell and buffer solution, realizes stable unicellular folder stream；
Cell/particle dielectric capture/release：Apply high-frequency voltage signal in electrode pair, make cell/particle capture to electricity
Polar region domain；
Raman spectrum gathers：The electrode zone of cell/particle is captured with raman laser alignment, collection cell/particle
Raman spectrum, cell/particle capture time and Raman acquisition time are by applying alive time control
The high-frequency voltage signal is 5-12 volts, and frequency is 0.5 million to 10 megahertz of voltage signal.
The present invention has advantages below and beneficial effect：
1. using the principle of cell/particle dielectric capture, the Integrated electrode pair on micro-fluidic chip microchannel, and periodically
Ground applies high-frequency voltage signal, and capture/release is unicellular/particle, its raman spectral signal is gathered during capture.
2. the unicellular capture of dielectric is easy to operate, response is rapid, harmless to cell, suitable for from nanometer to micro-meter scale
Raman spectrum under flow regime of cell and particle collection, especially Raman cell/particle airflow classification.
Brief description of the drawings
Fig. 1 is the micro-fluidic chip schematic diagram of continuous acquisition cell/particle Raman spectrum under dynamic state；
Wherein, 1, folder stream buffer inlet；2nd, cell suspension entrance；3rd, waste liquid outlet；4th, 5 electrode pair, with high-frequency ac
Signal generator output is connected；
Fig. 2 be unicellular dielectric capture/discharge schematic diagram；
Fig. 3 is yeast single cell dielectric capture collection Raman spectrogram.
Embodiment
Below in conjunction with the accompanying drawings and embodiment the present invention is described in further detail.
The specific content of the invention of the present invention is as follows：1, in order to reduce shadow of the Raman background of chip to cell/particle sizing
Ring, chip material involved in the present invention is quartz material；2, the chip is formed by upper and lower two layers, and upper strata is to be machined with 20-
150 microns wide, the quartzy cover plate of the deep microchannel network of 10-50 microns, to be machined with electrode structure, its electrode material is for lower floor
The quartz substrate of metal or other conductive materials；3, upper and lower two layers of quartz substrate is accurately directed at sealing-in into one piece of micro-fluidic core
Piece, electrode structure are exposed in microchannel；Chip is connected by interface with the sample and buffer container in the external world；4, on chip
Electrode pair is connected by wire with signal generator.5, continuous acquisition cell under flow regime according to claim 1/
Grain Raman optical spectrum method, it is characterised in that：When cell or particle flow through the electrode structure on chip, manually or automatically operation is believed
Number generator produces 5-24 volts, and frequency is 0.5 million to 10 megahertz of voltage signal, and cell is accurately captured in electrode district
Domain, raman laser hot spot are accurately aligned with capture electrode, start gather cell/particle Raman spectrum, the cell capture time and
Raman acquisition time is by applying alive time control.Cell can gather enough Raman signals during capture and be used for cell
Differentiate, and foundation is provided for sorting.
The following examples will be further described to 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 regime
Quartzy micro-fluidic chip constructed by this example is used for continuous acquisition cell/particle Raman spectrum under flow regime,
Its chip structure is shown in Fig. 1.Chip is made up of upper and lower two layers of quartz substrate, wide including 20-150 microns on cover plate, and 10-50 microns are deep
Microchannel network using wet etching and obtain, on lower floor's quartz substrate microelectrode structure using photoetching and electrode etch liquid etching
And obtain.Upper and lower quartz substrate is cleaned, under the microscope accurate alignment, then heating in vacuum pressurization bonding.Chip electrode passes through
Wire is connected with signal generator.
Embodiment 2：Yeast cells Raman spectrum gathers under flow regime
This example realizes that the unicellular capture of yeast cells is drawn under flow regime using the chip described in example 1 and correlation technique
The collection of graceful spectrum.Yeast cells is diluted to 106-107/milliliter through PBS, is promoted through static pressure or micro-injection pump
Flowed with 0.1-100 mm/seconds speed, enabling signal generator periodically produces 5-12 volts, and frequency is 0.5 million to 10 megahertz
High-frequency voltage signal hereby, when yeast cells flows through electrode pair, moment capture cell, starts Raman spectrum under dielectric effect
Capture program starts to gather yeast cells raman spectral signal 1 second, and acquisition time can be adjusted with basis signal requirement.Complete
After the collection of cell Raman spectrum, stop voltage and apply, cell release.Enter back into next cell capture Raman spectrum collection week
Phase.Yeast cells captures and gathers Raman spectrogram as shown in Figures 2 and 3.
Claims (4)
1. a kind of micro-fluidic chip for capturing collection cell Raman spectrum, it is characterised in that including upper and lower two layers, sealed by being aligned
Connect the microchannel to be formed and passed through for fluid；The upper strata is the cover plate that surface has microchannel, and the lower floor is with electrode
Substrate；
The electrode is an electrode pair (4,5), is connected by wire with HF signal generator；Distance is between the electrode pair
15-30 microns；
The microchannel, it is connected by interface with the sample and buffer container in the external world；It is slow that the microchannel interface includes folder stream
Fliud flushing entrance (1), cell suspension entrance (2), waste liquid outlet (3).
2. a kind of micro-fluidic chip for capturing collection cell Raman spectrum according to claim 1, it is characterised in that described
Microchannel is that 20-150 microns are wide, the deep microchannel network of 10-50 microns.
3. a kind of micro-fluidic chip for capturing collection cell Raman spectrum according to claim 1, it is characterised in that described
The material of cover plate and substrate is quartz.
A kind of 4. method for capturing collection cell Raman spectrum, it is characterised in that comprise the following steps：
Liquid injects：Miniflow syringe pump drives cell suspension and folder stream buffer solution slow through cell suspension entrance (2) and folder stream respectively
Fliud flushing entrance (1) flows in microchannel, adjusts the flow velocity of cell and buffer solution, realizes stable unicellular folder stream；
Cell dielectric capture/release：Apply high-frequency voltage signal in electrode pair (4,5), make cell capture to electrode zone；Institute
It is 5-12 volts to state high-frequency voltage signal, and frequency is 0.5 million to 10 megahertz of voltage signal；
Raman spectrum gathers：The electrode zone of cell is captured with raman laser alignment, gathers the Raman spectrum of cell, cell is caught
Time and Raman acquisition time are obtained by applying alive time control.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310613921.0A CN104677877B (en) | 2013-11-26 | 2013-11-26 | A kind of micro-fluidic chip and method for capturing collection cell/particle Raman spectrum |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310613921.0A CN104677877B (en) | 2013-11-26 | 2013-11-26 | A kind of micro-fluidic chip and method for capturing collection cell/particle Raman spectrum |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN104677877A CN104677877A (en) | 2015-06-03 |
| CN104677877B true CN104677877B (en) | 2017-11-28 |
Family
ID=53313225
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201310613921.0A Active CN104677877B (en) | 2013-11-26 | 2013-11-26 | A kind of micro-fluidic chip and method for capturing collection cell/particle Raman spectrum |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN104677877B (en) |
Families Citing this family (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP6839173B2 (en) * | 2015-08-18 | 2021-03-03 | ナショナル ヘルス リサーチ インスティテューツＮａｔｉｏｎａｌ Ｈｅａｌｔｈ Ｒｅｓｅａｒｃｈ Ｉｎｓｔｉｔｕｔｅｓ | Fluid Mechanics Shutling Chip Equipment and Methods for Capturing Separated Single Cells |
| CN105647799B (en) * | 2016-02-24 | 2018-08-10 | 西安交通大学 | A kind of circulating tumor cell separator of combined type field flow separation |
| CN109706053B (en) * | 2017-10-25 | 2021-04-23 | 中国科学院青岛生物能源与过程研究所 | Raman activated liquid drop sorting system and method |
| CN108728328B (en) * | 2018-05-30 | 2021-11-16 | 中北大学 | Microfluidic cell sorting chip integrated with single cell capture |
| CN113522378B (en) * | 2020-04-13 | 2023-03-17 | 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 |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1811389A (en) * | 2006-02-10 | 2006-08-02 | 厦门大学 | Micro-fluid control chip with surface enhanced Raman spectral active substrate and producing method thereof |
| CN1878875A (en) * | 2003-09-26 | 2006-12-13 | 英特尔公司 | Methods and device for DNA sequencing using surface enhanced raman scattering (SERS) |
| CN101221168A (en) * | 2008-01-08 | 2008-07-16 | 东南大学 | Microfluidic chip based on microsphere biological detection |
| US7476501B2 (en) * | 2002-03-26 | 2009-01-13 | Intel Corporation | Methods and device for DNA sequencing using surface enhanced raman scattering (SERS) |
| CN102019277A (en) * | 2010-10-29 | 2011-04-20 | 北京惟馨雨生物科技有限公司 | Sorter and sorting method for separating cells and particles |
| CN102788777A (en) * | 2011-05-19 | 2012-11-21 | 北京大学 | Microfluidic surface-enhanced Raman scattering detector and its preparation method and use |
-
2013
- 2013-11-26 CN CN201310613921.0A patent/CN104677877B/en active Active
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US7476501B2 (en) * | 2002-03-26 | 2009-01-13 | Intel Corporation | Methods and device for DNA sequencing using surface enhanced raman scattering (SERS) |
| CN1878875A (en) * | 2003-09-26 | 2006-12-13 | 英特尔公司 | Methods and device for DNA sequencing using surface enhanced raman scattering (SERS) |
| CN1811389A (en) * | 2006-02-10 | 2006-08-02 | 厦门大学 | Micro-fluid control chip with surface enhanced Raman spectral active substrate and producing method thereof |
| CN101221168A (en) * | 2008-01-08 | 2008-07-16 | 东南大学 | Microfluidic chip based on microsphere biological detection |
| CN102019277A (en) * | 2010-10-29 | 2011-04-20 | 北京惟馨雨生物科技有限公司 | Sorter and sorting method for separating cells and particles |
| CN102788777A (en) * | 2011-05-19 | 2012-11-21 | 北京大学 | Microfluidic surface-enhanced Raman scattering detector and its preparation method and use |
Non-Patent Citations (3)
| Title |
|---|
| Microfluidic Device for Electric Field-Driven Single-Cell Capture and Activation;Nicholas M. Toriello et al;《Anal. Chem.》;20051101;第77卷(第21期);第6935-6941页 * |
| Surface-enhanced Raman spectroscopy in living plant using triplex Au-Ag-C core-shell nanoparticles;Aiguo Shen et al;《J. Raman Spectrosc.》;20101007;第42卷;第879-884页 * |
| 阵列式对电极介电电泳芯片及其用于细胞分离富集研究;徐溢等;《高等学校化学学报》;20090531;第30卷;第1.2、1.3、2.1、2.2小节 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN104677877A (en) | 2015-06-03 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN104677877B (en) | A kind of micro-fluidic chip and method for capturing collection cell/particle Raman spectrum | |
| Adan et al. | Flow cytometry: basic principles and applications | |
| JP4047336B2 (en) | Cell sorter chip with gel electrode | |
| CN104941704B (en) | A kind of integrating cell focuses on method and micro mation system thereof with detection | |
| De Ninno et al. | High-throughput label-free characterization of viable, necrotic and apoptotic human lymphoma cells in a coplanar-electrode microfluidic impedance chip | |
| US20200376490A1 (en) | Method for forming and respectively exporting droplet wrapping single particle in micro-fluidic chip | |
| DK1941021T3 (en) | Method and apparatus for characterizing and counting particles, especially biological particles | |
| Kemna et al. | Label-free, high-throughput, electrical detection of cells in droplets | |
| CN104677808A (en) | Pressure sucking-based cell/particle sorting system and method | |
| CN107863286B (en) | A kind of Living single cell lysisin situ and online ionization detection mass spectrometer interface device | |
| Wang et al. | On-chip counting the number and the percentage of CD4+ T lymphocytes | |
| US7088116B1 (en) | Optoelectronic probe | |
| CN107818908B (en) | A kind of difference ion mobility spectrometry and High-Field asymmetric waveform ion mobility spectrometry combination device | |
| CN201548547U (en) | Flow cell analysis device based on microfluidic chip | |
| CN105300943B (en) | A kind of microscope integrated optical circuit system for drop fluorescence detection | |
| CN104877899A (en) | System for rapidly, directly, absolutely and quantitatively detecting microbes based on liquid drop, and method thereof | |
| CN108587902A (en) | Cell sorting devices based on dielectrophoresis and its screening technique | |
| CN102183504B (en) | Microfluidic unicellular active oxygen automatic analyzer | |
| US20170128939A1 (en) | ISOLATION AND DETECTION OF CIRCULATING TUMOR CELLS (CTCs) | |
| Chen et al. | Microfluidic methods for cell separation and subsequent analysis | |
| CN206666547U (en) | A kind of biochip for being used to screen rare cell in positioning and detection blood | |
| CN209652292U (en) | In micro-fluidic chip, analysis of the droplet whether containing particle or capture simple target object device | |
| CN206489050U (en) | A kind of impedance pulse grain count device based on inhomogeneous field | |
| CN110241018A (en) | A kind of cancer cell separation system and method | |
| CN114345428B (en) | Micro-fluidic chip for selecting single cells and detection method |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| TR01 | Transfer of patent right | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20211230 Address after: 266101 Shandong Province, Qingdao city Laoshan District Songling Road No. 189 Patentee after: Qingdao xingsai Biotechnology Co.,Ltd. Address before: 266101 Shandong Province, Qingdao city Laoshan District Songling Road No. 189 Patentee before: QINGDAO INSTITUTE OF BIOENERGY AND BIOPROCESS TECHNOLOGY, CHINESE ACADEMY OF SCIENCES |