CN100497653C - Method for analyzing single cell inclusion based on micro flow-controlled chip - Google Patents
Method for analyzing single cell inclusion based on micro flow-controlled chip Download PDFInfo
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- CN100497653C CN100497653C CNB021447721A CN02144772A CN100497653C CN 100497653 C CN100497653 C CN 100497653C CN B021447721 A CNB021447721 A CN B021447721A CN 02144772 A CN02144772 A CN 02144772A CN 100497653 C CN100497653 C CN 100497653C
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Abstract
The invention is a micro-flow control chip-based single-cell endosome analysis, made on a chip platform. First, exert voltage on cell pool and earth cell waste liquor one, the electric power drives the cells to flow from the cell pool to the cell waste liquor one, observe by microscope, and when the cells reach the exit of the detecting channel, make the electrodes of the two pools suspend; at the same time, exert high voltage on run buffer liquor pool and cell cracking liquor pool, and earth waste liquor pool; in this voltage mode, ensure the cells, the cell cracking liquor, screening agent, and run buffer liquor to enter separating channel in order to complete cracking and separating single cell; and detect the endosome on two ends of the channel. It can make single or parallel analysis on the endosome.
Description
Technical field:
The present invention relates to the analytical procedure of single-cell inclusion, a kind of analytical procedure based on micro-fluidic chip is provided especially.
Background technology:
Traditional analysis of cells inclusion method is a prerequisite with a large amount of cells, obtains the total value of a certain inclusion (such as protein, nucleic acid etc.) content, and the ratio of this value and cell quantity is extrapolated to the content of unicellular interior this material.For the cell mass of the relative homogeneous of character, this method can be accepted, but under the other occasion, such as the commitment of some major disease, only has the component of individual cells to change, and at this moment, traditional method will on average be fallen this special variation.Therefore, the analysis of individual cells aspect helps the early diagnosis of disease.In addition, compare with traditional method, single cell analysis is relatively accurate obtaining of information; Might short unstable intermediate product of detection of active cycle.
From technical standpoint, the analysis of individual cells can be divided into complete single cell analysis and broken single cell analysis.The present invention only relates to a kind of technology in back.Present existing technology comprises single cell gel electrophoresis and capillary electrophoresis in this technology category.The former object more is confined to the analysis of dna damage.The method of capillary electrophoresis technique analysis list cell inclusion is relatively ripe, but it exists operational difficulty, and flux is low, is difficult for shortcomings such as fully automated.
Micro-fluidic chip has advantages such as easy automatization, integrated degree height as a kind of novel analysis platform (having another name called chip lab, micro-total analysis system etc.), and its work aspect the single-cell inclusion analyzing and testing does not launch in the world wide as yet.Migration and the cracking in SDS in bibliographical information [Anal.Chem.1997,69,1564-1568, Paul C.H.Li and D.Jed.Harrison] the tbe buffer liquid of cell in glazing channel are only arranged, but do not finish the work that separates and detect.
Summary of the invention:
The object of the present invention is to provide a kind of is the analytical procedure of the single-cell inclusion of service platform with the micro-fluidic chip, nucleic acid, protein, amino acid, sugar and other material of this method in both can the separate analysis individual cells, also can the parallel parsing above-mentioned substance, this method has suitable flux simultaneously, has overcome the problem of capillary electrophoresis technique length consuming time.
The invention provides a kind of analytical procedure of the single-cell inclusion based on micro-fluidic chip, it is characterized in that; Whole process is carried out on a chip platform:
Chip platform is provided with cell pool, cell waste liquid pool, lysis liquid pool, runtime buffer liquid pool, waste liquid pool and sense channel, cell pool, cell waste liquid pool, lysis liquid pool, buffer pool communicate with the inlet of sense channel respectively, the outlet of sense channel connects waste liquid pool, in the passage in advance depended on pressure be full of screening agent and dyestuff;
At first with cell pool and cell waste liquid pool difference making alive and ground connection, under electrodynamic driving, cell flows to the cell waste liquid pool by cell pool, under microscopic examination, when individual cells arrives the inlet of sense channel, cell pool and cell waste liquid pool electrode are suspended;
Meanwhile runtime buffer liquid pool, lysis liquid pool are increased voltage, waste liquid pool ground connection; Under this kind voltage mode, ensure cell, cell pyrolysis liquid, screening agent, running buffer and be advanced into sense channel, finish the separation of single celled cracking and inclusion;
Two ends at sense channel are detected inclusion;
Wherein said cell concn should be 10
3Individual/mL-10
6Between individual/mL;
Unicellular cell pyrolysis liquid concentration should be between 0.01%-1%;
The screening agent concentration should be between 0.15%-10%.
Cell is preferably selected the various cells that do not have cell walls for use among the present invention.Cell pyrolysis liquid can be selected any known lysate for use, as sodium laurylsulfonate, sodium lauryl sulphate, Trombovar, Dodecyl trimethyl ammonium chloride, ten dimethyl trimethylammonium bromides, polyoxyethylene-23-lauryl ether (Brij35), Octyl glucoside, Triton X-100 etc.), the screening agent comprises Vltra tears (HPMC), Natvosol (HEC), polyacrylamide (PA), polypyrrole alkane ketone (PVP), polyethylene oxide (PEO), polyvinyl alcohol (PVA), methylcellulose gum (MC), glycan etc.
Used cell concn should be 10 among the present invention
3Individual/mL-10
6Between individual/mL, cell concn is lower than this scope, length consuming time.Be higher than this scope,, be difficult to accomplish single cell analysis because the chance that cell is in contact with one another is big.Unicellular cell pyrolysis liquid concentration should be between 0.01%-0.5%, and lysate concentration is lower than this scope, and cell still is kept perfectly when flowing through passage.Be higher than this scope, lysis is too fast.The screening agent concentration should be between 0.15%-10%, and the sieving medium of lower concentration does not reach screening effect, and the sieving medium viscosity of high density is big, is difficult in the injection gallery.
The present invention can be used for unicellular interior nucleic acid, protein, amino acid, the compartment analysis of materials such as sugar.
The present invention can be quartz, glass, silicon or PMMA, PDMS polymkeric substance with the micro-fluidic chip material.
Embodiment:
Fig. 1 is a kind of single-cell inclusion analysis micro-fluidic chip, certain this structure does not limit the present invention, and wherein 1 is that cell pool, 2 is that cell waste liquid pool, 3 is that lysis liquid pool, 4 is that runtime buffer liquid pool, 5 is that waste liquid pool, 6 is that check point I, 7 is check point II.
Method with laser induced fluorescence(LIF) detects: at first add damping fluid respectively in each Buffer Pool, cell pyrolysis liquid, screening agent (wherein comprising dyestuff), enchylema.Subsequently, insert platinum electrode in each Buffer Pool, electric high pressure (field intensity that this voltage produces in passage is between 50V/cm-250V/cm) is applied to each Buffer Pool by this electrode, and driving cell, cell pyrolysis liquid, screening agent flow to same sense channel.In this sense channel, finish cracking and the inclusion of release and the mark of dyestuff of individual cells.When mark the inclusion of dyestuff when the laser focal spot, the fluorescence that is gone out by laser excitation is by the photomultiplier collection.
See accompanying drawing 1 chip structure figure.Running buffer is 100mM TBE, and cell is PC12 (a rat suprarenal gland have a liking for cough up glucagonoma), concentration about 10
5Individual/mL.Cell pyrolysis liquid is 1%SDS.Used dyestuff is YOYO-I (0.3uM).Optical maser wavelength 488nm.Cracking in the passage of cell between cracking pond and waste liquid pool, the RNA that discharges is electronegative, so detect at check point I place.When detecting the material of positively charged, can detect at check point II place.The RNA that detects as shown in Figure 2.
See chip structure figure shown in the accompanying drawing 1.Running buffer is 100mM TBE, and cell is PC12 (a rat suprarenal gland have a liking for cough up glucagonoma), concentration about 10
5Individual/mL.Cell pyrolysis liquid is 1%SDS.The screening agent is 1%HPMC (50cp).Optical maser wavelength 488nm.At the cell liquid pool, the runtime buffer liquid pool, the lysis liquid pool applies 500V voltage, waste liquid pool ground connection.Detect at check point I place.The RNA that detects as shown in Figure 3.
Claims (6)
1, a kind of analytical procedure of the single-cell inclusion based on micro-fluidic chip is characterized in that; Whole process is carried out on a chip platform:
Chip platform is provided with cell pool, cell waste liquid pool, lysis liquid pool, runtime buffer liquid pool, waste liquid pool and sense channel, cell pool, cell waste liquid pool, lysis liquid pool, buffer pool communicate with the inlet of sense channel respectively, the outlet of sense channel connects waste liquid pool, in the passage in advance depended on pressure be full of screening agent and dyestuff;
At first with cell pool and cell waste liquid pool difference making alive and ground connection, under electrodynamic driving, cell flows to the cell waste liquid pool by cell pool, under microscopic examination, when individual cells arrives the inlet of sense channel, cell pool and cell waste liquid pool electrode are suspended;
Meanwhile runtime buffer liquid pool, lysis liquid pool are increased voltage, waste liquid pool ground connection; Under this kind voltage mode, ensure cell, cell pyrolysis liquid, screening agent, running buffer and be advanced into sense channel, finish the separation of single celled cracking and inclusion;
Two ends at sense channel are detected inclusion;
Wherein said cell concn should be 10
3Individual/mL-10
6Between individual/mL;
Unicellular cell pyrolysis liquid concentration should be between 0.01%-1%;
The screening agent concentration should be between 0.15%-10%.
2, according to the analytical procedure of the described single-cell inclusion based on micro-fluidic chip of claim 1, it is characterized in that: cell is selected the various cells that do not have cell walls for use.
3, according to the analytical procedure of the described single-cell inclusion based on micro-fluidic chip of claim 1, it is characterized in that: cell pyrolysis liquid is a kind of of sodium laurylsulfonate, sodium lauryl sulphate, Trombovar, Dodecyl trimethyl ammonium chloride, ten dimethyl trimethylammonium bromides, polyoxyethylene-23-lauryl ether Brij35, Octyl glucoside, Triton X-100.
4, according to the analytical procedure of the described single-cell inclusion based on micro-fluidic chip of claim 1, it is characterized in that: the screening agent is selected from a kind of of Vltra tears HPMC, Natvosol HEC, polyacrylamide PA, polypyrrole alkane ketone PVP, polyethylene oxide PEO, PVAC polyvinylalcohol, methylcellulose gum MC, glycan.
5, according to the analytical procedure of the described single-cell inclusion based on micro-fluidic chip of claim 1, it is characterized in that: used micro-fluidic chip material is quartz, glass, silicon or PMMA, PDMS polymkeric substance.
6, the analytical procedure of the described single-cell inclusion based on micro-fluidic chip of one of claim 1～5, be used for unicellular in nucleic acid, protein, amino acid, the compartment analysis of materials such as sugar.
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| Application Number | Priority Date | Filing Date | Title |
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| CNB021447721A CN100497653C (en) | 2002-12-13 | 2002-12-13 | Method for analyzing single cell inclusion based on micro flow-controlled chip |
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| CNB021447721A CN100497653C (en) | 2002-12-13 | 2002-12-13 | Method for analyzing single cell inclusion based on micro flow-controlled chip |
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Families Citing this family (7)
| Publication number | Priority date | Publication date | Assignee | Title |
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| US10753927B2 (en) | 2006-09-22 | 2020-08-25 | ALERE TECHNOLOGIES GmbH | Methods for detecting an analyte |
| CN101216458B (en) * | 2008-01-09 | 2011-04-20 | 浙江大学 | Sampling volume controllable micro-fluidic chip sieving electrophoresis analytical method |
| WO2011005776A1 (en) * | 2009-07-07 | 2011-01-13 | Sony Corporation | Microfluidic device |
| US9568723B2 (en) * | 2010-11-07 | 2017-02-14 | Csir | On-chip 4D lightfield microscope |
| US9506934B2 (en) * | 2013-04-29 | 2016-11-29 | Honeywell International Inc. | Polymer test cartridge mixer for cell lysis |
| CN103331097B (en) * | 2013-05-27 | 2015-04-08 | 陕西师范大学 | Application of polydimethylsiloxane micro fluidic chip in separating oligosaccharide and polysaccharide |
| CN107012220B (en) * | 2017-04-10 | 2019-11-05 | 杭州微著生物科技有限公司 | A method of utilizing the pairing unicellular content of micro-fluidic chip high throughput analysis |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| US6437551B1 (en) * | 1999-11-02 | 2002-08-20 | The Regents Of The University Of California | Microfabricated AC impedance sensor |
| CN1378485A (en) * | 1999-08-12 | 2002-11-06 | Ut-巴特勒有限公司 | Microfluidic device for controlled mianipulation of small volumes |
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| CN1378485A (en) * | 1999-08-12 | 2002-11-06 | Ut-巴特勒有限公司 | Microfluidic device for controlled mianipulation of small volumes |
| US6437551B1 (en) * | 1999-11-02 | 2002-08-20 | The Regents Of The University Of California | Microfabricated AC impedance sensor |
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