CN1313624C - Detecting miniflow controlled chip from single-cell wilting DNA fragmentation - Google Patents
Detecting miniflow controlled chip from single-cell wilting DNA fragmentation Download PDFInfo
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- CN1313624C CN1313624C CNB2004100207516A CN200410020751A CN1313624C CN 1313624 C CN1313624 C CN 1313624C CN B2004100207516 A CNB2004100207516 A CN B2004100207516A CN 200410020751 A CN200410020751 A CN 200410020751A CN 1313624 C CN1313624 C CN 1313624C
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- dna fragmentation
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Abstract
The present invention provides a micro flow control chip for detecting the unicell apoptosis DNA fragmentation, which mainly relates to a method for realizing the functions of sample introducing, cracking, fragmentation DNA marking, apoptosis cell DNA fragmentation electrophoretic separating, detecting, etc. of unicells in an integrated mode on the micro flow control chip. Each functional unit of the micro flow control chip is corresponding to a chip structure; the micro flow control chip has a structure in a double-T shape; the micro flow control chip is composed of a buffer solution tank, a cell sample introducing tank, a cracking solution tank, a waste solution tank and a detection region, wherein the solution tanks are connected by passages; the passage diameter is corresponding to the size of the detected unicells, and ensures the unicell accommodation transportation. The micro flow control chip of the present invention is used for screening various apoptosis causing factors (leading to the cell apoptosis) and detecting different kinds of cell apoptosis. The present invention has the advantages of convenient operation, easy implementation, high analysis flux and high analysis efficiency. The experimental functions are realized on one block of micro flow control chip, and the present invention has large intrinsic value in the fields of cell biology, etc.
Description
Technical field:
The present invention relates to the micro-total analysis technology, a kind of unicellular apoptosis dna fragmentation detection micro-fluidic chip is provided especially.
Background technology:
Apoptosis is the process of cell autonomy death in body growth growth, cytodifferentiation and the pathological state.Apoptosis and cell growth, the same cell incident or the process that belongs to each tool feature of differentiation, they are determining the essential characteristic and the destiny of cell and tissue.Because apoptosis has very important effect growth, immune response and many long-term puzzlements in human major disease such as the disease generating processes such as tumour, immune deficiency and neurodegeneration of fetal tissues and organ, in recent years, it had become the research focus in biology and the medical field.Based on the apoptosis critical role on many malignant tumour pathogenesiss unusually, be the elementary tactics that the apoptosis intervention techniques of purpose has become the treatment malignant tumour with inducing apoptosis of tumour cell optionally.
Studies show that in a large number the essence of apoptosis signal transduction path is a cover successive Biochemistry Reaction System, this system make reply in extracellular stimulus and the cell and the apoptosis effect between coupling, regulating and control the life of cell.The final final result of these effects shows as a series of morphocytologies and changes and biochemical change, and the chromatin dna fragmentation due to wherein being activated by endonuclease is the most important and distinctive biochemical indicator of tool of apoptosis.Traditional analytical procedure comprises agarose plate electrophoresis and capillary electrophoresis technique.The former can only carry out aggregate analysis (at least 1 * 10 to a large amount of colonies cell
6Cell), its resolving power, sensitivity are all lower, and operating process is loaded down with trivial details, analytic process is difficult to integrated.Though the latter can carry out the individual cells analysis at capillary electrophoresis, required time is longer relatively, and integrated degree is not high.These limitation limited to a certain extent the pair cell apoptotic process more deep, more fully study and be familiar with.
The micro-fluidic chip technology is a brand-new analytical technology that grew up in recent years, it has the advantage that unicellular horizontal pair cell is operated, it be on same chip with cell cultures, transport, clean and process integration one such as electrophoretic separation provides possibility.Simultaneously, the fluid network feature of micro-fluidic chip (two dimension or three-dimensional), make it and to carry out more complicated physics or chemical analysis by pair cell, cooperate highly sensitive detector can make this technology provide powerful technical support for the complicated cytobiology process of unicellular level research.
At present, only there is a laboratory report to utilize chip technology to carry out the fractional analysis of individual cells dna fragmentation in the world, detected object is a single stranded DNA in the cell, but owing to be subjected to the restriction of lysis mode (sodium hydroxide) that adopts, analytic process is longer relatively, and do not have chip channel interior to single celled operate continuously process, analysis throughput is relatively low.
Summary of the invention:
The objective of the invention is on micro-fluidic chip, to realize single celled sample introduction, cracking, fragmentation dna marker, apoptotic cell dna fragmentation electrophoretic separation and detection with integration mode.A kind of unicellular apoptosis dna fragmentation detection micro-fluidic chip is provided especially.
A kind of unicellular apoptosis dna fragmentation detection micro-fluidic chip of the present invention, it is characterized in that: this chip is made of three basic functional units, and wherein the 3rd basic functional units is connected with two other unit respectively:
First elementary cell is the cell sample injection unit;
Second lysate sample injection unit that elementary cell is a cell;
The 3rd elementary cell be lysis, fragmentation dna marker, dna fragmentation divide from and detecting unit.
A kind of unicellular apoptosis dna fragmentation detection micro-fluidic chip of the present invention, it is characterized in that: the used sample introduction solution cell density of described first elementary cell is 10
4～10
6Individual/mL; Adopt low electric field electricity to drive the cell sample introduction, sample introduction voltage is 10～100V/cm.
A kind of unicellular apoptosis dna fragmentation detection micro-fluidic chip of the present invention is characterized in that: described second elementary cell adopts low electric field electricity to drive the cell pyrolysis liquid sample introduction, and sample introduction voltage is 10～100V/cm.
A kind of unicellular apoptosis dna fragmentation detection micro-fluidic chip of the present invention is characterized in that: described the 3rd elementary cell is the connecting passage between chip buffer pool and the waste liquid pool:
The chemical cracking method is adopted in lysis, and the apoptotic cell cracking is online finishing in chip channel.
It is 0.5%～5% Vltra tears 50cp that dna fragmentation divides from used sieving medium, viscosity 5～30kD, and the dna fragmentation electrophoretic separation is 100～250V/cm.
A kind of unicellular apoptosis dna fragmentation detection micro-fluidic chip of the present invention is characterized in that: described the 3rd elementary cell detects by the laser induced fluorescence(LIF) one-point method and realizes.
A kind of unicellular apoptosis dna fragmentation detection micro-fluidic chip of the present invention, it is characterized in that: the used labeling dye of described fragmentation dna marker is the fluorescence dye that is pre-applied in running buffer or the cell pyrolysis liquid; The excitation wavelength of fluorescence dye, emission wavelength and laser-Induced Fluorescence Detection adapt with the optical maser wavelength of laser apparatus.
A kind of unicellular apoptosis dna fragmentation detection micro-fluidic chip of the present invention, it is characterized in that: it is 1uM that fragmentation DNA is carried out the online dynamically labeled labeling dye concentration that adopts.
A kind of unicellular apoptosis dna fragmentation detection micro-fluidic chip of the present invention, it is characterized in that: the isolating separation voltage of described dna fragmentationization is 170V/cm.
A kind of unicellular apoptosis dna fragmentation detection micro-fluidic chip of the present invention, it is characterized in that: between the lysate sample intake passage and cell sample intake passage of described detection position in reaction channel, with the ratio of the distance of lysate sample intake passage and cell sample intake passage be 1 to 2.
A kind of unicellular apoptosis dna fragmentation detection micro-fluidic chip of the present invention is characterized in that:
Described chip is double-T shaped structure, and it is made up of following components: buffer pool (1), cell sample inlet pool (2), lysate sample inlet pool (3), waste liquid pool (4), surveyed area (5); Connected by passage between each solution pool of double-T shaped structure chip, channel diameter adapts with the cell size of being studied, and can guarantee to hold the transportation of individual cells;
The double-T shaped structure of chip is corresponding with each basic functional units of chip: corresponding first elementary cell of cell sample inlet pool (2); Corresponding second elementary cell of lysate sample inlet pool (3); Cell sample inlet pool and corresponding the 3rd elementary cell of reaction channel between the lysate sample inlet pool.
The present invention is applicable to causing the various screenings that cause antiapoptotic factors of apoptosis, and dissimilar cell such as tumour cell, neurone, and the detection of apoptosis of many kinds such as embryo.The present invention is easy to operate, cell sample introduction, cracking, fragmentation dna marker, dna fragmentation divided from, testing process be integrated on the micro-fluidic chip and finish, and have higher analysis throughput and analysis efficiency, have great potential value in fields such as cytobiology, medical science.
Description of drawings:
The unicellular apoptosis dna fragmentation of Fig. 1 detection microfluidic chip structure synoptic diagram;
Fig. 2 microchip electrophoresis figure.
Embodiment:
Embodiment
The structure of the drug-induced model of cell apoptosis of 1 present embodiment:
With acute children's grain leukon morning of people (NB4 cells) is object, and cell cultures is in the RPMI1640 nutrient solution that contains 10% new-born calf serum.With representative antitumor drug (white arsenic, As
2O
3) cell death inducing.With certain density As
2O
3Making the medicine final concentration in the adding cell culture fluid is 1uM, continues then to cultivate 48 hours.Collecting cell cleans once with PBS solution after 1000rpm is centrifugal 5 minutes, and is stand-by after resuspended again.
2 chip designs:
Chip design that present embodiment adopts as shown in Figure 1, chip is double-T shaped structure, the chip split tunnel is wide 50 microns, dark 20 microns.Because the size of cells of mamma animals is usually in 10-25um diameter scope, designed chip split tunnel diameter and cell size be at an order of magnitude, and can guarantee to hold the transportation of individual cells.Four circular holes are represented the different solutions pond respectively among the figure, and wherein 1,4 is damping fluid and waste liquid pool, and 2 and 3 are respectively cell sample inlet pool and lysate sample inlet pool, and surveyed area is in the connecting passage between 1,4.
The double-T shaped structure of chip is corresponding with each basic functional units of chip: the corresponding cell sample injection unit of cell sample inlet pool (2); The corresponding lysate sample injection unit of lysate sample inlet pool; Cell sample inlet pool and the corresponding lysis of reaction channel, fragmentation dna marker, dna fragmentation between the lysate sample inlet pool divide from and detecting unit.
3 lysis modes and fragmentation dna marker mode
Under low electric field driven (50V/cm), respectively by pond 2 and 3 sample introductions, when entering split tunnel, cell mixes with lysate, in the rapid cracking of 10ms left and right sides cell through the cell of drug treating and cell pyrolysis liquid (2%SDS).The fragmentation DNA that discharges after the cracking combines with the fluorescence dye in the running buffer, carries out online dynamically labeled.The optimum dye label concentration that is adopted is 1uM.Dna fragmentation behind the significant notation is realized detecting by the laser induced fluorescence(LIF) one-point method at surveyed area under the effect that separates electric field and sieving medium.Used cell density is 10 in the experiment
5About individual/mL, can avoid sticking together between the cell in the high-density cells suspension, influence the operation of individual cells in passage.
4 dna fragmentation microchip electrophoresis separation conditions
A. sieving medium
Main effective constituent in the present embodiment in the used running buffer is that sieving medium is the 1.5% Vltra tears 50cp (1.5%HPMC-50cp that optimizes gained voluntarily, viscosity 15kD), specific viscosity is lower mutually with conventional media for this sieving medium, be particularly suitable for the microsize of micro-fluidic chip passage, passage cleans convenient, be easy to change solution, and can be satisfied with the separation requirement of apoptosis process chips dna fragmentationization.
B. separation voltage
In the microchip electrophoresis process, the selection of separation voltage directly has influence on the disintegrate-quality of fragmentation DNA, high separation voltage significantly reduce separating effect (＞250V/cm), low voltage then obviously prolong disengaging time (＜100V/cm), be unfavorable for real-time analysis.The optimum separation voltage of present embodiment through optimizing is 170V/cm.
5. apoptotic cell electrophoresis result
As shown in Figure 2, through As
2O
3NB4 cell generation apoptosis after the effect dna fragmentationization occurs, and can detect through microchip electrophoresis.Preceding 3 small peaks among the figure are fragmentation DNA, relatively confirm that clip size is 180bp, 360bp and 540bp in the back with the DNA standard substance, and the 4th climax is genomic dna.
Claims (7)
1, a kind of unicellular apoptosis dna fragmentation detection micro-fluidic chip, it is characterized in that: this chip is made of three basic functional units, and wherein the 3rd basic functional units is connected with two other unit respectively:
First elementary cell is the cell sample injection unit;
Second lysate sample injection unit that elementary cell is a cell;
The 3rd elementary cell be lysis, fragmentation dna marker, dna fragmentation divide from and detecting unit,
Chip has double-T shaped structure, and it is made up of following components: buffer pool (1), cell sample inlet pool (2), lysate sample inlet pool (3), waste liquid pool (4), surveyed area (5); Connected by passage between each solution pool of double-T shaped structure chip, channel diameter adapts with the cell size of being studied, and can guarantee to hold the transportation of individual cells;
The double-T shaped structure of chip is corresponding with described each basic functional units: corresponding first elementary cell of cell sample inlet pool (2); Corresponding second elementary cell of lysate sample inlet pool (3); Cell sample inlet pool and corresponding the 3rd elementary cell of reaction channel between the lysate sample inlet pool.
2, according to the described a kind of unicellular apoptosis dna fragmentation detection micro-fluidic chip of claim 1, it is characterized in that: the used sample introduction solution cell density of described first elementary cell is 10
4～10
6Individual/mL; Adopt low electric field electricity to drive the cell sample introduction, sample introduction voltage is 10～100V/cm.
3, according to the described a kind of unicellular apoptosis dna fragmentation detection micro-fluidic chip of claim 1, it is characterized in that: described second elementary cell adopts low electric field electricity to drive the cell pyrolysis liquid sample introduction, and sample introduction voltage is 10～100V/cm.
4, according to the described a kind of unicellular apoptosis dna fragmentation detection micro-fluidic chip of claim 1, it is characterized in that: described the 3rd elementary cell is the connecting passage between chip buffer pool and the waste liquid pool:
It is 0.5%～5% Vltra tears 50cp that dna fragmentation divides from used sieving medium, viscosity 5～30kD, and the dna fragmentation electrophoretic separation is 100～250V/cm.
5, according to the described a kind of unicellular apoptosis dna fragmentation detection micro-fluidic chip of claim 1, it is characterized in that: described the 3rd elementary cell detects by the laser induced fluorescence(LIF) one-point method and realizes.
6, according to the described a kind of unicellular apoptosis dna fragmentation detection micro-fluidic chip of claim 1, it is characterized in that: the used labeling dye of described fragmentation dna marker is the fluorescence dye that is pre-applied in running buffer or the cell pyrolysis liquid; The excitation wavelength of fluorescence dye, emission wavelength and laser-Induced Fluorescence Detection adapt with the optical maser wavelength of laser apparatus.
7, according to the described a kind of unicellular apoptosis dna fragmentation detection micro-fluidic chip of claim 6, it is characterized in that: it is 1uM that fragmentation DNA is carried out the online dynamically labeled labeling dye concentration that adopts.
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| CN103529195B (en) * | 2013-10-24 | 2014-08-06 | 山东大学 | Detection method applied to measurement of trace target materials |
| CN106754873B (en) * | 2016-12-05 | 2019-11-26 | 四川大学 | DNA separating kit |
| CN111304077B (en) * | 2020-03-03 | 2023-10-24 | 广东工业大学 | Integrated microfluidic chip for extracting nucleic acid and extraction method thereof |
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| JP2003075437A (en) * | 2001-09-04 | 2003-03-12 | Tsukuba Bio Syst:Kk | Detection method of fragmentation of dna by cell apoptosis derivation due to near-infrared spectrochemical analysis method |
| WO2003102578A2 (en) * | 2002-06-03 | 2003-12-11 | Pamgene B.V. | High throughput cellular response assay using microarrays |
| CN1469123A (en) * | 2002-07-18 | 2004-01-21 | 中国科学院大连化学物理研究所 | Microflow control chip for protein analysis and its application in protein analysis |
| CN1477206A (en) * | 2002-08-23 | 2004-02-25 | 中国科学院大连化学物理研究所 | Cell difference expression and gene test composit microcurrent chip |
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2003075437A (en) * | 2001-09-04 | 2003-03-12 | Tsukuba Bio Syst:Kk | Detection method of fragmentation of dna by cell apoptosis derivation due to near-infrared spectrochemical analysis method |
| WO2003102578A2 (en) * | 2002-06-03 | 2003-12-11 | Pamgene B.V. | High throughput cellular response assay using microarrays |
| CN1469123A (en) * | 2002-07-18 | 2004-01-21 | 中国科学院大连化学物理研究所 | Microflow control chip for protein analysis and its application in protein analysis |
| CN1477206A (en) * | 2002-08-23 | 2004-02-25 | 中国科学院大连化学物理研究所 | Cell difference expression and gene test composit microcurrent chip |
Non-Patent Citations (3)
| Title |
|---|
| Differential effects of human papillomavirus DNA types on p53tumor-suppressor gene apoptosis in sperm. Lee CA,et al,Gynecol.Oncol.,Vol.85 No.3 2002 * |
| 微流控芯片实验室及其功能化 林炳承，中国药科大学学报，第34卷第1期 2003 * |
| 微流控芯片实验室在基因分析研究中的应用 秦建华，色谱，第21卷第5期 2003 * |
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