CN101923053A - Device and method for continuously analyzing single-cell contents by miniflow control chip at high speed - Google Patents
Device and method for continuously analyzing single-cell contents by miniflow control chip at high speed Download PDFInfo
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- CN101923053A CN101923053A CN 201010230276 CN201010230276A CN101923053A CN 101923053 A CN101923053 A CN 101923053A CN 201010230276 CN201010230276 CN 201010230276 CN 201010230276 A CN201010230276 A CN 201010230276A CN 101923053 A CN101923053 A CN 101923053A
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Abstract
The invention provides a miniflow control chip which has a simple structure and can continuously analyzing single-cell contents at high speed and an operation method. The invention is characterized in that a sheath flow passageway is arranged at each side of a sample injection passageway of a cross-miniflow control chip; through adjusting static pressure difference between liquid storage tanks, cell suspension and sheath flow liquid simultaneously flow out of a sample reservoir and a sheath flow liquid reservoir; under the action of sheath flow, single cells in the cell suspension rank in a row to enter into a separation passageway from the sample injection passageway in turn, contact with a film dissolving agent in the process of movement and conduct rapid film dissolution at the inlet of the separation passageway; the film-dissolved cell contents fully enter into the separation passageway to be continuously separated at high speed under the action of an field stress generated at two ends of the separation passageway; and laser induces fluorescence detection. As the solution entering into the separation passageway is a mixed solution of the sheath flow liquid and normal saline in the cell suspension, the invention can also greatly reduce the concentration of the normal saline entering into the separation passageway and markedly lower band broadening caused by Joule heat when electrophoresis is carried out.
Description
(1) technical field
The present invention relates to a kind of device and method that utilizes micro-fluidic chip continuous high speed analysis list intracellular matter, belong to the single cell analysis technical field.
(2) background technology
Single cell analysis is significant to the research of major disease early diagnosis, treatment, drug screening and cell physiologicals such as cancer, pathologic process.(diameter 8 μ m～200 μ m, volume fL～nL), sample size is few, and (zmol～fmol), component is very complicated in the cell, and it is very big to analyze difficulty because cell is small.Capillary electrophoresis technique is to carry out unicellular multicomponent analysis at present to use maximum methods.But capillary electrophoresis technique is subjected to the restriction of kapillary one-dimentional structure, the unicellular diameter that need use glass capillary to be drawn into when entering Capillary Electrophoresis is that micron-sized tip holds individual cells, cooperation is with the fine motion manipulater of precision, just can finish the single-cell injection operation, therefore complicated and time consumption requires very high to operating personnel.So analysis speed is very slow.Can only analyze tens cells every day.
Because the network structure and the micron-sized channel size of micro-fluidic chip, and the characteristic of chip capillary cataphoresis high efficiency separation, the compartment analysis of single celled sample introduction, molten film and intracellular organic matter can be realized on a micro-fluidic chip.On cross and double T micro-fluidic chip, by automatically controlled, hydraulic pressure in conjunction with automatically controlled or laser tweezers, handle unicellular sample intake passage and enter split tunnel from microfluidic analysis chip, cell is accurately rested in the split tunnel on the injection port tube wall, behind the static molten film, measure the content of intracellular organic matter by the chip capillary cataphoresis compartment analysis.But fixed cell needs the time also longer on split tunnel import tube wall and the complicated operation of static molten film, has therefore limited the speed of single cell analysis greatly.The single celled speed of this methods analyst is at per hour 25 below the cell.
Up to now, had only three pieces of bibliographical informations and on micro-fluidic chip, carried out the method for continuous high speed analysis list intracellular matter content.But physiological saline during the cell continuous sample introduction in the cell suspending liquid and the unicellular split tunnel that enters together, Joule heat increases when causing electrophoresis, the separating property variation.For fear of the increase of Joule heat, need to use equipment (Analytical Chemistry 2003,75,5646～5655) such as expensive pulse ac electricity galvanic particular power source of stack and micro-injection pump.Owing to these reasons, on micro-fluidic chip, carry out the method for continuous high speed analysis list intracellular matter and be not used widely.
(3) summary of the invention
The invention provides a kind of micro-fluidic chip and method of operating of energy continuous high speed analysis list intracellular matter simple in structure.
The technical solution used in the present invention is:
A kind of device that utilizes micro-fluidic chip continuous high speed analysis list cellular content, mainly comprise micro-fluidic chip, high-voltage DC power supply and laser induced fluorescence detector, described micro-fluidic chip is provided with sample intake passage and the split tunnel that is cross, in the sample intake passage both sides, respectively be provided with a sheath circulation road, article two, sheath circulation road and sample intake passage flow to along sample introduction liquid and intersect at right-angled intersection point upstream, with right-angled intersection point distance be 10～1000 microns, sample intake passage one end is provided with sample cell, the other end is provided with the sample waste liquid pool, split tunnel one end is provided with buffer pool, the other end is provided with the damping fluid waste liquid pool, is provided with the sheath flow liquid pond at two sheath circulation road other ends.Usually, sample intake passage, sheath circulation road width and the degree of depth need more than or equal to the width and the degree of depth of split tunnel.High-voltage DC power supply voltage is generally 500～10000V, and positive pole links to each other with buffer pool, negative pole links to each other with the damping fluid waste liquid pool, forms DC electric field in split tunnel.In the split tunnel of check point operated by rotary motion between damping fluid waste liquid pool and right-angled intersection point of laser induced fluorescence detector, be used to detect test substance sends fluorescence under induced with laser light intensity.
The present invention respectively adds a sheath circulation road by two sides at cross micro-fluidic chip sample intake passage, sheath flow liquid provides a kind of micro-fluidic chip and method of operating of energy continuous high speed analysis list intracellular matter simple in structure by molten film and electrophoresis buffer solution mixed preparing.Poor by the liquid level of regulating between sample waste liquid pool and other liquid storage tanks, the cell suspension and the sheath stream that place sample cell are flowed out from sample cell and sheath flow liquid pond simultaneously.Behind the unicellular point of crossing in the cell suspension by sample intake passage and sheath circulation road, being subjected to sheath stream is in line automatically from the extruding of two sides, cell in operational process be added in sheath flow liquid in molten film contact and fast instant film in the split tunnel porch, under the electric field force effect of split tunnel two ends, cellular content behind the molten film enters split tunnel from sample intake passage, flowing through is arranged at laser induced fluorescence detector on the split tunnel, analyzes by fluorescence intensity pair cell content content.In the present invention, the solution that enters in the micro-fluidic chip split tunnel is the mixed solution of physiological saline in sheath flow liquid and the cell suspension.It both can make unicellular in the split tunnel porch fast instant film, can also make to enter in the split tunnel ratio of cell suspension and dwindle greatly, make that the concentration of physiological saline reduces greatly in the electrophoretic buffer, significantly reduce the band broadening that Joule heat causes.
Described two sheath circulation roads are 15～75 ° of angles with sample intake passage along the sample introduction liquid flow direction respectively and intersect.
Buffer pool (B), sample cell (S), sample waste liquid pool (SW), two sheath flow liquid pond (SF are arranged on the micro-fluidic chip referring to Fig. 1 and Fig. 2 continuous high speed analysis list provided by the invention intracellular matter
1And SF
2) and damping fluid waste liquid pool (BW) and sample intake passage, sheath circulation road and split tunnel.Passage between described sample liquid storage tank and the sample waste liquid pool is sample intake passage S-SW; Two sides at sample intake passage respectively have a sheath circulation road that communicates with the sheath flow liquid pond, and described split tunnel B-BW and sample intake passage intersect vertically, and sample intake passage and split tunnel cross be in the O point, and two sheath circulation roads and sample intake passage intersect at O point upstream.Laser spots places the split tunnel below in O point downstream, is used to detect intracellular matter sends fluorescence under induced with laser light intensity.
The invention still further relates to a kind of method of utilizing described device continuous high speed analysis list cellular content, described method comprises: add electrophoretic buffer in buffer pool and damping fluid waste liquid pool, it is sheath flow liquid that the sheath flow liquid pond adds the electrophoretic buffer that is dissolved with molten film, add cell suspension to be measured in the sample cell, cell suspension and sheath flow liquid are flowed out from sample cell and sheath flow liquid pond simultaneously, cell suspension and sheath flow liquid converge the back along passage and form unicellular stream, cell in operational process be added in sheath flow liquid in molten film contact and fast instant film in the split tunnel porch, under the electric field force effect of split tunnel two ends, cellular content behind the molten film all enters split tunnel from sample intake passage, flowing through is arranged at laser induced fluorescence detector on the split tunnel, analyzes by fluorescence intensity pair cell content content.Kind by changing molten film, concentration and two sheath circulation roads and sample intake passage flow to the distance that intersects at right-angled intersection point upstream along sample introduction liquid, can make cell contact in the 50ms fast instant film with sheath stream solution in the split tunnel porch, cellular content behind the molten film separates under the effect of the electric field force that electric field produced at split tunnel two ends, all enters split tunnel.
The key of this method is to utilize the sheath flow liquid pair cell suspension side of containing molten film to push and makes it to be in line automatically, and make it fast instant film, concrete molten film and electrophoretic buffer can be selected by this area conventional method, those of ordinary skills can be according to general knowledge, according to the different molten film of different choice of cell to be measured.Usually, cell concentration is 0.5～10 * 10
5Cells/mL, molten film concentration in damping fluid is 0.1～10% (w/w).
The single celled analytical procedure of micro-fluidic chip is as follows among the present invention:
On micro-fluidic chip, add electrophoretic buffer among buffer pool B and the damping fluid waste liquid pool BW, sheath flow liquid pond SF
1And SF
2The middle electrophoresis buffer solution that contains molten film that adds adds cell suspension in sample cell S, SW is vacant for the sample waste liquid pool.Under the driving of the static pressure that the liquid level difference causes, cell suspension in the sample cell and sheath stream flow out from sample cell and sheath flow liquid pond simultaneously, after cell liquid stream can flow with the sheath circulation road, the unicellular extruding that is subjected to sheath stream in the cell suspension is in line and continues operation, and be in operation and contact with molten film in the sheath flow liquid, separate at split tunnel two ends under the effect of the electric field force that electric field produced, 150 cells of per minute may command are continuous, individually enter split tunnel from sample intake passage, and being in fast instant film in the 50ms at split tunnel inlet, it is continuous by the chip capillary voltage that the cellular content behind the molten film all enters split tunnel, separation detection at high speed.
The inventive method is easy to operate, poor by the liquid level of regulating between sample waste liquid pool and other liquid storage tanks, unicellular stream under the extruding at sheath in the cell suspension is in line automatically, improve single celled sample rate greatly, avoided a plurality of cells to enter split tunnel from sample intake passage simultaneously; By molten film being added in the electrophoretic buffer as sheath flow liquid, can make unicellular in the split tunnel porch fast instant film, and make cellular content behind the molten film all enter split tunnel to give separation detection continuously and in high speed.Because the solution that enters in the micro-fluidic chip split tunnel is the mixed solution of physiological saline in sheath flow liquid and the cell suspension, enter in the time of can also making the cell continuous sample introduction that the concentration of physiological saline reduces greatly in the split tunnel, significantly reduce the band broadening that Joule heat causes.
(4) description of drawings
Fig. 1 analyzes single celled micro-fluidic chip synoptic diagram continuously for sheath stream focuses on;
Fig. 2 analyzes single celled installation drawing continuously for sheath stream focuses on;
Fig. 3 focuses on continuous analysis list cell synoptic diagram for micro-fluidic chip sheath stream;
Fig. 4 is unicellular content electrophoresis detection figure as a result.
(5) embodiment
The present invention is described further below in conjunction with specific embodiment, but protection scope of the present invention is not limited in this:
Embodiment 1:
Referring to Fig. 1, buffer pool (B), sample cell (S), sample waste liquid pool (SW), two sheath stream liquid storage tank (SF are arranged on the micro-fluidic chip
1And SF
2) and damping fluid waste liquid pool (BW).Sample intake passage is S-SW, and length is 12mm, channel width 95 μ m, dark 35 μ m, and passage S-O length is 6mm; Split tunnel is B-BW, and length is 46mm, channel width 65 μ m, and dark 20 μ m, channel B-O length is 6mm; Sample intake passage and split tunnel cross are in the O point.Two sides at sample intake passage respectively have a sheath circulation road, intersect at 200nm place, O point top with sample intake passage, the wide 95 μ m of sheath circulation road, and dark 35 μ m, length is 6mm.At sample intake passage, the end points place of split tunnel and sheath circulation road respectively spiles, and uses adhesive bonds micro plastics liquid storage tank on aperture.
Referring to Fig. 2,100 μ L, 100 μ L add respectively among liquid storage tank B and the BW with electrophoretic buffer (as borate buffer, N-glycine amide damping fluid etc.), the cell suspension (1.2 * 10 that 100 μ L were hatched with fluorescent reagent
5Cells/mL) add sample cell S.The electrophoretic buffer 100 μ L that will contain molten film (conventional molten film is as triton X-100, sodium dodecylsulphonate, digitonin etc.), 100 μ L add sheath stream liquid storage tank SF respectively
1And SF
2, do not add solution among the liquid storage tank SW.In the B of split tunnel two ends and BW liquid storage tank, apply the 2000V DC voltage.
Referring to Fig. 3, because the liquid level difference, the cell in the cell suspension flows to SW from liquid storage tank S.Contain molten film electrophoretic buffer simultaneously from sheath flow liquid pond SF
1And SF
2Flow out.After cell liquid stream can flow with the sheath circulation road, the cell of sheath stream from the extruding cell suspension of two sides was in line cell.Cell is in operation and contacts with molten film in the sheath flow liquid, run to the point of crossing O of sample intake passage and split tunnel when cell after, executed at split tunnel two ends and separated under the effect of the electric field force that electric field produced, make unicellular turning round from sample intake passage successively enter split tunnel and at the fast instant film in the split tunnel porch, the cellular content behind the molten film all enters split tunnel.Concentration by cell in liquid level difference between adjusting sample waste liquid pool and other liquid storage tanks and the cell suspension, 150 cells of per minute may command individually enter split tunnel from sample intake passage continuously, and being in fast instant film in the 50ms at split tunnel inlet, the cellular content matter that discharges behind the molten film is by chip Capillary Electrophoresis separation detection continuously and in high speed.
Embodiment 2: the mensuration of single blood erythrocyte by mocro glutathion inside and active oxygen
The micro-fluidic chip that present embodiment uses is consistent with embodiment 1, and sample is a red cell suspension.With glutathione and active oxygen in naphthalene dicarbaldehyde and the two hydrogen rhodamine 123 difference labeled cells, be 1.2 * 10 with physiological saline dilution configuration density
5Behind the cells/mL cell suspension, add sample cell; 20mmol borate buffer solution (pH 9.2) is added among liquid storage tank B and the BW, and (w/w) electrophoretic buffer (20mmol borate buffer solution, pH 9.2) adds sheath stream liquid storage tank SF for tritonX-100, concentration 1% will to contain molten film
1And SF
2In, the liquid volume added in each liquid storage tank is consistent with embodiment 1.In the B of split tunnel two ends and BW liquid storage tank, apply the 2000V DC voltage.
Because the liquid level difference, the cell in the red cell suspension flows to SW from liquid storage tank S.Contain molten film electrophoretic buffer simultaneously and flow out, behind the point of crossing by sample intake passage and sheath circulation road, red blood cell is in line from the sheath flow liquid pond.Cell contacts at the volley with molten film triton X-100 in being added on sheath flow liquid.Run to the point of crossing O of sample intake passage and split tunnel when cell after, separate at split tunnel two ends under the effect of the electric field force that electric field produced, unicellular successively from sample intake passage turn round enter split tunnel and in the split tunnel porch fast instant film, glutathion inside cell behind the molten film and active oxygen all enter split tunnel.By chip Capillary Electrophoresis separation detection continuously and in high speed.Measurement result is seen Fig. 4.By controlling the length of single celled sample introduction speed and split tunnel, can make same unicellular glutathion inside and active oxygen reach baseline separation, and different unicellular measurement result does not interfere with each other yet.From Fig. 4 as seen, glutathione and active o content in 12 cells in 19 seconds, have been measured.
Claims (3)
1. device that utilizes micro-fluidic chip continuous high speed analysis list cellular content, mainly comprise micro-fluidic chip, direct supply and laser induced fluorescence detector, described micro-fluidic chip is provided with sample intake passage and the split tunnel that is cross, in the sample intake passage both sides, respectively be provided with a sheath circulation road, article two, sheath circulation road and sample intake passage flow to along sample introduction liquid and intersect at right-angled intersection point upstream, with right-angled intersection point distance be 10～1000 microns, sample intake passage one end is provided with sample cell, the other end is provided with the sample waste liquid pool, split tunnel one end is provided with buffer pool, the other end is provided with the damping fluid waste liquid pool, and two sheath circulation road other ends are provided with the sheath flow liquid pond.
2. device as claimed in claim 1 is characterized in that described two sheath circulation roads are 15～75 ° of angles with sample intake passage along the sample introduction liquid flow direction respectively and intersect.
3. method of utilizing the described device continuous high speed of claim 1 analysis list cellular content, described method comprises: add electrophoretic buffer in buffer pool and damping fluid waste liquid pool, it is sheath flow liquid that the sheath flow liquid pond adds the electrophoretic buffer that is dissolved with molten film, add cell suspension to be measured in the sample cell, poor by the static pressure of regulating between sample waste liquid pool and other liquid storage tanks, cell suspension and sheath flow liquid are flowed out from sample cell and sheath flow liquid pond simultaneously, cell suspension and sheath flow liquid are in line after passage converges automatically, form unicellular stream, cell in operational process be added in sheath flow liquid in molten film contact and fast instant film in the split tunnel porch, separate at split tunnel two ends under the effect of the electric field force that electric field produced, cellular content behind the molten film all enters split tunnel by continuous, separate at high speed, separate each component in the cellular content of back and flow through and be arranged at laser induced fluorescence detector on the split tunnel, analyze by the content of each component in the fluorescence intensity pair cell content.
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| CN102183504A (en) * | 2011-01-25 | 2011-09-14 | 山东师范大学 | Microfluidic unicellular active oxygen automatic analyzer |
| CN102680679A (en) * | 2011-03-15 | 2012-09-19 | 中国科学院上海生命科学研究院 | Cell microporous chip for detecting specific antibody secretion of single cell and preparation method thereof |
| CN104388300A (en) * | 2014-11-27 | 2015-03-04 | 山东师范大学 | Micro-fluidic continuous sampling method and device for unicellular quantitative analysis |
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