CN103018437A - Immunofluorescence microfluidic chip based on quantum dots, as well as preparation method and use of chip - Google Patents
Immunofluorescence microfluidic chip based on quantum dots, as well as preparation method and use of chip Download PDFInfo
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Abstract
The invention discloses an immunofluorescence microfluidic chip, which is composed of two pieces of polydimethylsiloxane (PDMS) in a superposing manner, wherein the upper layer is composed of a PDMS film integrated with an inlet microfluid path and a cell culture micro-cavity; the lower layer is composed of the PDMS film integrated with a micro-pit array; the micro-pit array is composed of 40+/-10 rows and 35+/-10 columns of micro pits, and is 2.5+/-0.5mm long and 2+/-0.5mm wide; the micro pits are round, the diameters are 30+/-5 microns and the depths are 25+/-5 microns; the cell culture micro-cavity of the PDMS film at the upper layer is leveled with the micro-pit array of the PDMS film at the lower layer; and the PDMS films at two layers are irreversibly sealed after being subjected to oxygen plasma treatment, so as to form the immunofluorescence microfluidic chip based on quantum dots. The immunofluorescence microfluidic chip disclosed by the invention can be applied to glycosyl expression analysis of the surface of a single-cell horizontal membrane. The preparation method of the immunofluorescence microfluidic chip is also disclosed by the invention.
Description
Technical field
The present invention relates to the unicellular horizontal thin after birth of quantum dot immune fluorescent micro-fluidic chip surface glycosyl analytical approach.
Background technology
Obtaining of unicellular behavioural information is very important.[referring to: (a) Sethuraman, N.; Stadheim, T. A Curr. Opin. Biotechnol. 2006,17,341 346. (b) Rao, C. V.; Wolf, D. M.; Arkin, A. P. Nature 2002,420,231 237.].Flow cytometry can realize that as one of the most frequently used technology the high flux of unicellular data obtains, yet its analysis ability to a small amount of cell sample is subject to certain restrictions.The light tongs technology, though the dielectrophoresis technology can effectively catch and control unicellular and be used for to analyze, thereby these technology flux are all not high and be not suitable for the Cell Biology Experiment chamber.The micro-fluidic chip technology with its easily cell control accurate reagent transmission, and low sample consumption just is being subject to extensive concern [referring to (c) Carlo, D. D.; Aghdam, N.; Lee, L. P. Anal. Chem. 2006,78,4,925 4930. (d) Wlodkowic, D.; Faley, S.; Zagnoni, M.; Wikswo, J. P.; Cooper, J. M. Anal. Chem. 2009,81,5,517 5523. (e) Figueroa, X. A.; Cooksey, G. A.; Votaw, S. V.; Horowitz, L. F.; Folch, A. Lab Chip 2010,10,1,120 1127. (f) Hosokawa, M.; Hayashi, T.; Mori, T.; Yoshino, T.; Nakasono, S.; Matsunaga, T. Anal. Chem. 2011,83,3,648 3654.].
Immunofluorescence technique all is widely used in the middle of scientific research and clinical diagnosis.It is used to assessment tissue, cultured cell colony, unicellular specific proteins [referring to (g) Behrens, M.; Born, S.; Redel, U.; Voigt, N.; Schuh, V.; Raguse, J.-D.; Meyerhof, W. PLoS One 2012,7, e40304. (h) Fitzpatrick, E.; Mcbride, S.; Yavelow, J.; Najmi, S.; Zanzucchi, P.; Wieder, R. Clin. Chem. 2006,52,1,080 1088.], biological micromolecule is [referring to (i) Falsey, J. R.; Renil, M.; Park, S.; Li, S.; Lam, K. S. Bioconjugate Chem. 2001,12,346 353.], saccharide residue is [referring to (j) Venkatesan, C.; Chrzaszcz, M.; Choi, N.; Wainwright, M. S. J. Neuroinflammation 2010,7,32.] etc.Wherein, adherent, the differentiation of the expression of cell surface saccharide residue and cell and immunity identification are all closely related.It usually express to change with disease association, particularly cancer [referring to (k) Radhakrishnan, P.; Lin, M. F.; Cheng, P. W. Glycoconjugate J. 2009,26,75 81.].At present commonly used glycosyl analytical approach comprises that mass spectroscopy is [referring to (l) Yamada, K.; Hyodo, S.; Kinoshita, M.; Hayakawa, T.; Kakehi, K. Anal. Chem. 2010,82,7,436 7443.], chromatography [referring to: (m) Klapoetke, S.; Zhang, J.; (n) Becht, S.; Gu, X.; Ding, X. J. Pharm. Biomed. Anal. 2010,53,315 324.], nuclear magnetic resonance [referring to: (o) Gustafsson, A.; Sjoblom, M.; Strindelius, L.; Johansson, T.; Fleckenstein, T.; Chatzissavidou, N.; Lindberg, L.; Ngstrom, J.; Rova, U.; Holgersson, J. Glycobiology 2011,21,1,071 1086.] etc.Yet these methods usually need expensive instrument and equipment, and specialized operating personnel limit its application thereby need that especially cell sample is carried out the destructiveness processing.And usually face the problem of a photobleaching based on the immunofluorescence analysis method of organic dyestuff or fluorescin.Quantum dot becomes a class novel fluorescence probe as the nano material of a kind of high quantum production rate and high stability, receives increasing concern.But the report that is used for unicellular flat membrane surface glycosyl expression analysis method based on the immunofluorescence chip of quantum dot is arranged not yet so far.
Summary of the invention
The purpose of this invention is to provide a kind of immunofluorescence micro-fluidic chip based on quantum dot, can carry out analysis highly sensitive, that the unicellular flat membrane of high flux surface glycosyl is expressed with this chip.
A kind of immunofluorescence micro-fluidic chip, as shown in Figure 1, it is to be formed by stacking by two dimethyl silicone polymers (PDMS), the upper strata is made of the PDMS film that is integrated with entrance microfluidic circuit and cell cultivation microcavity, PDMS thickness 2 ± 0.5mm, described entrance microfluidic circuit is to connect many entrance branch streams under the entrance stream by long a 5 ± 0.5mm to consist of, many entrance branch streams and long a 2.5 ± 0.5mm, the cell of wide 2 ± 0.5mm is cultivated microcavity and is communicated, the relative direction of cultivating the entrance microfluidic circuit of microcavity at cell has the outlet microfluidic circuit, the outlet microfluidic circuit is corresponding with the entrance microfluidic circuit, also be made of with the outlet stream many outlet branch streams that communicate with cell cultivation microcavity, many entrance branch streams and many outlet branch streams can guarantee the inflow of uniform liquid ground and flow out cell cultivation microcavity; Lower floor is made of the PDMS film that is integrated with micro-pit array, described PDMS thickness 200 ± 20 μ m, that micro-pit array is that 30-40 is capable, the little hole of 25 ± 45 row forms, long 2.5 ± 0.5mm, wide 2 ± 0.5mm micro-pit array, little hole is circular, diameter is 30 ± 5 μ m, the degree of depth is 25 ± 5 μ m, adjacent two little hole frontier distances are 25 ± 5 μ m, the cell of upper strata PDMS film is cultivated microcavity and is alignd with the micro-pit array of the PDMS of lower floor film, two-layer PDMS film carries out irreversible sealing-in after by oxygen plasma treatment, consists of the immunofluorescence micro-fluidic chip based on quantum dot.
Above-mentioned immunofluorescence micro-fluidic chip, described entrance microfluidic circuit can be to connect two branch's streams by an entrance stream, and every branch's stream connects again two secondary branch streams, and four secondary branch streams are cultivated microcavity with cell and communicated.
Above-mentioned immunofluorescence micro-fluidic chip, described outlet branch stream is corresponding with the entrance branch stream.
Above-mentioned immunofluorescence micro-fluidic chip, described microfluidic circuit are the fluid pipes of wide 0.2 ± 0.05mm, dark 60 ± 5 μ m, and described cell is cultivated dark 60 ± 5 μ m of microcavity.
A kind of method for preparing above-mentioned immunofluorescence micro-fluidic chip, it comprises the steps:
The preparation of step 1. immunofluorescence micro-fluidic chip mould:
Parameter by above-mentioned immunofluorescence chip, single throwing silicon chip with diameter 52mm is template, (2000 turn the SU-8 2050 negative photoresists that spin coating one deck 60 ± 5 μ m are thick in the above, 30s), through 65 ℃ of front bakings, 3min and 95 ℃, 5min, then carry out photoetching (24mW/cm by the mask plate that is integrated with microchannel and cultivation microcavity
2, 8s),
Then through later drying by the fire 65 ℃, 3 min and 95 ℃, 5min, develop, obtain preparing the silicon chip mould that is integrated with microchannel and cultivates the upper strata PDMS film of microcavity behind the solid film; Single throwing silicon chip with diameter 52mm is template, (5000 turn the thick photoresist of spin coating one deck 25 ± 5 μ m in the above, 30s), front baking, exposure and rear baking parameter are the same, obtain preparing the silicon chip mould of the PDMS of the lower floor film that is integrated with micro-pit array behind development, solid film;
The preparation of step 2. immunofluorescence chip:
Be that the PDMS of 20:1 fully mixes with monomer and hardening agent mass ratio, after vacuumizing 10min, what be cast in that step 1 obtains has microfluidic circuit and cultivates microcavity and have on the silicon chip mould of micro-pit array, be positioned over baking 2h in 70 ℃ of baking ovens after leaving standstill 10min, peel off the PDMS film from the silicon chip mould, thickness 2 ± 0.5mm is cut into suitable size, makes the PDMS chip that has microfluidic circuit and cultivate microcavity; The silicon chip mould that will have micro-pit array is placed on the glue evenning table, then the about 3g of PDMS that with monomer and hardening agent mass ratio is 10:1 is poured on the silicon chip mould, 1000 turn, 30s, after be positioned over baking 2h in 70 ℃ of baking ovens, peel off the PDMS film from the silicon chip mould, be cut into suitable size and make the PDMS chip with micro-pit array, chip thickness 200 ± 20 μ m, the integrated microfluidic circuit of gained and cultivation microcavity PDMS film place with the PDMS film that is integrated with micro-pit array processes 1min in the oxygen plasma washer, with involution after two film alignment, after 75% ethanolic solution disinfection, use the PBS(phosphate buffered solution) flushing after namely get the immunofluorescence micro-fluidic chip.
A kind of above-mentioned immunofluorescence micro-fluidic chip is used for unicellular flat membrane surface glycosyl expression analysis method, and it comprises the steps:
Step 3. makes liquid flow to reverse direction to flow by raising the outlet liquid level, and repeating step 2 is realized cell capture again;
Step 4. repeating step 2 and 3 obtains satisfied cell capture efficient;
After the flushing of step 6. Tri(Hydroxymethyl) Amino Methane Hydrochloride damping fluid-polysorbas20, place and take pictures under the laser confocal microscope and analyze.
Cell capture efficient of the present invention obtains by the inverted fluorescence microscope analysis of accounts.The result shows that cell can be caught in little hole of 90%, and can obtain about 70% the unicellular little hole that contains.
Cytoactive of the present invention characterizes by calcein Calcein AM and propidium iodide PI.The result shows that cell still kept high activity after cell carried out cultivating in 12 hours in microchip.
The present invention observes the immunofluorescent staining effect by laser confocal microscope.The result shows, presents highlighted red fluorescence in unicellular level.
Description of drawings
Fig. 1 is the used micro-fluidic chip Platform Designing of the present invention synoptic diagram.
Fig. 2 is cell capture figure of the present invention.
Fig. 3 is cytoactive characterization result calcein Calcein AM cytoactive dyeing of the present invention (figure A), propidium iodide PI cell death dyeing (figure B).
Fig. 4 is that the present invention is used for slender after birth surface glycosyl immuning fluorescent dyeing analysis result.Figure A is double dish inner cell fluorescence picture at the bottom of the burnt special glass of copolymerization, and figure B and C are immunofluorescence micro-fluidic chip inner cell fluorescence picture.
Embodiment
Further specify particular content of the present invention below in conjunction with the accompanying drawing illustrated embodiment:
The mould preparation of embodiment 1. immunofluorescence micro-fluidic chips
Chip apparatus adopts dimethyl silicone polymer material (PDMS) by the preparation of standard soft lithographic method.It is as follows to set little hole chip parameter: be integrated with microchannel and cultivate shown in design parameter Figure 1A of PDMS film of microcavity; Little hole of micro-pit array chip arranges by 40 row, 35 row, totally 1400 little holes, hole spacing 25 ± 5 μ m(two hole frontier distances), diameter 30 ± 5 μ m, dark 25 ± 5 μ m, throwing silicon chip with the list of diameter 52mm is template, (2000 turn the SU-8 2050 negative photoresists that spin coating one deck 60 ± 5 μ m are thick in the above, 30s), then carry out photoetching (24mW/cm by the mask plate that is integrated with microchannel and cultivation microcavity through front baking (65 ℃ of 3min, 95 ℃ of 5min)
2, 8s), then through later baking (65 ℃ of 3min, 95 ℃), develop, admittedly make the silicon chip mould that is integrated with microchannel and cultivates microcavity behind the film; Single throwing silicon chip with diameter 52 mm is template, and (5000 turn the photoresist that spin coating one deck 25 ± 5 μ m are thick in the above, and 30s), front baking, exposure and rear baking parameter are the same, make the silicon chip mould that is integrated with micro-pit array after the baking after developing.
The preparation of embodiment 2. immunofluorescence micro-fluidic chips
Be that the PDMS of 20:1 fully mixes with monomer and hardening agent mass ratio, vacuumize 10min after, what be cast in that step 1 obtains has microfluidic circuit and cultivates on the silicon chip mould of microcavity, is positioned over baking 2h in 70 ℃ of baking ovens after leaving standstill 10min.Peel off the PDMS film from the silicon chip mould, the PDMS thickness is 2 ± 0.5mm, is cut into suitable size, makes the PDMS chip that has microfluidic circuit and cultivate microcavity; The silicon chip mould that will have micro-pit array is placed on the glue evenning table, then the about 3g of PDMS that with monomer and hardening agent mass ratio is 10:1 is poured on the silicon chip mould, 1000 turn, 30s, after be positioned over baking 2 h in 70 ℃ of baking ovens, peel off the PDMS film from the silicon chip mould, be cut into suitable size and make the PDMS chip with micro-pit array, chip thickness 200 ± 20 μ m.After two PDMS films clean with detergent, respectively at ultrasonic 10min in ethanol, the intermediate water, after drying up, nitrogen is put in 70 ℃ of baking 1h in the baking oven.The integrated microfluidic circuit of oven dry and the PDMS film cultivating the PDMS film of microcavity and be integrated with micro-pit array placed in the oxygen plasma washer process 1min.With involutions after the alignment of two chips, with 75% ethanolic solution disinfection after, use the PBS(phosphate buffered solution) wash for subsequent use.
Embodiment 3. gravity settling effects change unicellular catching in conjunction with the gateway liquor capacity
With 1 * 10
6~ 5 * 10
6Cells mL
-1Cell suspension introduce the chip entrance by micro-syringe, reduce the outlet liquid level, rely on gravitational difference that cell is introduced the microfluidic circuit pipeline, end liquid flow by balance gateway volume, because the sedimentation in little hole of Action of Gravity Field cell, the liquid level of increasing export, fluid flow direction is with opposite before this at this moment, and the cell in the outlet is again regional by micro-pit array, the repetition aforesaid operations, cell sedimentation in little hole obtains satisfied unicellular capture rate.Add PBS(phosphate buffered solution at inlet tube this moment) solution, cell flushing at large in microfluidic circuit and the little culture chamber is used the micro-syringe sucking-off to the exit.Characterization result is seen Fig. 2.
Cytoactive test on the embodiment 4. micro-fluidic chip platforms
On the microchip platform, after cell is hatched 12h, calcein Calcein AM and propidium iodide PI are introduced the microfluidic circuit pipeline, carry out microexamination after hatching 10min, the result is presented on this platform cell and hatches through 12h, still keeps high cytoactive.Analysis result is seen Fig. 3.
Immuning fluorescent dyeing analysis on the embodiment 5. micro-fluidic chip platforms
4% formalin is introduced the microfluidic circuit pipeline cell is fixed, times 10 min at room temperature carries out.After cell is fixing with Tri(Hydroxymethyl) Amino Methane Hydrochloride damping fluid flushing 10min, and with surperficial non-specific site 30 min of bovine serum albumin(BSA) closing cell.Agglutinin solution is introduced chip, at room temperature hatch 10 min with cell.Wash cell with Tri(Hydroxymethyl) Amino Methane Hydrochloride damping fluid-polysorbas20 solution, and seal 10 min with bovine serum albumin(BSA).Antiagglutinin antibody is introduced 37 ℃ of chips hatch 1 h, repeat above-mentioned flushing sealing step.CdTe quantum dot probe is introduced chip, hatch 1 h for 37 ℃.Place under the laser confocal microscope with Tri(Hydroxymethyl) Amino Methane Hydrochloride damping fluid-polysorbas20 solution flushing chip and with chip and to take pictures, obtain individual cells film surface glycosyl expressing information.
Claims (8)
1. immunofluorescence micro-fluidic chip, it is characterized in that: it is to be formed by stacking by two dimethyl silicone polymers (PDMS), the upper strata is made of the PDMS film that is integrated with entrance microfluidic circuit and cell cultivation microcavity, the PDMS film thickness is 2 ± 0.5mm, described entrance microfluidic circuit is to connect many entrance branch streams under the entrance stream by long a 5 ± 0.5mm to consist of, many entrance branch streams and long a 2.5 ± 0.5mm, the cell of wide 2 ± 0.5mm is cultivated microcavity and is communicated, the relative direction of cultivating the entrance microfluidic circuit of microcavity at cell has the outlet microfluidic circuit, the outlet microfluidic circuit is corresponding with the entrance microfluidic circuit, also be made of with the outlet stream many outlet branch streams that communicate with cell cultivation microcavity, many entrance branch streams and many outlet branch streams can guarantee the inflow of uniform liquid ground and flow out cell cultivation microcavity; Lower floor is made of the PDMS film that is integrated with micro-pit array, described PDMS film thickness is 200 ± 20 μ m, micro-pit array is that 40 ± 10 row, the little hole of 35 ± 10 row form, little hole is circular, diameter is 30 ± 5 μ m, the degree of depth is 25 ± 5 μ m, adjacent two little hole frontier distances are 25 ± 5 μ m, the cell of upper strata PDMS film is cultivated microcavity and is alignd with the micro-pit array of the PDMS of lower floor film, two-layer PDMS film carries out irreversible sealing-in after by oxygen plasma treatment, consists of the immunofluorescence micro-fluidic chip based on quantum dot.
2. immunofluorescence micro-fluidic chip according to claim 1, it is characterized in that: described entrance microfluidic circuit is to connect two branch's streams by an entrance stream, every branch's stream connects again two secondary branch streams, and four secondary branch streams are cultivated microcavity with cell and communicated.
3. immunofluorescence micro-fluidic chip according to claim 2 is characterized in that: described outlet branch stream is corresponding with the entrance branch stream.
4. immunofluorescence micro-fluidic chip according to claim 1, it is characterized in that: described microfluidic circuit is the fluid pipe of wide 0.2 ± 0.05mm, dark 60 ± 5 μ m, described cell is cultivated dark 60 ± 5 μ m of microcavity.
5. a method for preparing immunofluorescence micro-fluidic chip claimed in claim 1 is characterized in that it comprises the steps:
The preparation of step 1. immunofluorescence micro-fluidic chip mould:
Parameter by above-mentioned immunofluorescence chip, single throwing silicon chip with diameter 52mm is template, the SU-8 2050 negative photoresists that spin coating one deck 60 ± 5 μ m are thick in the above, through 65 ℃ of front bakings, 3min and 95 ℃, 5min, then carry out photoetching by the mask plate that is integrated with microchannel and cultivation microcavity
Then through later drying by the fire 65 ℃, 3min and 95 ℃, 5min, develop, obtain preparing the silicon chip mould that is integrated with microchannel and cultivates the upper strata PDMS film of microcavity behind the solid film; Throwing silicon chip with diameter 52mm single is template, the thick photoresist of spin coating one deck 25 ± 5 μ m in the above, and front baking, exposure and rear baking parameter are the same, through developing, obtain preparing behind the solid film silicon chip mould of the PDMS of the lower floor film that is integrated with micro-pit array;
The preparation of step 2. immunofluorescence chip:
Be that the PDMS of 20:1 fully mixes with monomer and hardening agent mass ratio, after vacuumizing 10min, what be cast in that step 1 obtains has microfluidic circuit and cultivates on the silicon chip mould of microcavity, be positioned over baking 2h in 70 ℃ of baking ovens after leaving standstill 1 min, peel off the PDMS film from the silicon chip mould, thickness 2 ± 0.5mm is cut into suitable size, makes the PDMS chip that has microfluidic circuit and cultivate microcavity; The silicon chip mould that will have micro-pit array is placed on the glue evenning table, then the about 3g of PDMS that with monomer and hardening agent mass ratio is 10:1 is poured on the silicon chip mould, 1000 turn, 30s, after be positioned over baking 2h in 70 ℃ of baking ovens, peel off the PDMS film from the silicon chip mould, be cut into suitable size and make the PDMS chip with micro-pit array, chip thickness 200 ± 20 μ m, after two PDMS films clean with detergent, respectively at ethanol, ultrasonic 10min in the intermediate water, be put in the interior 70 ℃ of baking 1h of baking oven after nitrogen dries up, the integrated microfluidic circuit of drying and the PDMS film of cultivating microcavity placed in the oxygen plasma washer with the PDMS film that is integrated with micro-pit array process 1min, with two rear involutions of chips alignment, after 75% ethanolic solution disinfection, use the PBS(phosphate buffered solution) flushing after namely get the immunofluorescence micro-fluidic chip.
6. the method for preparing the immunofluorescence micro-fluidic chip according to claim 3 is characterized in that: step 1 is described, and to carry out photoetching be that power is 24 mW/cm
2, the time is 8s.
7. the application of immunofluorescence micro-fluidic chip claimed in claim 1 in the glycosyl expression analysis of unicellular flat membrane surface.
8. the application of immunofluorescence micro-fluidic chip according to claim 5 in the glycosyl expression analysis of unicellular flat membrane surface is characterized in that concrete steps are as follows:
Step 1. is with 1 * 10
6~ 5 * 10
6Cells mL
-1Cell suspension enters cell by Action of Gravity Field from the inlet streams pass and cultivates in the microcavity;
Step 2. makes liquid stream keep static by balance gateway liquid level, allows cell enter Wei Keng by Action of Gravity Field;
Step 3. makes liquid flow to reverse direction to flow by raising the outlet liquid level, and repeating step 2 is realized cell capture again;
Step 4 repeating step 2 and 3 obtains satisfied cell capture efficient;
Step 5. is through the PBS(phosphate buffered solution) wash not the sedimentation cell after, introduce the formaldehyde fixed cell, wash through the Tri(Hydroxymethyl) Amino Methane Hydrochloride damping fluid, introducing agglutinin after the bovine serum albumin(BSA) sealing hatches, Tri(Hydroxymethyl) Amino Methane Hydrochloride damping fluid-polysorbas20 flushing, after the bovine serum albumin(BSA) sealing, introduce the agglutinin antibody incubation, introduce CdTe quantum dot probe after the flushing sealing;
After the flushing of step 6. Tri(Hydroxymethyl) Amino Methane Hydrochloride damping fluid-polysorbas20, place and take pictures under the laser confocal fluorescence microscope and analyze.
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