CN103185802B - Heterogeneous micro-fluidic Western blotting chip and its production and use - Google Patents
Heterogeneous micro-fluidic Western blotting chip and its production and use Download PDFInfo
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Abstract
The invention provides a kind of heterogeneous micro-fluidic Western blotting chip and its production and use, described chip comprises substrate and the lamellar body of fixing separative protein band, a surface of described lamellar body is provided with at least one miniflow groove, described miniflow groove and described polymer film form at least one microchannel circulated for antibody jointly, and another of described lamellar body is respectively equipped with perforation with the feeder connection and the channel outlet that form described microchannel in the position of two ports corresponding to described microchannel on the surface; The preparation method of described chip comprises the following steps: the substrate of the fixing separative protein band of preparation; Preparation is provided with the lamellar body of at least one miniflow groove; The substrate of assembling preparation and lamellar body; Purposes is it in protein detection or for the preparation of the application in the kit of protein detection.Single immunization trace reaction of the present invention can determine the existence of multiple target protein, and detection sensitivity is high, simple to operate, improves detection efficiency, reduces costs.
Description
Technical field
The present invention relates to a kind of chip, particularly relate to a kind of heterogeneous micro-fluidic Western blotting chip and its production and use, belong to biotechnology and field of tissue engineering technology.
Background technology
Micro-fluidic (Microfluidics) is a kind of analytical technology utilizing the small pipeline of micron level, the structure micro fluid to microlitre magnitude to finely control, corresponding technology carrier and micro-fluidic chip.The soft etching technique that it is derived with microelectronic technique is for relying on, microflow path system is built by the micro component such as integrated micro-channels, micro-reaction chamber on chip, load biological sample or chemical reaction liquid, microfluidic circuit is formed as power using forcing pump or electroosmotic flow, thus on chip, carry out one or more meticulous operation or reactions, reach the multiple objects such as biological detection, chemosynthesis or Growth of Cells.
Western blotting (immunoblotting), also known as Western blotting (Westernblotting), is that the people such as Towbin are in a kind of effective ways by specific antibody qualification antigenic information of invention in 1979.This method is a kind of immune biochemical technology grown up in gel electrophoresis and solid-phase immunoassay technical foundation.First after being used by sample containing target protein (antigen) sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) or native polyacrylamide gel electrophoresis (Native-PAGE) etc. to be separated, then by transfer techniques that electric current guides, original position is transferred to nitrocellulose filter, or the surface of polyvinylidene fluoride film or other film, then the protein on film surface is carried out specific detection with antigen-antibody reaction again.Such as, the protein belt be separated transferred to after on film through SDS-PAGE, film confining liquid process, then react with first antibody, film resists with enzyme labeling or fluorescein-labeled two after rinsing again, can demonstrate the position of target protein.
Existing Western blotting Problems existing is: (1) is once tested and can only be detected a kind of protein expression situation in biological sample.Needs can not to be met in present biological study simultaneously to the object that multiple protein in cell or tissue detects.(2) antibody-solutions of primary immune response Blot experiment needs more (a few hectolambda is to several milliliters), and antibody is expensive, makes immunoblot experiment cost higher.
In order to allow once experiment detect multiple protein, existing technology has multicolor fluorescence element mark two anti-methods, color quantum point marks two anti-method (Bakalova.R.; Zhelev, Z.; Ohba, H.; BabaYJ.Am.Chem.Soc.2005,27,9328-9329.), surface-enhanced Raman Western blot (Han.X.X.; Jia, H.Y; Wang, Y.F.; Lu, Z.C.; Wang, C.X.; Xu, W.Q.; Zhao, B.; Ozaki, Y.Anal.Chem.2008,80,2799-2804.).But, the albumen number that multicolor fluorescence element mark and color quantum point labelling method can detect is restricted, because the exciting light of the material (fluorescein or quantum dot) of isolabeling and radiative range of wavelengths do not have overlap, the Protein Detection number of report is mostly below 5 kinds now.The defect of surface-enhanced Raman Western blot is, when detecting the potpourri of multiple proteins, spectrum spectrogram is complicated and be difficult to distinguish.
Also there is now the multiple protein that some reports utilize microflow control technique to be formed to detect electrophoresis, such as by whole electrophoresis, the process of transfer focuses on (He, M. in a small passage, Herr, A.E., Nat.Prot.2010,5,1844-1856), but the problem of this technology is the information that can not provide target protein molecules amount, and the means of reading more complicated, be difficult to for general biology laboratory adopts; And for example, invented a kind of system before us, utilized parallel microchannel to introduce antibody test target protein (Pan, W.Y.; Chen, W.; Jiang, X.Y.; Anal.Chem.2010,82,3974-3976), problem above can be solved, but this method once can only process a sample, obviously can not meet needs in biological experiment and process the demand of a lot of sample simultaneously.
Summary of the invention
Therefore, the object of the invention is, the deficiency of a sample once can only be processed for current micro-fluidic Protein Detection, and the demand of a large amount of sample in biological experiment, will be processed simultaneously, a kind of heterogeneous micro-fluidic Western blotting chip of invention design, can carry out Protein Detection by microflow control technique high flux to reaching.
For above-mentioned purpose, technical scheme of the present invention is as follows:
On the one hand, the invention provides a kind of heterogeneous micro-fluidic Western blotting chip, comprise the substrate of fixing separative protein band and the lamellar body stacked with it, the described lamellar body surface mutually stacked with substrate is provided with at least one miniflow groove, described miniflow groove and described substrate form at least one microchannel circulated for fluid jointly, and another of described lamellar body is respectively equipped with perforation with the feeder connection and the channel outlet that form described microchannel in the position of two ports corresponding to described microchannel on the surface.
Preferably, at least one miniflow groove described comprises the first groove of multiple parallel distribution, the end of the first groove of described multiple parallel distribution is connected successively by the second groove, and the first groove and the suprabasil protein band of described multiple parallel distribution are perpendicular.
Preferably, the first groove of described multiple parallel distribution and the second groove perpendicular.
Preferably, described fluid comprises antibody.
Above-mentioned multiple first groove and suprabasil protein band perpendicular, described multiple first indention parallel distribution, and the first groove and the second groove are perpendicular, can ensure the accurate of measurement result and accuracy, make result more clear.
Preferably, the first groove of described multiple parallel distribution is 5 ~ 15.
Preferably, described substrate is made up of PVDF membrane, polyvinylidene fluoride Electrospun film, the surperficial gold-plated glass sheet through chemical modification, polyaniline film or film of poly pyrrole, and more preferably, described substrate is made up of PVDF membrane.
Preferably, described protein band for transfer to suprabasil protein band from polyacrylamide gel, and preferably, described transfer is electrotransfer, more preferably, described protein band is transferred in the substrate of being made up of PVDF membrane.
Preferably, described lamellar body is macromolecular material through the lamellar body of thermoplastic or heat curing, and preferably, described lamellar body is made up of dimethyl silicone polymer or polymethylmethacrylate, more preferably, described lamellar body is made up of dimethyl silicone polymer (PDMS).
Preferably, the width of the miniflow groove on lamellar body of the present invention is 50-200 μm, and the degree of depth is 50-200 μm, and length is 10cm-50cm.
On the other hand, the present invention also provides a kind of preparation method of heterogeneous micro-fluidic Western blotting chip, comprises the following steps:
Step 1: the substrate of the fixing separative protein band of preparation;
Step 2: preparation is provided with the lamellar body of at least one miniflow groove, preferably, described miniflow groove comprises the first groove of multiple parallel distribution, the end of the first groove of described multiple parallel distribution is connected successively by the second groove, first groove and the suprabasil protein band of described multiple parallel distribution are perpendicular, preferably, the first groove of described multiple parallel distribution and the second groove perpendicular;
Step 3: substrate prepared by number of assembling steps 1 and lamellar body prepared by step 2, to obtain final product.
Preferably, in step 1, described substrate is made up of PVDF membrane, polyvinylidene fluoride Electrospun film, the surperficial gold-plated glass sheet through chemical modification, polyaniline film or film of poly pyrrole, and preferably, described substrate is made up of PVDF membrane.
Preferably, described protein band for transfer to suprabasil protein band from polyacrylamide gel, and preferably, described transfer is electrotransfer, and more preferably, described protein band is transferred in the substrate of being made up of PVDF membrane.
Preferably, in step 2, described lamellar body is macromolecular material through the lamellar body of thermoplastic or heat curing, and preferably, described lamellar body is made up of dimethyl silicone polymer or polymethylmethacrylate, and more preferably, described lamellar body is made up of dimethyl silicone polymer.
Preferably, the width of the miniflow groove on described lamellar body is 50-200 μm, and the degree of depth is 50-200 μm, and length is 10cm-50cm.
Preferably, in step 2, when described lamellar body is made up of dimethyl silicone polymer, carry out especially by the method comprised the following steps: dimethyl silicone polymer is mixed with hardening agent and pours the template with microchannel into, after being dried again, the dimethyl silicone polymer of solidification is cut from template, in pre-designed injection port and sample outlet position punching, to obtain final product.
Preferably, in step 2, described dimethyl silicone polymer and hardening agent by 18: 1-24: 1 mass ratio mix; Preferably, at 80 DEG C, toast 10-50 minute, more preferably, described dimethyl silicone polymer and hardening agent mix by the mass ratio of 20: 1, and toast 25 minutes at 80 DEG C, most preferably, described template is silicon chip.
Preferably, in step 3, carry out especially by the method comprised the following steps:
Step 3.1: fixing poly-substrate of pressing the protein band that molecular weight is separated dried, described drying condition is preferably 37 DEG C, 60 minutes;
Step 3.2: the lamellar body being provided with at least one miniflow groove is covered in the substrate obtained in step 3.1, preferably, the lamellar body being provided with at least one miniflow groove is covered by the direction that the first groove of multiple parallel distribution is vertical with suprabasil protein band in the substrate obtained in step 3.1, flicking compresses, and to obtain final product.
Again on the one hand, the application of heterogeneous micro-fluidic Western blotting chip in protein detection that the present invention also provides a kind of said method to obtain, or the application in the kit of preparation protein detection, preferably, described protein detection is for optimizing immune response antibody concentration.
On the other hand, the present invention also provides a kind of kit for detecting protein, and described kit comprises the obtained heterogeneous micro-fluidic Western blotting chip of heterogeneous micro-fluidic Western blotting chip described above or method described above.
The usage range of micro-fluidic Western blotting chip provided by the present invention comprises the multiple protein simultaneously detected in cell, detect the molecular weight standard of protein and to optimize in immune response antibody concentration etc., compared with prior art, micro-fluidic Western blotting chip provided by the present invention and corresponding detection method have obvious advantage, can save time significantly, traditional immune detection system is 5h to whole night detection time, and is less than or equal to 1h the detection time of chip of the present invention; Detect the sample size needed few, the detection sample that traditional immune detection system detects ten kinds of samples to be needed is 100 ~ 200 μ g, and the detection sample size that chip detection of the present invention ten kinds of samples need is 5 ~ 10 μ g; Consumption antibody is few, the antibody-solutions volume that traditional immune detection system detects the consumption of a kind of albumen is 5-10mL, and the antibody-solutions volume that a kind of albumen of chip detection of the present invention consumes is 20 ~ 40 μ L, reduce costs, improve effect and the quality of engram analysis, the immunoblotting assay that tradition is taken time and effort enters the high throughput automated analysis phase.
Accompanying drawing explanation
Below, describe embodiment of the present invention in detail by reference to the accompanying drawings, wherein:
Fig. 1 is the schematic diagram of heterogeneous micro-fluidic Western blotting chip of the present invention and corresponding pictorial diagram, and in figure, A is the schematic diagram of the substrate of fixing separative protein band, and in figure, B is the pictorial diagram of the substrate of fixing separative protein band; In figure, C is the schematic diagram of heterogeneous micro-fluidic Western blotting chip, and in figure, D is the pictorial diagram of heterogeneous micro-fluidic Western blotting chip; In figure, E is the test findings schematic diagram after heterogeneous micro-fluidic Western blotting chip detection albumen; In figure, F is the test findings after heterogeneous micro-fluidic Western blotting chip detection albumen, and wherein 1 for substrate, and 101 is suprabasil protein labeling, and 102 is the protein band of suprabasil separation; 2 is lamellar body, and 201 is the perforation forming feeder connection, and 202 is the perforation forming channel outlet, and 3 is miniflow groove, and 301 is the first groove, and 302 is the second groove;
Fig. 2 is the partial enlarged drawing after the test findings after heterogeneous micro-fluidic Western blotting chip detection albumen of the present invention, in figure 1,1 '; 2,2 '; 3,3 '; 4,4 '; 5,5 '; 6,6 '; 7, the 7 ' testing results all representing symmetric position place in heterogeneous micro-fluidic Western blotting chip, arrow is depicted as the antibody that corresponding microchannel adds, and described antibody is respectively anti-actin antibodies, anti-annexin antibody and anti-Pan-14-3-3 antibody;
Fig. 3 is the test findings of the antibody detection protein of heterogeneous micro-fluidic Western blotting chip variable concentrations protein band of the present invention and variable concentrations, in figure, 3-1 represents that the cracking holoprotein band of four NIH3T3 cells adds the test findings of the antibody test of variable concentrations, 1 testing result representing the protein band that is separated to be concentration be the cracking holoprotein of the NIH3T3 cell of 10 μ g in Fig. 3-1, the testing result of 2 represent the protein band that is separated to be concentration the be cracking holoprotein of the NIH3T3 cell of 5 μ g, the testing result of 3 represent the protein band that is separated to be concentration the be cracking holoprotein of the NIH3T3 cell of 2.5 μ g, the testing result of 4 represent the protein band that is separated to be concentration the be cracking holoprotein of the NIH3T3 cell of 1.25 μ g, in figure, 3-2 to represent in Fig. 3-1 the 1 testing result change adding the antibody of variable concentrations, and in figure, 3-3 to represent in Fig. 3-1 1,2,3, the change of testing result thirdly in 4,
Fig. 4 is the test findings compared with heterogeneous micro-fluidic Western blotting chip of the present invention detects albumen with traditional immunologic detection method, in figure, A represents heterogeneous micro-fluidic Western blotting chip of the present invention, B represents the partial enlarged drawing of figure A, what C represented common immunologic detection method hatches box, D represents that heterogeneous micro-fluidic Western blotting chip of the present invention detects comparing of albumen result with traditional immunologic detection method, wherein Mic-WB represents heterogeneous micro-fluidic Western blotting chip method of protein detection of the present invention, tradition-WB represents traditional immunologic detection method, in figure, arrow is depicted as protein band,
Fig. 5 is the result of the close albumen of heterogeneous micro-fluidic Western blotting chip isolated molecule amount of the present invention, and in figure, 1-7 represents the testing result of adding different antibody in heterogeneous micro-fluidic Western blotting chip respectively.
Embodiment
Term used in the present invention, unless otherwise specified, generally has the implication that those of ordinary skill in the art understand usually.
Below in conjunction with concrete preparation embodiment and Application Example, and comparable data describes the present invention in further detail.These embodiments just in order to demonstrate the invention, but not limit the scope of the invention by any way.
Anti-beta-actin antibody (anti-β-actin antibody), anti-annexin antibody (anti-Annexin antibody) used in following examples, anti-Pan-14-3-3 antibody is purchased from SantaCruz company.
embodiment 1
The present embodiment illustrates the preparation method of heterogeneous micro-fluidic Western blotting chip of the present invention
1. the preparation of micro-fluidic chip template
The main process of preparation is photoetching, namely utilizes the feature of photoresist convertibility matter under Ultraviolet radiation to make and the figure on all four photoresist silicon chip template on the mask designed.Concrete preparation method can see YXia, G.Whitesides, AnnualReviewofMaterialsScience1998, and 28,15.
2. be provided with the preparation of the lamellar body of multiple miniflow groove
Preparation method is Soft lithograph technology, preparing material is dimethyl silicone polymer (PDMS, poly-dimethylsiloxane), it is transparent and the liquid of thickness under common state, through reacting with hardening agent (184siliconeelastomercuringagent, purchased from American Dow Corning Corporation) and heating rear curable.Utilize PDMS the raised image in silicon chip template can be converted to corresponding pipeline figure, thus obtain the lamellar body being provided with multiple miniflow groove.When forming temperature remains 80 DEG C, the present invention is provided with the raw material mass mixture ratio of the lamellar body of multiple PDMS miniflow groove to preparation and the high-temperature molding time is optimized, shown in actual conditions table 1.
The each group of material that table 1 dimethyl silicone polymer is different with the proportioning of hardening agent
Concrete experimental procedure is as follows:
(1) according to PDMS monomer (the SYLGARD184 silicon rubber shown in table 1, purchased from American Dow Corning Corporation) and hardening agent (SYLGARD184 silicon rubber hardening agent, purchased from American Dow Corning Corporation) quality proportioning, take raw material respectively and mix, stir about reduces pressure pump bubble to evenly for 10 minutes in vacuum pump.
(2) in the silicon chip template with microchannel, pour the potpourri of PDMS and hardening agent into, be positioned in vacuum pump and pump bubble.
(3) put into 80 DEG C, baking oven, toast according to the time shown in table 1, PDMS can be stamped groove by photoresist outstanding on silicon chip.
(4) PDMS of solidification is cut from template, and punch at the syringe of pre-designed injection port place band syringe needle, clean PDMS miniflow groove surface with Scotch (purchased from 3M) white glue band.
(5) be placed on microslide by PVDF membrane, cover on PVDF membrane by the lamellar body being provided with multiple PDMS miniflow groove, the lamellar body size being provided with multiple PDMS miniflow groove is greater than PVDF membrane.With hand, PVDF membrane and the lamellar body being provided with multiple PDMS miniflow groove are compressed by flicking.
(6) 30 μ L blue inks are dripped with liquid-transfering gun at the aperture place of chip upper channel end, at the other end syringe pump of passage, record and observe the complexity of liquid inlet channel and the leakage scenarios of passage, result shows, when the quality proportioning of 20: 1 pressed by PDMS monomer and hardening agent, dry the condition of 25 minutes at 80 DEG C under, liquid easily passes into passage, and do not have Seepage, effect is better.Miniflow groove specification on the made lamellar body got ready is: width: 50-200 μm, the degree of depth: 50-200 μm, length: 10cm-50cm, table 2 specific as follows.
The microchannel specification that table 2 group number 1-9 is obtained
3. electrophoresis
By electrophoresis apparatus, protein mixture is separated into band by molecular size range in polyacrylamide gel, experimental procedure is identical with the electrophoresis step in traditional immunoblot experiment, specifically can see H.Towbin, T.Staehelin, J.Gordon, ProceedingsoftheNationalAcademyofSciencesoftheUnitedStat esofAmerica1979,76,4350.
4. electrotransfer method
Under electric field action, by albumen by transferring to PVDF membrane (i.e. trace perforated membrane in polyacrylamide gel, to be volatilized pore-forming by solvent, aperture is 0.2-0.45 μm, purchased from milipore company), or polyvinylidene fluoride Electrospun film (nonwoven fabrics that nano-scale fiber is formed, aperture is nanoscale, concrete preparation method can see Yang, D.Y.; Niu, X.; Liu, YY; Wang, Y; Gu, X.; Song, L.S.; Zhao, R.; Ma, L.Y; Shao, Y.M.; Jiang, X.YAdvMate:2008,20,4770-4775.) on, experimental procedure goes to step identical with the electricity in traditional immunoblot experiment, specifically can see H.Towbin, T.Staehelin, J.Gordon, ProceedingsoftheNationalAcademyofSciencesoftheUnitedStat esofAmerica1979,76,4350.
5. the assembling of micro-fluidic chip
Namely chip assembling is the PVDF membrane securing albumen and the lamellar body being provided with multiple PDMS miniflow groove are combined, the passage that composition is closed, and makes liquid can suction passage under negative pressure, and passage there will not be seepage.The assembling of micro-fluidic chip specifically comprises the following steps:
(1) PVDF membrane of upper for transfer protein band is put dry in an oven, 37 DEG C, 60 minutes.
(2) PVDF membrane is placed on microslide, the lamellar body being provided with multiple PDMS miniflow groove is covered on PVDF membrane.
(3) compress by by PVDF membrane and the lamellar body being provided with multiple PDMS miniflow groove with have gentle hands.Because PDMS has elasticity and viscosity, so be provided with lamellar body and the PVDF membrane energy good seal of multiple PDMS groove.
6. micro-fluidic chip Immunofluorescence test
By antigen-antibody identification, in different passages, hatch different antibody, detect the multiple protein on PVDF membrane.Specifically comprise the following steps:
(1) passage inner sealing: drip 3 μ L confining liquids (5% volume bovine serum albumin BSA at the aperture place of chip upper channel end with liquid-transfering gun, phosphate buffer PBS), at the other end syringe pump of passage, solution is made to be full of passage, after hatching 10 minutes, extract solution out with syringe.
(2) primary antibodie is hatched: diluted by primary antibodie antibody diluent, and optimizes the formula of this antibody diluent, and the volume fraction of regulation and control Tween-20 is 0.01%, 0.02%, 0.05%, 0.1%, 0.2%, 0.4%, 0.5%, obtain different ratio phosphate buffer.Respectively to passing into the antibody diluent of 40 μ L for different albumen in different passages, hatching after 30 minutes and extracting out.Open PDMS passage, with deionized water rinsing PVDF membrane surface.
(3) shake and wash: shake with PBST (0.2% volume Tween-20) and wash PVDF membrane three times, each 5 minutes.
(4) membrane closure: film is placed in confining liquid (its formula synantibody dilution) and sways 20 minutes.
(5) two anti-hatch: PVDF membrane is put into fluorescently-labeled two anti-dilutions (its formula synantibody dilution) lucifuge and sway 30 minutes.
(6) shake and wash: shake by PBST (0.2% volume Tween-20) lucifuge and wash PVDF membrane three times, each 10 minutes.
(7) fluoroscopic examination: PVDF membrane is put into TyphoonTrioTM Multifunctional imaging analytic system (Amersham company) detection signal, optimum configurations is as follows: launch optical filter 520nm, laser 488nm, PMT480, susceptibility is medium.
(8) observe and record the fluorescence signal and background often organized, result shows, time in the phosphate buffer of primary antibodie dilution containing 0.2% volume Tween-20, signal to noise ratio (S/N ratio) is the strongest.
embodiment 2
The present embodiment is for illustration of the heterogeneous detectability of heterogeneous micro-fluidic Western blotting chip of the present invention.Implementation step is as follows:
As described in example 1 above, the cracking holoprotein of 80 μ gNIH3T3 cells is loaded in two swimming lanes respectively, electrophoresis is carried out according to the preferred time, transferring film, then the film of load protein is dried, use PDMS monomer and hardening agent to press the quality proportioning of 20: 1, the condition of toasting 25 minutes at 80 DEG C prepares PDMS chip, film and chip is combined by light pressure.Pass into different antibody respectively in the channel from left to right, particularly, 1 and 2: the potpourri passing into anti-annexin antibody and anti-Pan-14-3-3 antibody two kinds of antibody; 3 and 4: the potpourri passing into anti-actin antibodies (anti-actin antibody) and anti-Pan-14-3-3 antibody two kinds of antibody; 5 and 6: the potpourri passing into anti-actin antibodies (anti-actin antibody) and anti-annexin antibody two kinds of antibody, 7: pass into anti-actin antibodies (anti-actin antibody), anti-annexin antibody (anti-Annexin antibody), the potpourri of anti-Pan-14-3-3 antibody three kinds of antibody, from right to left 1 '-7 ' order of antibody that passes into is with from left to right 1-7 is identical, the preferred concentration of antibody is 1: 80, the fluorescently-labeled rabbit anti-mouse IgG of two anti-uses, concentration is 1: 2000.Result as shown in Figure 2.
embodiment 3
The present embodiment is used for the ability of half-quantitative detection for illustration of heterogeneous micro-fluidic Western blotting chip of the present invention.
As described in example 1 above, upper 10 μ g will be contained in respectively in four swimming lanes, 5 μ g, 2.5 μ g, the cracking holoprotein of 1.25 μ gNIH3T3 cells, electrophoresis is carried out according to the preferred time, transferring film, then dries the film of load protein, uses PDMS monomer and hardening agent to press the quality proportioning of 20: 1, the condition of drying 25 minutes at 80 DEG C prepares PDMS chip, film and chip is combined by light pressure.Take the anti-annexin antibody of SantaCruz company, initial concentration is taken as 1: 20, and then gradient dilution antibody-solutions is respectively 1: 40, and 1: 80,1: 160,1: 320,1: 640,1: 1280.Passed into by the antibody of these seven kinds of concentration among 7 microchannels respectively, two concentration resisted are 1: 2000.The dot chart that the antibody obtaining variable concentrations acts on respectively with the antigen of different amount.As shown in accompanying drawing 3-1, different reaction results is neatly clearly arranged in a result picture.These results are done to the quantitative test of fluorescence intensity, the curve of fluorescence intensity with the antigen-antibody change of setting can be obtained easily.Respectively, as shown in figure 3-2, for a certain amount of antigen amount, use the antibody of variable concentrations, obtain antibody titer curve, this curve illustrates and is fixed on suprabasil quantify antigens for the binding ability of antibody and degree of saturation, for judging the preferred concentration in follow-up test; As shown in figure 3-2, for a certain amount of antibody concentration, use not antigen as such, obtain the titration curve of antigen, this curve can illustrate the amount of antigen number, for judging the concentration of this specific objective albumen in unknown sample in follow-up test.This dot chart illustrates that the present invention obtains these two useful information in once experiment.
embodiment 4
The present embodiment shortens the ability of immune response time for illustration of heterogeneous micro-fluidic Western blotting chip of the present invention.
As described in Example 1, NIH-3T3 cell (purchased from ATCC) the cracking holoprotein of 20 μ g is loaded in albumen swimming lane, according to the preferred time, voltage, temperature conditions carries out electrophoresis, transferring film, drying, the concentration of primary antibodie is the primary antibodie passing into same concentrations in 1: 200,7 passages, its incubation time is set as 2.5min respectively, 5min, 10min, 20min, 30min, 60min, 90min, two concentration resisted are 1: 2000, and incubation time is set as 60min.As comparative experiments, for traditional protein immunoblot method, equally the concentration of primary antibodie is set as 1: 200, prepares 7 and same hatch ware, the primary antibodie incubation time of hatching 7 in ware is also set as 2.5min respectively, 5min, 10min, 20min, 30min, 60min, 90min, two concentration resisted and incubation time are also respectively 1: 2000 and 60min.React respectively, the time dependent degree of fluorescence intensity of the present invention and traditional immunization immunoblot method is compared after rinsing, as shown in Figure 4, can see that the fluorescence intensity in the present invention can reach capacity within the relatively short time (20min), therefore obviously can reduce the time needed for normal reaction.
embodiment 5
The present embodiment illustrates that heterogeneous micro-fluidic Western blotting chip of the present invention is used for the ability of adjacent protein band in the resolved immuno marking.
After the electrophoretic separation and electrotransfer of routine, to choose in holoprotein the close protein annexin (36kD) of two molecular weight and GAPDH (35.8kD), according to the reaction of traditional immunoblotting analysis, because two bands are separated by too near, cannot distinguish, can only adopt twice independently detect realize qualification.And in the present invention, because different antibody can be separated by different microchannels, so it is close to detect multiple molecular weight in once testing simultaneously, the protein band that position is adjacent.Such as, by the microchannel bedding in the present invention on a band to be distinguished, by passing into the antibody of anti-annexin in a passage, in another adjacent passage, pass into the antibody of anti-GAPDH, concrete: in figure 1 and 7: anti-Pan-14-3-3 antibody; 2 and 6: anti-annexin antibody (anti-Annexin antibody); 3 and 5: anti-GAPDH antibody; 4: anti-actin antibodies (anti-actin antibody), with reference to the step in embodiment 1 through reaction, after development, as shown in Figure 5, there is signal in the place of passage 2 and passage 3 correspondence, then illustrate that this band is composited by the band of anti-annexin and the band of GAPDH.
embodiment 6
The present embodiment illustrates microchannel strict separation antibody-solutions in heterogeneous micro-fluidic Western blotting chip of the present invention, ensures the ability of sealing.
Use PDMS monomer and hardening agent by 20: 1 quality proportioning, at 80 DEG C, the baking condition of 25 minutes prepares PDMS chip, is fitted by the PVDF membrane of itself and load protein.Then utilize the flat board of plastics by PVDF membrane and PDMS chip pressing, take off plastic plate, the two contact position lighter, both explanations are fitted all right.Now pass into antibody-solutions, can antibody-solutions be made easily to flow to channel outlet smoothly from feeder connection, the leakage of liquid does not occur.
embodiment 7
The present embodiment illustrates that the microchannel in heterogeneous micro-fluidic Western blotting chip of the present invention has the ability of auto injection.
First PDMS chip and clean glass sheet are fitted, in passage, then pass into the skim milk powder solution dissolved with phosphate buffer, employ different skimmed milk power different ratio in the solution, its massfraction is made to be 1%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, in passage, hatch 5 minutes, then extract out.The PDMS chip of being hatched by skim milk powder solution and PVDF membrane are fitted, compresses, drip antibody-solutions at feeder connection place.Result shows, the antibody-solutions at feeder connection place can under the driving not having external force in automatic flow channel.Learnt by comparative experiments, when in the skim milk powder solution selected, the massfraction of milk powder is 5%, auto injection effect is the most obvious.
Claims (32)
1. a heterogeneous micro-fluidic Western blotting chip, comprise the substrate of fixing separative protein band and the lamellar body stacked with it, the surface mutually stacked with substrate of described lamellar body is provided with at least one miniflow groove, at least one miniflow groove described and described substrate form at least one microchannel circulated for fluid jointly, and another of described lamellar body is respectively equipped with perforation with the feeder connection and the channel outlet that form described microchannel in the position of two ports corresponding to described microchannel on the surface;
Wherein, described miniflow groove comprises the first groove of multiple parallel distribution, the end of the first groove of described multiple parallel distribution is connected successively by the second groove, first groove and the suprabasil protein band of described multiple parallel distribution are perpendicular, and the first groove and second groove of described multiple parallel distribution are perpendicular;
Wherein, described heterogeneous micro-fluidic Western blotting chip is high-throughout;
Wherein, each described microchannel is all arranged on different protein swimming lanes;
First groove of described multiple parallel distribution is 5 ~ 15.
2. heterogeneous micro-fluidic Western blotting chip according to claim 1, it is characterized in that, described fluid comprises antibody.
3. heterogeneous micro-fluidic Western blotting chip according to claim 1 and 2, is characterized in that, described substrate is made up of PVDF membrane, the surperficial gold-plated glass sheet through chemical modification, polyaniline film or film of poly pyrrole.
4. heterogeneous micro-fluidic Western blotting chip according to claim 3, it is characterized in that, described substrate is made up of PVDF membrane.
5. heterogeneous micro-fluidic Western blotting chip according to claim 3, is characterized in that, described PVDF membrane is polyvinylidene fluoride Electrospun film.
6. heterogeneous micro-fluidic Western blotting chip according to claim 1 and 2, it is characterized in that, described protein band for transfer to suprabasil protein band from polyacrylamide gel.
7. heterogeneous micro-fluidic Western blotting chip according to claim 6, it is characterized in that, described transfer is electrotransfer.
8. heterogeneous micro-fluidic Western blotting chip according to claim 6, is characterized in that, described protein band is transferred in the substrate of being made up of PVDF membrane.
9. heterogeneous micro-fluidic Western blotting chip according to claim 1 and 2, is characterized in that, described lamellar body is macromolecular material through the lamellar body of thermoplastic or heat curing.
10. heterogeneous micro-fluidic Western blotting chip according to claim 9, it is characterized in that, described lamellar body is made up of dimethyl silicone polymer or polymethylmethacrylate.
11. heterogeneous micro-fluidic Western blotting chips according to claim 10, it is characterized in that, described lamellar body is made up of dimethyl silicone polymer.
12. heterogeneous micro-fluidic Western blotting chips according to claim 1 and 2, it is characterized in that, the width of the miniflow groove on described lamellar body is 50-200 μm, and the degree of depth is 50-200 μm, and length is 10cm-50cm.
The preparation method of 13. heterogeneous micro-fluidic Western blotting chips according to any one of claim 1 to 12, comprises the following steps:
Step 1: the substrate of the fixing separative protein band of preparation;
Step 2: preparation is provided with the lamellar body of at least one miniflow groove, described miniflow groove comprises the first groove of multiple parallel distribution, the end of the first groove of described multiple parallel distribution is connected successively by the second groove, first groove and the suprabasil protein band of described multiple parallel distribution are perpendicular, and the first groove and second groove of described multiple parallel distribution are perpendicular;
Step 3: substrate prepared by number of assembling steps 1 and lamellar body prepared by step 2, to obtain final product.
The preparation method of 14. heterogeneous micro-fluidic Western blotting chips according to claim 13, it is characterized in that, in step 1, described substrate is the substrate of PVDF membrane, the surperficial gold-plated glass sheet through chemical modification, polyaniline film or film of poly pyrrole.
The preparation method of 15. heterogeneous micro-fluidic Western blotting chips according to claim 14, it is characterized in that, described substrate is the substrate of PVDF membrane.
The preparation method of 16. heterogeneous micro-fluidic Western blotting chips according to claims 14 or 15, it is characterized in that, described PVDF membrane is polyvinylidene fluoride Electrospun film.
The preparation method of 17. heterogeneous micro-fluidic Western blotting chips according to claim 13, it is characterized in that, described protein band for transfer to suprabasil protein band from polyacrylamide gel.
The preparation method of 18. heterogeneous micro-fluidic Western blotting chips according to claim 17, it is characterized in that, described transfer is electrotransfer.
The preparation method of 19. heterogeneous micro-fluidic Western blotting chips according to claim 17 or 18, is characterized in that, described protein band is transferred in the substrate of being made up of PVDF membrane.
20., according to claim 13 to the preparation method of the heterogeneous micro-fluidic Western blotting chip described in 15,17 and 18 any one, is characterized in that, in step 2, described lamellar body is macromolecular material through the lamellar body of thermoplastic or heat curing.
The preparation method of 21. heterogeneous micro-fluidic Western blotting chips according to claim 20, it is characterized in that, described lamellar body is made up of dimethyl silicone polymer or polymethylmethacrylate.
The preparation method of 22. heterogeneous micro-fluidic Western blotting chips according to claim 21, it is characterized in that, described lamellar body is made up of dimethyl silicone polymer.
23. according to claim 13 to the preparation method of the heterogeneous micro-fluidic Western blotting chip described in 15,17 and 18 any one, and it is characterized in that, the width of the miniflow groove on described lamellar body is 50-200 μm, and the degree of depth is 50-200 μm, and length is 10cm-50cm.
The preparation method of 24. heterogeneous micro-fluidic Western blotting chips according to claim 20, it is characterized in that, in step 2, when described lamellar body is made up of dimethyl silicone polymer, carry out especially by the method comprised the following steps: dimethyl silicone polymer is mixed with hardening agent and pours the template with microchannel into, then after being dried, the dimethyl silicone polymer of solidification is cut from template, in pre-designed injection port and sample outlet position punching, to obtain final product.
The preparation method of 25. heterogeneous micro-fluidic Western blotting chips according to claim 24, it is characterized in that, preferably, described dimethyl silicone polymer mixes with the mass ratio of hardening agent by 18:1-24:1, at 80 DEG C, toast 10-50 minute.
The preparation method of 26. heterogeneous micro-fluidic Western blotting chips according to claim 25, it is characterized in that, described dimethyl silicone polymer mixes with the mass ratio of hardening agent by 20:1, toasts 25 minutes at 80 DEG C.
The preparation method of 27. heterogeneous micro-fluidic Western blotting chips according to claim 24, it is characterized in that, described template is silicon chip.
28., according to claim 13 to the preparation method of the heterogeneous micro-fluidic Western blotting chip described in 15,17 and 18 any one, is characterized in that, in step 3, carry out especially by the method comprised the following steps:
Step 3.1: fixing substrate of pressing the protein band that molecular weight is separated dried, described drying condition is 37 DEG C, 60 minutes;
Step 3.2: covered by the lamellar body being provided with at least one miniflow groove in the substrate obtained in step 3.1, flicking compresses, and to obtain final product.
The preparation method of 29. heterogeneous micro-fluidic Western blotting chips according to claim 28, it is characterized in that, in step 3.2, the lamellar body being provided with at least one miniflow groove is covered by the direction that the first groove direction of multiple parallel distribution is vertical with suprabasil protein band in the substrate obtained in step 3.1, flicking compresses, and to obtain final product.
The application of the heterogeneous micro-fluidic Western blotting chip that according to any one of 30. heterogeneous micro-fluidic Western blotting chips according to any one of claim 1 to 12 or claim 13 to 29, method obtains in protein detection or for the preparation of the application in the kit of protein detection.
31. application according to claim 30, is characterized in that, described protein detection is for optimizing immune response antibody concentration.
32. 1 kinds for detecting the kit of protein, described kit comprises the heterogeneous micro-fluidic Western blotting chip that according to any one of heterogeneous micro-fluidic Western blotting chip according to any one of claim 1 to 12 or claim 13 to 29, method is obtained.
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| WO2019054500A1 (en) * | 2017-09-15 | 2019-03-21 | Ａｇｃ株式会社 | Microchannel chip |
| KR101965299B1 (en) * | 2017-11-14 | 2019-04-03 | 주식회사 수젠텍 | Assembly type blot strip device |
| CN111686826B (en) * | 2019-03-15 | 2023-05-23 | 国家纳米科学中心 | Microfluidic chip with layered structure and application thereof |
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