CN103185802A - Multiphase microfluidic immunoblotting chip, and preparation method and application thereof - Google Patents
Multiphase microfluidic immunoblotting chip, and preparation method and application thereof Download PDFInfo
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Abstract
The invention provides a multiphase microfluidic immunoblotting chip, and a preparation method and applications thereof. The chip comprises a substrate fixed with a separate protein band, and a chip body, wherein at least one micro fluid concave groove is arranged on one surface of the chip body, the micro fluid concave groove and the a polymer film together form at least one microflow passage for antibody circulation, and a via hole is respectively arranged on each end port, corresponding the microflow passage, on another surface, to form a passage inlet and a passage outlet of the microflow passage. The chip preparation method comprises the following steps of: preparing the substrate fixed with the separate protein band; and assembling the prepared substrate and the clip body. The chip is used for protein detection and for preparing a kit for protein detection. Single-time immunoblotting reaction of the invention can determine existence of multiple target proteins, and the chip and the preparation method has advantages of high detection sensitivity, simple operation, high detection efficiency and low cost.
Description
Technical field
The present invention relates to a kind of chip, relate in particular 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 small pipeline of micron level, analytical technology that structure is carried out meticulous control to the micro fluid of microlitre magnitude utilized, and corresponding technology carrier is micro-fluidic chip.The soft etching technique that it derives out with microelectronic technique is support, make up microflow path system by micro component such as integrated micro-channels, little reaction chamber on chip, load biological sample or chemical reaction liquid, form microfluidic circuit with forcing pump or electroosmotic flow as power, thereby carry out one or more meticulous operation or reactions at chip, reach multiple purposes such as biological detection, chemosynthesis or cell growth.
Western blotting (immunoblotting) claims Western blotting (Western blotting) again, is that people such as Towbin is in a kind of effective ways of identifying antigenic information by specific antibody of invention in 1979.This method is a kind of immune biochemical technology that grows up in gel electrophoresis and solid-phase immunoassay technical foundation.After the sample that will contain target protein (antigen) at first uses sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) or native polyacrylamide gel electrophoresis (Native-PAGE) etc. to separate, transfer techniques by electric current guiding then, original position is transferred to nitrocellulose filter, or the surface of polyvinylidene fluoride film or other film, the protein with the film surface carries out specific detection with antigen-antibody reaction more then.For example, will be after the protein belt that SDS-PAGE separates to be transferred on the film, film is handled with confining liquid, and then with the first antibody reaction, film resists with enzyme labeling or fluorescein-labeled two after rinsing again, can demonstrate the position of target protein.
The problem that existing Western blotting exists is: (1) once test can only the detection of biological sample in a kind of protein expression situation.Can not satisfy the purpose that needs detect multiple protein in the cell or tissue simultaneously in the present biological study.The antibody-solutions of (2) immunoblot experiment needs more (several hectolambdas are to several milliliters), and antibody is expensive, makes the immunoblot experiment cost higher.
Experiment can detect multiple protein in order to allow once, and existing technology has the plain mark two anti-methods of multicolor fluorescence, color quantum point mark two anti-method (Bakalova.R.; Zhelev, Z.; Ohba, H.; Baba Y J.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 the plain mark of multicolor fluorescence and color quantum point labelling method can detect is restricted, because the exciting light of the material of isolabeling (fluorescein or quantum dot) and radiative range of wavelengths do not have overlappingly, now the Protein Detection number of report is mostly below 5 kinds.The defective of surface-enhanced Raman Western blot is, when detecting the potpourri of multiple proteins, spectrum spectrogram is complicated and be difficult to distinguish.
Now also there are some reports to utilize microflow control technique to detect the multiple protein that electrophoresis forms, for example with whole electrophoresis, the process of transfer focuses on (He, M. in the small passage, Herr, A.E., Nat.Prot.2010,5,1844-1856), but the problem of this technology is the information that the target protein molecular weight can not be provided, and reads the means more complicated, is difficult to adopt for general biology laboratory; And for example, invent 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), can solve top problem, but this method once can only handle a sample, obviously can not satisfy the demand that needs in the biological experiment are handled a lot of samples simultaneously.
Summary of the invention
Therefore, the objective of the invention is, once can only handle the deficiency of a sample at present micro-fluidic Protein Detection, and the demand that will handle a large amount of samples in the biological experiment simultaneously, a kind of heterogeneous micro-fluidic Western blotting chip of invention design carries out Protein Detection in the hope of being able to by microflow control technique high flux ground.
At 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 mutually stacked surface of described lamellar body and substrate is provided with at least one miniflow groove, described miniflow groove and described substrate form at least one microchannel for fluid circulation jointly, and another surface of described lamellar body is gone up in the position corresponding to two ports of described microchannel and is respectively equipped with feeder connection and the channel outlet of perforation to form described microchannel.
Preferably, described at least one miniflow groove comprises first groove of a plurality of parallel distributions, the end of first groove of described a plurality of parallel distributions connects successively by second groove, and first groove and the suprabasil protein band of described a plurality of parallel distributions are perpendicular.
Preferably, first groove of described a plurality of parallel distributions and second groove are perpendicular.
Preferably, described fluid comprises antibody.
Above-mentioned a plurality of first groove and suprabasil protein band are perpendicular, the parallel distribution of described a plurality of first grooves, and first groove and second groove are perpendicular, can guarantee measurement result accurately and accuracy, make the result more clear.
Preferably, first groove of described a plurality of parallel distributions is 5～15.
Preferably, described substrate is made through gold-plated glass sheet, polyaniline film or the film of poly pyrrole of chemical modification by PVDF membrane, polyvinylidene fluoride electrospinning silk film, surface, and more preferably, described substrate is made by PVDF membrane.
Preferably, described protein band is for to 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 by PVDF membrane.
Preferably, the described lamellar body lamellar body that to be macromolecular material form through thermoplastic or heat curing, preferably, described lamellar body is made by dimethyl silicone polymer or polymethylmethacrylate, and more preferably, described lamellar body is made by dimethyl silicone polymer (PDMS).
Preferably, the width of the miniflow groove on the lamellar body of the present invention is 50-200 μ m, and the degree of depth is 50-200 μ m, and length is 10cm-50cm.
On the other hand, the present invention also provides a kind of heterogeneous micro-fluidic Western blotting chip production method, may further comprise the 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 first groove of a plurality of parallel distributions, the end of first groove of described a plurality of parallel distributions connects successively by second groove, first groove and the suprabasil protein band of described a plurality of parallel distributions are perpendicular, preferably, first groove of described a plurality of parallel distributions and second groove are perpendicular;
Step 3: the lamellar body of the substrate of number of assembling steps 1 preparation and step 2 preparation, namely.
Preferably, in step 1, described substrate is made through gold-plated glass sheet, polyaniline film or the film of poly pyrrole of chemical modification by PVDF membrane, polyvinylidene fluoride electrospinning silk film, surface, and preferably, described substrate is made by PVDF membrane.
Preferably, described protein band is for to 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 by PVDF membrane.
Preferably, in step 2, the described lamellar body lamellar body that to be macromolecular material form through thermoplastic or heat curing, preferably, described lamellar body is made by dimethyl silicone polymer or polymethylmethacrylate, and more preferably, described lamellar body is made by dimethyl silicone polymer.
Preferably, the width of the miniflow groove on the described lamellar body is 50-200 μ m, and the degree of depth is 50-200 μ m, and length is 10cm-50cm.
Preferably, in step 2, when described lamellar body is made by dimethyl silicone polymer, specifically undertaken by the method that may further comprise the steps: dimethyl silicone polymer is mixed with hardening agent and pour the template with miniflow pipeline into, again with after its oven dry, the dimethyl silicone polymer that solidifies is downcut from template, in injection port and the sample outlet position punching of pre-design, namely.
Preferably, in step 2, described dimethyl silicone polymer and hardening agent are by 18: 1-24: 1 mass ratio mixes; Preferably, toasted 10-50 minute down in 80 ℃, more preferably, described dimethyl silicone polymer mixes with the mass ratio of hardening agent by 20: 1, toasts 25 minutes down in 80 ℃, and most preferably, described template is silicon chip.
Preferably, in step 3, specifically undertaken by the method that may further comprise the steps:
Step 3.1: will fix the poly-substrate oven dry of the protein band that separates by molecular weight, described drying condition is preferably 37 ℃, 60 minutes;
Step 3.2: the lamellar body that will be provided with at least one miniflow groove covers in the substrate that step 3.1 obtains, preferably, the lamellar body that is provided with at least one miniflow groove is covered in the substrate that step 3.1 obtains by first groove of a plurality of parallel distributions direction vertical with suprabasil protein band, flicking compresses, namely.
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 make, or the application in the kit of preparation protein detection, preferably, described protein detection is for optimizing the immune response antibody concentration.
On the other hand, the present invention also provides a kind of kit for detection of protein, and described kit comprises the heterogeneous micro-fluidic Western blotting chip that heterogeneous micro-fluidic Western blotting chip described above or method described above make.
The usage range of micro-fluidic Western blotting chip provided by the present invention comprises the multiple protein that detects simultaneously in the cell, detect the molecular weight standard of protein and optimize in the immune response antibody concentration etc., compared with prior art, micro-fluidic Western blotting chip provided by the present invention and corresponding detection method have remarkable advantages, can save time significantly, traditional immune detection system is 5h to whole night detection time, and the detection time of chip of the present invention is smaller or equal to 1h; It is few to detect the sample size that needs, and the test sample that traditional immune detection system detects ten kinds of samples to be needed is 100～200 μ g, and the test sample amount that ten kinds of samples of chip detection of the present invention need is 5～10 μ g; Consumption antibody is few, the antibody-solutions volume that traditional immune detection system detects a kind of albumen consumption 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 cost, 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.
Description of drawings
Below, describe embodiment of the present invention by reference to the accompanying drawings in detail, wherein:
Fig. 1 is synoptic diagram and the corresponding pictorial diagram of heterogeneous micro-fluidic Western blotting chip of the present invention, and A is the synoptic diagram of the substrate of fixing separative protein band among the figure, and B is the pictorial diagram of the substrate of fixing separative protein band among the figure; C is the synoptic diagram of heterogeneous micro-fluidic Western blotting chip among the figure, and D is the pictorial diagram of heterogeneous micro-fluidic Western blotting chip among the figure; E is the test findings synoptic diagram behind the heterogeneous micro-fluidic Western blotting chip detection albumen among the figure; F is the test findings behind the heterogeneous micro-fluidic Western blotting chip detection albumen among the figure, and wherein 1 is substrate, and 101 is suprabasil protein labeling, and 102 is the protein band of suprabasil separation; 2 is lamellar body, and 201 for forming the perforation of feeder connection, and 202 for forming the perforation of channel outlet, and 3 is the miniflow groove, and 301 is first groove, and 302 is second groove;
Fig. 2 be behind the heterogeneous micro-fluidic Western blotting chip detection albumen of the present invention test findings after partial enlarged drawing, among the figure 1,1 '; 2,2 '; 3,3 '; 4,4 '; 5,5 '; 6,6 '; 7,7 ' all represent the testing result at symmetric position place in the heterogeneous micro-fluidic Western blotting chip, and arrow is depicted as the antibody that corresponding microchannel adds, described antibody is respectively anti--actin antibody, anti-annexin antibody and anti-Pan-14-3-3 antibody;
Fig. 3 is the test findings that heterogeneous micro-fluidic Western blotting chip of the present invention is used the antibody detection protein of variable concentrations protein band and variable concentrations, 3-1 represents that the cracking holoprotein band of four NIH 3T3 cells adds the test findings of the antibody test of variable concentrations among the figure, the protein band that 1 expression separates among Fig. 3-1 is that concentration is the testing result of cracking holoprotein of the NIH 3T3 cell of 10 μ g, the protein band that 2 expressions separate is that concentration is the testing result of cracking holoprotein of the NIH 3T3 cell of 5 μ g, the protein band that 3 expressions separate is that concentration is the testing result of cracking holoprotein of the NIH 3T3 cell of 2.5 μ g, and the protein band that 4 expressions separate is that concentration is the testing result of cracking holoprotein of the NIH 3T3 cell of 1.25 μ g; The 1 detection of antibodies results change that adds variable concentrations among the 3-2 presentation graphs 3-1 among the figure, among the figure among the 3-3 presentation graphs 3-1 testing result thirdly in 1,2,3,4 change;
The test findings that Fig. 4 compares with traditional immunologic detection method detection albumen for heterogeneous micro-fluidic Western blotting chip of the present invention, A represents heterogeneous micro-fluidic Western blotting chip of the present invention among the figure, the partial enlarged drawing of B presentation graphs A, C represents the box of hatching of common immunologic detection method, D represents that heterogeneous micro-fluidic Western blotting chip of the present invention and traditional immunologic detection method detect albumen result's comparison, 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, and arrow is depicted as protein band among the figure;
Fig. 5 is the result of the close albumen of heterogeneous micro-fluidic Western blotting chip isolated molecule amount of the present invention, and 1-7 represents to add in the heterogeneous micro-fluidic Western blotting chip different detection of antibodies results respectively among the figure.
Embodiment
Employed term unless other explanation is arranged, generally has the implication of those of ordinary skills' common sense in the present invention.
Below in conjunction with concrete preparation embodiment and Application Example, and comparable data is described the present invention in further detail.These embodiment are in order to demonstrate the invention, but not limit the scope of the invention by any way.
Used anti-beta-actin antibody (anti-β-actin antibody), anti-annexin antibody (anti-Annexin antibody) in following examples, anti-Pan-14-3-3 antibody is available from SantaCruz company.
Present embodiment illustrates heterogeneous micro-fluidic Western blotting chip production method of the present invention
1. the preparation of micro-fluidic chip template
The main process of preparation is photoetching, namely utilizes the figure on all four photoresist silicon chip template of photoresist on the mask that the characteristics of convertibility matter under the ultraviolet ray irradiation are made and designed.Concrete preparation method can be referring to Y Xia, G.Whitesides, Annual Review of Materials Science 1998,28,15.
2. be provided with the preparation of the lamellar body of a plurality of miniflow grooves
The preparation method is soft lithographic technique, the preparation material is dimethyl silicone polymer (PDMS, poly-dimethylsiloxane), it is transparent and liquid thickness under common state, through curable with hardening agent (184 silicone elastomer curing agent are available from Dow corning company) reaction and heating back.Utilize PDMS the projection figure on the silicon chip template can be converted to corresponding pipeline figure, thereby obtain being provided with the lamellar body of a plurality of miniflow grooves.Remain at forming temperature under 80 ℃ the situation, the present invention is provided with raw material mass mixture ratio and the high-temperature molding time of the lamellar body of a plurality of PDMS miniflow grooves and optimizes to preparation, shown in the actual conditions table 1.
The proportioning of table 1 dimethyl silicone polymer and hardening agent is different respectively organizes material
Concrete experimental procedure is as follows:
(1) according to (SYLGARD 184 silicon rubber of the PDMS monomer shown in the table 1, available from Dow corning company) and hardening agent (SYLGARD 184 silicon rubber hardening agent, available from Dow corning company) the quality proportioning, take by weighing raw material and mixing respectively, stir about reduced pressure in vacuum pump and pumps bubble to evenly in 10 minutes.
(2) pour the potpourri of PDMS and hardening agent in the silicon chip template with miniflow pipeline, be positioned over and pump bubble in the vacuum pump.
(3) put into 80 ℃ in baking oven, toast according to the time shown in the table 1, photoresist outstanding on the silicon chip can stamp groove with PDMS.
(4) PDMS that solidifies is downcut from template, and use the syringe punching of band syringe needle at the injection port place of pre-design, with Scotch (available from 3M) white glues band cleaning PDMS miniflow groove surface.
(5) PVDF membrane is placed on the microslide, the lamellar body that is provided with a plurality of PDMS miniflow grooves is covered on PVDF membrane, be provided with the lamellar body size of a plurality of PDMS miniflow grooves greater than PVDF membrane.With hand flicking is compressed PVDF membrane and the lamellar body that is provided with a plurality of PDMS miniflow grooves.
(6) drip 30 μ L blue inks with liquid-transfering gun at the place, aperture of chip upper channel end, other end syringe pump at passage, the complexity of record and observation liquid admission passage and the seepage situation of passage, the result shows, when PDMS monomer and the hardening agent quality proportioning by 20: 1, at 80 ℃ down under 25 minutes the condition of oven dry, liquid feeds passage easily, and do not have the seepage phenomenon, effect is better.Miniflow groove specification on the made lamellar body of getting ready is: width: 50-200 μ m, the degree of depth: 50-200 μ m, length: 10cm-50cm, table 2 specific as follows.
The microchannel specification that table 2 group number 1-9 makes
3. electrophoresis
In polyacrylamide gel, be band by the molecular weight size separation with protein mixture by electrophoresis apparatus, experimental procedure is identical with the electrophoresis step in traditional immunoblot experiment, specifically can be referring to H.Towbin, T.Staehelin, J.Gordon, Proceedings of the National Academy of Sciences of the United States of America 1979,76,4350.
4. electrotransfer method
Under electric field action, (be the trace perforated membrane with albumen by transferring to PVDF membrane in the polyacrylamide gel, by the solvent evaporates pore-forming, the aperture is 0.2-0.45 μ m, available from milipore company), perhaps polyvinylidene fluoride electrospinning silk film (nonwoven fabrics that nano-scale fiber forms, the aperture is nanoscale, concrete preparation method can be referring to Yang, D.Y.; Niu, X.; Liu, Y Y; Wang, Y; Gu, X.; Song, L.S.; Zhao, R.; Ma, L.Y; Shao, Y.M.; Jiang, X.Y Adv Mate:2008,20,4770-4775.) on, it is identical that experimental procedure is changeed step with the electricity in traditional immunoblot experiment, specifically can be referring to H.Towbin, T.Staehelin, J.Gordon, Proceedings of the National Academy of Sciences of the United States of America 1979,76,4350.
5. the assembling of micro-fluidic chip
Chip assembling namely is will fix the PVDF membrane of albumen to combine with the lamellar body that is provided with a plurality of PDMS miniflow grooves, forms the passage of sealing, makes the liquid can be at suction passage under the negative pressure, and passage seepage can not occur.The assembling of micro-fluidic chip specifically may further comprise the steps:
(1) PVDF membrane that will shift protein band is placed in the baking oven and dries, and 37 ℃, 60 minutes.
(2) PVDF membrane is placed on the microslide, the lamellar body that is provided with a plurality of PDMS miniflow grooves is covered on PVDF membrane.
(3) with have gentle hands by PVDF membrane and the lamellar body that is provided with a plurality of PDMS miniflow grooves are compressed.Because PDMS has elasticity and viscosity, can good seal so be provided with lamellar body and the PVDF membrane of a plurality of PDMS grooves.
6. the micro-fluidic chip immunofluorescence detects
By antigen-antibody identification, in different passages, hatch different antibody, detect the multiple protein on the PVDF membrane.Specifically may further comprise the steps:
(1) passage inner sealing: drip 3 μ L confining liquid (5% volume bovine serum albumin BSA at the place, aperture of chip upper channel end with liquid-transfering gun, phosphate buffer PBS), the other end syringe pump at passage makes solution be full of passage, after hatching 10 minutes, extract solution out with syringe.
(2) primary antibodie is hatched: primary antibodie is diluted with antibody diluent, and optimized the prescription of this antibody diluent, 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 proportionings phosphate buffer.In different passages, feed 40 μ L at the antibody diluent of different albumen respectively, hatch after 30 minutes and extract out.Open the 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 the confining liquid (its prescription synantibody dilution) swayed 20 minutes.
(5) two anti-hatching: PVDF membrane is put into fluorescently-labeled two anti-dilutions (its prescription synantibody dilution) lucifuge swayed 30 minutes.
(6) shake and wash: shake with PBST (0.2% volume Tween-20) lucifuge and wash PVDF membrane three times, each 10 minutes.
(7) fluoroscopic examination: PVDF membrane is put into the multi-functional image analysis system of Typhoon Trio TM (Amersham company) detection signal, and parameter arranges as follows: emission optical filter 520nm, and laser 488nm, PMT480, susceptibility is medium.
(8) observe and record every group fluorescence signal and background, the result shows that when containing 0.2% volume Tween-20 in the phosphate buffer of primary antibodie dilution, signal to noise ratio (S/N ratio) is the strongest.
Present embodiment is used for the heterogeneous detectability of explanation 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 g NIH 3T3 cells is gone up respectively in sample to two swimming lane, carry out electrophoresis according to the preferred time, change film, then the film of load protein is dried, the quality proportioning of using PDMS monomer and hardening agent to press 20: 1 is prepared the PDMS chip 80 ℃ of conditions of toasting 25 minutes down, and film and chip are combined by light pressure.In passage, feed different antibody from left to right respectively, particularly, 1 and 2: the potpourri that feeds anti-annexin antibody and two kinds of antibody of anti-Pan-14-3-3 antibody; 3 and 4: the potpourri that feeds anti--actin antibody (anti--actin antibody) and two kinds of antibody of anti-Pan-14-3-3 antibody; 5 and 6: the potpourri that feeds anti--actin antibody (anti--actin antibody) and two kinds of antibody of anti-annexin antibody, 7: feed anti--actin antibody (anti--actin antibody), anti-annexin antibody (anti-Annexin antibody), the potpourri of three kinds of antibody of anti-Pan-14-3-3 antibody, the order of the antibody of 1 '-7 ' feeding is with from left to right 1-7 is identical from right to left, the preferred concentration of antibody is 1: 80, the anti-mouse IgG of the fluorescently-labeled rabbit of two anti-uses, and concentration is 1: 2000.The result as shown in Figure 2.
Present embodiment is used for the ability that explanation heterogeneous micro-fluidic Western blotting chip of the present invention is used for half-quantitative detection.
As described in example 1 above, last 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 g NIH 3T3 cells, carry out electrophoresis according to the preferred time, change film, with the film oven dry of load protein, use PDMS monomer and hardening agent are pressed 20: 1 quality proportioning then, prepare the PDMS chip 80 ℃ of conditions of drying 25 minutes down, film and chip are combined by light pressure.Take the anti-annexin antibody of SantaCruz company, initial concentration is taken as 1: 20, and the gradient dilution antibody-solutions was respectively 1: 40 then, and 1: 80,1: 160,1: 320,1: 640,1: 1280.The antibody of these seven kinds of concentration is fed respectively among 7 microchannels, and two anti-concentration are 1: 2000.Obtain the dot chart that the antigen of the antibody of variable concentrations and different amounts acts on respectively.Shown in accompanying drawing 3-1, different reaction results neatly clearly is arranged in one as a result in the picture.These results are done the quantitative test of fluorescence intensity, can obtain the curve that fluorescence intensity changes with the antigen-antibody of setting at an easy rate.Respectively, shown in Fig. 3-2, for a certain amount of antigen amount, use the antibody of variable concentrations, obtain antibody titer curve, this curve shows is fixed on suprabasil quantitative antigen for binding ability and the degree of saturation of antibody, is used for judging the preferred concentration of follow-up test; Shown in Fig. 3-2, for a certain amount of antibody concentration, use not antigen as such, obtain the titration curve of antigen, this curve can illustrate what of amount of antigen, is used for judging the concentration of this specific objective albumen of follow-up test in unknown sample.This dot chart explanation the present invention is once obtaining this two Useful Informations in the experiment.
Present embodiment is used for the ability that explanation heterogeneous micro-fluidic Western blotting chip of the present invention shortens the immune response time.
As described in embodiment 1, NIH-3T3 cell (available from ATCC) the cracking holoprotein of 20 μ g is loaded in the albumen swimming lane, according to the preferred time, voltage, temperature conditions carries out electrophoresis, changes film, drying, the concentration of primary antibodie are 1: 200,7 interior primary antibodies that feed same concentrations of passage, its incubation time is set at 2.5min, 5min, 10min respectively, 20min, 30min, 60min, 90min, two anti-concentration are 1: 2000, incubation time is set at 60min.Experiment as a comparison, for traditional protein immunoblot method, equally the concentration with primary antibodie is set at 1: 200, prepares 7 same wares of hatching, 7 primary antibodie incubation times of hatching in the ware also are set at 2.5min respectively, 5min, 10min, 20min, 30min, 60min, 90min, two anti-concentration and incubation times also were respectively 1: 2000 and 60min.Reaction respectively, the time dependent degree of fluorescence intensity that compares the present invention and traditional western blotting method after the rinsing, as shown in Figure 4, can see that the fluorescence intensity among the present invention can reach capacity, and therefore can obviously reduce the required time of normal reaction in the relatively short time (20min).
Present embodiment illustrates that heterogeneous micro-fluidic Western blotting chip of the present invention is used for differentiating the ability of the adjacent protein band of the immune marking.
After the electrophoretic separation and electrotransfer of routine, choose two protein annexin (36kD) and GAPDH (35.8kD) that molecular weight is close in the holoprotein, if adopt the reaction of traditional immune marking, because two bands are separated by too near, can't distinguish, can only adopt independently to detect for twice and realize identifying.And in the present invention, owing to different microchannels can be separated different antibody, so it is close to detect a plurality of molecular weight simultaneously in once testing, the protein band that the position is adjacent.For example, the microchannel bedding among the present invention on a band to be distinguished, will be in the passage be fed the antibody of anti-annexin, feed the antibody of anti-GAPDH in another adjacent passage, concrete: among the 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 antibody (anti--actin antibody), with reference to the reaction of the process of the step among the embodiment 1, after the development, as shown in Figure 5, there is signal in the place of passage 2 and passage 3 correspondences, illustrates that then this band is to be composited by the band of anti-annexin and the band of GAPDH.
Present embodiment illustrates the strict antibody-solutions of separating of microchannel in the heterogeneous micro-fluidic Western blotting chip of the present invention, guarantees the ability of sealing.
Use PDMS monomer and the quality proportioning of hardening agent by 20: 1,80 ℃ down 25 minutes conditions of baking prepare the PDMS chip, with the PVDF membrane applying of itself and load protein.Utilize the flat board of plastics with PVDF membrane and PDMS chip pressing then, take off plastic plate, the two contact position color shoals, and illustrates that the two applying is all right.Feed antibody-solutions this moment, can make antibody-solutions flow to channel outlet smoothly from feeder connection easily, and the leakage of liquid does not take place.
Present embodiment illustrates that the microchannel in the heterogeneous micro-fluidic Western blotting chip of the present invention has the ability of auto injection.
At first PDMS chip and clean glass sheet are fitted, in passage, feed the skimmed milk power solution with the phosphate buffer dissolving then, used the different proportionings of different skimmed milk powers in solution, making its massfraction is 1%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, in passage, hatched 5 minutes, extract out then.To be fitted by PDMS chip and PVDF membrane that skimmed milk power solution was hatched, compress, drip antibody-solutions at the feeder connection place.The result shows that the antibody-solutions at feeder connection place can be under the driving that does not have external force in the automatic flow channel.Learn that by comparative experiments when the massfraction of milk powder in the skimmed milk power solution of selecting for use was 5%, the auto injection effect was the most obvious.
Claims (13)
1. heterogeneous micro-fluidic Western blotting chip, comprise the substrate of fixing separative protein band and the lamellar body stacked with it, described lamellar body be provided with at least one miniflow groove with the mutually stacked surface of substrate, described at least one miniflow groove and described substrate form at least one microchannel for fluid circulation jointly, and another surface of described lamellar body is gone up in the position corresponding to two ports of described microchannel and is respectively equipped with feeder connection and the channel outlet of perforation to form described microchannel.
2. heterogeneous micro-fluidic Western blotting chip according to claim 1, it is characterized in that, described miniflow groove comprises first groove of a plurality of parallel distributions, the end of first groove of described a plurality of parallel distributions connects successively by second groove, first groove and the suprabasil protein band of described a plurality of parallel distributions are perpendicular, preferably, first groove and second groove of described a plurality of parallel distributions are perpendicular, more preferably, described fluid comprises antibody, more preferably, first groove of described a plurality of parallel distributions is 5～15.
3. heterogeneous micro-fluidic Western blotting chip according to claim 1 and 2, it is characterized in that, described substrate is made through gold-plated glass sheet, polyaniline film or the film of poly pyrrole of chemical modification by PVDF membrane, polyvinylidene fluoride electrospinning silk film, surface, preferably, described substrate is made by PVDF membrane.
4. according to each described heterogeneous micro-fluidic Western blotting chip in the claim 1 to 3, it is characterized in that, described protein band is for transferring to suprabasil protein band from polyacrylamide gel, preferably, described transfer is electrotransfer, more preferably, described protein band is transferred in the substrate of being made by PVDF membrane.
5. according to each described heterogeneous micro-fluidic Western blotting chip in the claim 1 to 4, it is characterized in that, the described lamellar body lamellar body that to be macromolecular material form through thermoplastic or heat curing, preferably, described lamellar body is made by dimethyl silicone polymer or polymethylmethacrylate, more preferably, described lamellar body is made by dimethyl silicone polymer.
6. according to each described heterogeneous micro-fluidic Western blotting chip in the claim 1 to 5, it is characterized in that the width of the miniflow groove on the described lamellar body is 50-200 μ m, the degree of depth is 50-200 μ m, and length is 10cm-50cm.
7. according to each described heterogeneous micro-fluidic Western blotting chip production method of claim 1 to 6, may further comprise the 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 first groove of a plurality of parallel distributions, the end of first groove of described a plurality of parallel distributions connects successively by second groove, first groove and the suprabasil protein band of described a plurality of parallel distributions are perpendicular, preferably, first groove of described a plurality of parallel distributions and second groove are perpendicular;
Step 3: the lamellar body of the substrate of number of assembling steps 1 preparation and step 2 preparation, namely.
8. according to claim 7 mutually micro-fluidic Western blotting chip production method, it is characterized in that, in step 1, described substrate is PVDF membrane, polyvinylidene fluoride electrospinning silk film, the surface is through the gold-plated glass sheet of chemical modification, the substrate of polyaniline film or film of poly pyrrole, preferably, described substrate is the substrate of PVDF membrane, preferably, described protein band is for transferring to suprabasil protein band from polyacrylamide gel, preferably, described transfer is electrotransfer, and more preferably, described protein band is transferred in the substrate of being made by PVDF membrane.
9. according to claim 7 or 8 described mutually micro-fluidic Western blotting chip production method, it is characterized in that, in step 2, the described lamellar body lamellar body that to be macromolecular material form through thermoplastic or heat curing, preferably, described lamellar body is made by dimethyl silicone polymer or polymethylmethacrylate, and more preferably, described lamellar body is made by dimethyl silicone polymer, preferably, the width of the miniflow groove on the described lamellar body is 50-200 μ m, and the degree of depth is 50-200 μ m, and length is 10cm-50cm.
10. according to claim 9 mutually micro-fluidic Western blotting chip production method, it is characterized in that, in step 2, when described lamellar body is made by dimethyl silicone polymer, specifically undertaken by the method that may further comprise the steps: dimethyl silicone polymer is mixed with hardening agent and pour the template with miniflow pipeline into, with after its oven dry, the dimethyl silicone polymer that solidifies is downcut from template again, in injection port and the sample outlet position punching of pre-design, namely;
Preferably, described dimethyl silicone polymer and hardening agent are by 18: 1-24: 1 mass ratio mixes; Toasted 10-50 minute down in 80 ℃, more preferably, described dimethyl silicone polymer mixes with the mass ratio of hardening agent by 20: 1, toasts 25 minutes down in 80 ℃, and most preferably, described template is silicon chip.
11. according to each described heterogeneous micro-fluidic Western blotting chip production method in the claim 7 to 10, it is characterized in that, in step 3, specifically undertaken by the method that may further comprise the steps:
Step 3.1: will fix the substrate oven dry of the protein band that separates by molecular weight, described drying condition is 37 ℃, 60 minutes;
Step 3.2: the lamellar body that will be provided with at least one miniflow groove covers in the substrate that step 3.1 obtains, preferably, the lamellar body that is provided with at least one miniflow groove is covered in the substrate that step 3.1 obtains by first groove direction of a plurality of parallel distributions direction vertical with suprabasil protein band, flicking compresses, namely.
12. the heterogeneous micro-fluidic Western blotting chip that makes according to each described method in each described heterogeneous micro-fluidic Western blotting chip or the claim 7 to 11 in the claim 1 to 6 in protein detection application or for the preparation of the application in the kit of protein detection, preferably, described protein detection is for optimizing the immune response antibody concentration.
13. the kit for detection of protein, described kit comprise the heterogeneous micro-fluidic Western blotting chip that each described method makes in each described heterogeneous micro-fluidic Western blotting chip in the claim 1 to 6 or the claim 7 to 11.
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