CN1212543C - Method for fixing biological macro molecule pattern on polymer active surface by micro transfer technology - Google Patents
Method for fixing biological macro molecule pattern on polymer active surface by micro transfer technology Download PDFInfo
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- CN1212543C CN1212543C CN 02160335 CN02160335A CN1212543C CN 1212543 C CN1212543 C CN 1212543C CN 02160335 CN02160335 CN 02160335 CN 02160335 A CN02160335 A CN 02160335A CN 1212543 C CN1212543 C CN 1212543C
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Abstract
The present invention discloses a method for patternizing and fixing biologic macromolecules on the active surfaces of polymers by microtransfer technology, which comprises the following steps that firstly, biologic macromolecules are selectively loaded in the microwell of a polydimethylsiloxane stamp; one layer of condensed water is formed on the surface of the stamp, the biologic macromolecules are dissolved again by utilizing the original water in the microwell and water transversely diffused in contact areas after the stamp and the modified polymer film are pressed, and thus, the biologic macromolecules in a solution form chemically react with surface base groups so as to realize that biologic macromolecules are patternized and fixed on the active surfaces of the polymers. The present invention has the advantages of simple and flexible technology, favorable repeatability, high contrast degree and firmness of the obtained micropatterns, and low requirements to the surface property and the microwell depth of the stamp, and the preparation method is a simple cheap effective method for patternizing and fixing the biologic macromolecules, particularly applicable to biomolecule / active surface system with low reactivity. The present invention has a favorable application prospect in the fields of biologic device construction based on the cells, tissue engineering, basic research of cell biology, etc.
Description
Technical field
The present invention relates to little transmission technology in the macromolecular method of polymer active patterned surface fixed biologically.Be specifically " splendid attire " in the little well of dimethyl silicone polymer (PDMS) seal surface array thus the protide biological macromolecule solns be delivered to the polymer active surface and make the reaction of itself and surperficial reactive group can carry out under solution state realizing more easily that the protide biomacromolecule fixes at the patterning of polymer surfaces.
Background technology
Along with the development of modern biotechnology, the fixing extensive concern that caused of the patterning of biomacromolecule.At the big molecule of surface of polymer material patterning fixed biologically and cell to specific Biological Detection, make up and the reparation of ANN all has great practical value based on the biology sensor of living cells.At present, adopt the micro-contact printing technology can be on surfaces such as gold, glass, silicon and polymkeric substance to realize that by the mode of physisorption the patterning of biomacromolecule fixes.But the shortcoming that has easy desorption and poor stability.How to realize that fixedly securing of biomacromolecule pattern is the problem that needs solution at present.
Polymeric material becomes the fixing desirable base material of biomacromolecule patterning because of its good physical property and chemical property.Can introduce various reactive groups easily at polymer surfaces, the mode by covalent bonding realizes that the fastness of biomacromolecule fixes.Traditional reactive micro-printing technology can realize easily that the patterning fastness of biological micromolecule fixes.But be " pollution " of avoiding the solvent transverse dispersion to cause, generally require to dry up template or keep the minute quantity solvent before the bat printing, or require the reaction time short.This is disadvantageous to the biomacromolecule of non-liquid state and the reaction of surperficial reactive group, particularly to the biomacromolecule/active surface system of low reaction activity.
Summary of the invention
The purpose of this invention is to provide the little transmission technology of a kind of usefulness in the macromolecular method of polymer surfaces patterning fixed biologically.
Method of the present invention is earlier the biomacromolecule selectivity " to be packed into " in little well of dimethyl silicone polymer (PDMS) seal, and then at its surface formation one deck condensate water, the water that utilizes seal and modifying polymer film to compress original water and contact area transverse dispersion in the little well in back dissolves biomacromolecule again, thereby form and surface group generation chemical reaction with solution are implemented in the big molecule of polymer active patterned surface fixed biologically.Specifically may further comprise the steps:
1) polymer-modified with conventional chemical modification or plasma modification method, polymer surfaces introduce can with hydroxyl, carboxyl, the amino group of biomacromolecule reaction;
2) the preparation surface contains the PDMS seal of little well, and at it biological macromolecule solns of one side dip-coating 0.1～20mg/ml concentration of little well is arranged, and slowly dries up with nitrogen stream;
3) get above-mentioned thin polymer film a slice and impressed in the seal surface pressure 10～200g/cm 0.1～10 hour
2, remove the biomacromolecule that is adsorbed on zone between little well with selectivity;
4) seal was kept 0.1～5 minute under-18～0 ℃ of temperature, again under 20～37 ℃ of temperature, relative humidity is to leave standstill 0.1～5 minute in 50～99% the environment, forms one deck condensation droplet on its surface;
5) under 0～100 ℃, immediately this seal is impressed in the reforming polymer surface of step 1) gained 0.1～10 hour pressure 10～200g/cm
2, 4～50 ℃ of preferred temperature;
6) uncover seal, the damping fluid or the aqueous solution of the corresponding pH value of usefulness biomacromolecule are washed, and soak to dry up after 24 hours, promptly obtain little pattern of biomacromolecule.
Among the present invention, described polymkeric substance be can by chemical modification or plasma modification its surperficial generation can with the polymkeric substance of biomacromolecule reactive activity group, as poly-(L-lactic acid) (PLLA), poly-(D-lactic acid) (PDLA), polycaprolactone (PCL), poly-(D, L-lactic acid-altogether-glycollic acid) (PLGA), polyurethane (PU), polyethylene terephthalate (PET), polypropylene (PP), polystyrene (PS) etc.
Above-mentioned steps 3) saidly impressing in the thin polymer film on seal surface to be the polymkeric substance identical with step 1), can be the polymkeric substance that does not pass through modification, also can be the polymkeric substance through modification.Selectivity is removed biomacromolecule and is meant contact with the thin polymer film of the impression biomacromolecule in zone of seal jut is shifted and keeps biomacromolecule in little well.
Preparation surface contains the PDMS seal of little well in the invention, be earlier by conventional bed die that contains the micron order columnar protrusions of " photoetching " technique construction, again at this bed die surface casting PDMS prepolymer, uncovers after the heat cross-linking curing and makes.Generally be glass or silicon face spin coating ultraviolet type photoresist in smooth cleaning, has ultraviolet photoetching under the optical mask plate of characteristic pattern again, develop, make the original template that contains the micron order columnar protrusions, make bed die with this then, pour into a mould the potpourri of PDMS prepolymer and crosslinking chemical more thereon, uncover after being heating and curing and make, 40～100 ℃ of general solidification temperatures, 6～24 hours time.
Among the present invention, described biomacromolecule is meant protide biomacromolecule and derivant thereof, comprises albumin, fibronectin, laminin, poly-D-lysine, collagen and gelatin etc.Step 2) said biological macromolecule solns is meant the buffer soln or the aqueous solution of its corresponding pH value, as the PBS solution of albuminous phosphate buffer (PBS) solution, gelatin, and acetic acid solution of 0.3% of collagen or shitosan etc.
Little pattern for the biomacromolecule that obtains with the inventive method, can detect its existence with laser confocal microscope (CLSM) or fluorescent microscope, for ease of observing, at this moment used biomacromolecule can be used fluorescent dye such as marks such as fluorescein, rhodamine in advance.
Adopt the inventive method, by the diameter of regulating little well in the PDMS seal and the little pattern of biomacromolecule that spacing can obtain corresponding size and spacing.The amount of regulating condensate water can be controlled the quality of the little pattern of biomacromolecule, and condensation water quantity can be regulated from condensation time and two factors of relative humidity.
Technology simple and flexible of the present invention, good reproducibility, the little pattern that is obtained has very high contrast and firmness, is a kind of macromolecular effective ways of patterning fixed biologically of cheap and simple.Adopt the method for " drying up-shift-condensation " to realize " splendid attire " of biological macromolecule solns in little well more conveniently.Each little well becomes a microreactor, makes that the reaction between biomacromolecule and surface group can be carried out the pattern " pollution " of having avoided the solvent transverse dispersion to cause again simultaneously with the speed of working as with the interface crosslinking reacting phase that carries out in solution.Than traditional reactive micro-printing technology, the present invention is also lower to seal surface hydrophilic and hydrophobic and the requirement of little well depth, and the PDMS seal need not carry out the plasma water wettability to be handled, and little well depth need not be too dark, can make with conventional " photoetching " technology.The present invention is specially adapted to have the big molecule of polymer surfaces patterning fixed biologically of general reactivity.In specific Biological Detection, make up and the fields such as reparation of ANN have a good application prospect based on the biology sensor of living cells.
Description of drawings
Fig. 1 is at the macromolecular process chart of polymer-modified active surface patterning fixed biologically with little transmission technology.1 is illustrated in one side dip-coating 0.1～20mg/ml biological macromolecule solns that the PDMS seal has little well among the figure; 2 expressions slowly dry up seal with nitrogen stream; 3 are illustrated in seal surface imprint thin polymer film is removed zone between little well with selectivity biomacromolecule; 4 are illustrated in the seal surface forms condensate water; 5 expressions impress seal in reforming polymer surface.6 expressions are uncovered seal and are washed the polymer film that patterning has been fixed biomacromolecule.
Fig. 2 is the atomic force microscope photo that the PDMS seal of the little well of array is contained on used surface in the invention.
Fig. 3 (a) is that albumin shows light tone because of the mark of fluorescein isothiocynate (FITC) with laser co-focusing micro-(CLSM) photo of the inventive method at the fixing albumin (BSA) of the polycaprolactone surface of aldehyde radicalization (PCL-CHO) patterning; (b) be (a) corresponding fluorescence intensity analysis.
Fig. 4 be with the inventive method at the fixing CLSM photo of PDMS seal before and after the albumin of polycaprolactone (PCL-CHO) patterned surface of aldehyde radicalization, wherein (a) be patterning fixing before, be after patterning is fixed (b).Albumin shows light tone because of the mark of FITC.
Fig. 5 (a) is that albumin shows light tone because of the mark of FITC with the CLSM picture of existing reactive micro-printing technology at the fixing albumin (BSA) of PCL-CHO patterned surface; (b) be (a) corresponding fluorescence intensity analysis.
Fig. 6 is at polycaprolactone (PCL-CHO) patterned surface of aldehyde radicalization fixedly during albumin with the inventive method, the defect pattern that causes because of the condensation water quantity discomfort, (a) circular loop pattern of obtaining when not enough for condensate water wherein, (b) pattern " pollution " that causes when excessive for condensate water.
Embodiment
Below with instantiation the present invention is elaborated.
Example 1
The little transmission technology of usefulness of the present invention may further comprise the steps in the macromolecular method of polymer active patterned surface fixed biologically:
1. preparation surface is with the PCL film of free aldehyde radical: it is the aqueous isopropanol of the hexane diamine of 10% (wt) that the PCL film is placed concentration, 37 ℃ of following aminolysis 10 minutes, and the PCL surface is a large amount of free aminos because of aminolysis produces.The PCL film that amido is rich on the surface is put into 2% glutaraldehyde solution again, and 37 ℃ of following coupling reactions 30 minutes promptly make the PCL film that free aldehyde radical is rich on the surface.This moment, film presented light red or orange red because of the aldehyde radical reaction.
2. the PDMS seal of the little well of array is contained on the preparation surface: prepare an original bed die with conventional " photoetching " technology earlier, promptly at 3 a clean * 3cm
2Slide surface with 2000 rev/mins speed spin coating BP218 ultraviolet eurymeric photoresist, with being placed in 90 ℃ the baking oven baking 30 minutes, rotation exposure 150 seconds under 1000 watts uviol lamp, photomask was again developed 45 seconds in 2% NaOH solution then, noted the homogeneity of developing.Because of used photomask contains the blind hole of microarray, the projection post of corresponding microarray is promptly contained on the bed die surface that finally makes.At this original bed die surface casting PDMS prepolymer, this prepolymer has added 10: 1 crosslinking chemical of mass ratio.Crosslinking curing is uncovered after 12 hours and is promptly made the soft seal of PDMS in 60 ℃ of baking ovens, and the little well of the array of the correspondingly-sized that its surface is contained can be with atomic force microscope observation to (see figure 2).
3. the selectivity splendid attire of albumin (BSA) in little well: the albumin of marked by fluorescein isothiocyanate (FITC-BSA) is made into the PBS buffer soln that concentration is 1.0mg/ml, drip the one side that little well is arranged in above-mentioned PDMS, catch up with evenly with glass bar back and forth, and slowly dry up with nitrogen stream.Gently place the PDMS seal to scribble the one side of FITC-BSA, and use 100g/cm a slice PCL film
2Pressure compress, kept under the room temperature 1 hour.Uncover the PCL film, the BSA that is adsorbed in zone between little well just has been transferred the BSA of only remaining splendid attire in the little well of array.
4. the formation of condensate water: this PDMS seal that fills BSA is placed-12 ℃ refrigerator and cooled but 2 minutes, is that (25 ℃) left standstill 1 minute in 80～90% the atmospheric environment in relative humidity again, promptly forms the skim condensate water on its surface.
5. this seal is inverted in rapidly on the aldehyde radical PCL film, and uses 100g/cm
2Pressure compress, kept under the room temperature 3 hours.Thereby the water of original water and contact area transverse dispersion makes biomacromolecule dissolve form and surface group generation chemical reaction with solution again in the little well in the tight back of seal and aldehyde radical PCL mould, and each little well becomes a microreactor.
6. uncover the PDMS seal gently, fixed the PCL film of biomacromolecule with a large amount of PBS damping fluids flushing patternings, rinsing 24 hours in PBS again, nitrogen dries up.
With laser confocal microscope (CLSM) observations: Fig. 3 a is the BSA that obtains with the said method microarray pattern on aldehyde radical PCL film surface, and b is the fluorescence intensity analysis of this pattern correspondence, illustrates to use little transmission method
Can obtain very high pattern contrast, confirm the validity of little transmission method.The a of Fig. 4 and b are respectively the BSA pattern in the PDMS seal before and after little transmit, confirm that BSA has been delivered to PCL film surface fully from the little well of PDMS.
Example 2
This example is and comparative example of the present invention.Other step is with example 1, but uses fixedly albumin of existing reactive micro-contact printing technology patterning instead.Promptly at the PDMS seal one side dip-coating concentration of little well being arranged is the PBS buffer soln of the FITC-BSA of 1.0mg/ml, and slowly dries up with nitrogen stream, and bat printing is on the PCL of aldehyde radicalization film rapidly.Pressure, time, temperature reach with post-flush all with example 1.CLSM observes discovery, the BSA pattern that obtains with the method (Fig. 5 a) than the BSA pattern dimness that obtains with the present invention many, its corresponding fluorescence intensity is analyzed also low many (Fig. 5 b) of display pattern contrast.
Example 3
Other step is with example 1, but the PDMS seal is that (25 ℃) time of repose is 0.1 minute or 3 minutes in 80～90% the atmospheric environment in relative humidity, observes its influence to pattern quality.(Fig. 6 a), latter Ze Yin condensate water is excessive and caused pattern " pollution " (Fig. 6 b) because of the condensate water deficiency has formed circular loop pattern to find the former.This explanation is with big minute period of the day from 11 p.m. to 1 a.m of little transmission method patterning fixed biologically, and the condensation water quantity that forms on the PDMS surface has a significant impact the quality of final pattern, and mistake is low or too highly all will cause pattern defect.
Claims (5)
- With little transmission technology in the macromolecular method of polymer surfaces patterning fixed biologically, it is characterized in that may further comprise the steps:1) polymer-modified with conventional chemical modification or plasma modification method, polymer surfaces introduce can with hydroxyl, carboxyl, the amino group of biomacromolecule reaction;2) the preparation surface contains the dimethyl silicone polymer seal of little well, and at it biological macromolecule solns of one side dip-coating 0.1～20mg/ml concentration of little well is arranged, and slowly dries up with nitrogen stream;3) get above-mentioned thin polymer film a slice and impressed in the seal surface pressure 10～200g/cm 0.1～10 hour 2, remove the biomacromolecule that is adsorbed on zone between little well with selectivity;4) seal was kept 0.1～5 minute under-18～0 ℃ of temperature, again under 20～37 ℃ of temperature, relative humidity is to leave standstill 0.1～5 minute in 50～99% the environment, forms one deck condensation droplet on its surface;5) under 0～100 ℃, immediately this seal is impressed in the reforming polymer surface of step 1) gained 0.1～10 hour pressure 10～200g/cm 26) uncover seal, the damping fluid or the aqueous solution of the corresponding pH value of usefulness biomacromolecule are washed, and soak to dry up after 24 hours, promptly obtain little pattern of biomacromolecule.
- 2. press the little transmission technology of the described usefulness of claim 1 in the macromolecular method of polymer surfaces patterning fixed biologically, it is characterized in that said polymkeric substance is poly (l-lactic acid), poly-D-lactic acid, polycaprolactone, poly-D, L-lactic acid-be total to-glycollic acid, polyurethane, polyethylene terephthalate, polypropylene, polystyrene.
- 3. press the little transmission technology of the described usefulness of claim 1 in the macromolecular method of polymer surfaces patterning fixed biologically, it is characterized in that the surface contains the dimethyl silicone polymer seal of little well, be earlier by conventional bed die that contains the micron order columnar protrusions of " photoetching " technique construction, again at this bed die surface casting dimethyl silicone polymer prepolymer, uncover after heat cross-linking solidifies and to make.
- 4. press the little transmission technology of the described usefulness of claim 1 in the macromolecular method of polymer surfaces patterning fixed biologically, it is characterized in that said biomacromolecule is meant protide biomacromolecule and derivant thereof, comprises albumin, fibronectin, laminin, poly-D-lysine, collagen and gelatin etc.
- By the little transmission technology of the described usefulness of claim 1 in the macromolecular method of polymer surfaces patterning fixed biologically, it is characterized in that step 2) said biological macromolecule solns is meant the buffer soln or the aqueous solution of its corresponding pH value.
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| EP2180042A1 (en) * | 2008-10-24 | 2010-04-28 | Commissariat A L'energie Atomique | Methods and device to constrain multicellular arrangements in stable, stationary and reproducible spatial configuration |
| CN104387837B (en) * | 2014-11-05 | 2016-09-28 | 广西师范学院 | A kind of water solublity bilayer sandwich type Sc Metalloporphyrins and the application in microcontact printing techniques thereof |
| CN104527254B (en) * | 2015-01-04 | 2017-02-01 | 浙江农林大学 | Method for printing double-protein composite micro pattern on surface of material |
| CN105504327B (en) * | 2016-02-03 | 2018-07-10 | 华中科技大学 | A kind of biomaterial with the micro- patterns of 3D and preparation method thereof |
| CN105772119B (en) * | 2016-03-04 | 2018-08-14 | 北京乐普医疗科技有限责任公司 | A kind of PDMS micro-fluidic chips surface polylysine modification method |
| CN106381263A (en) * | 2016-08-30 | 2017-02-08 | 上海浦美生物医药科技有限公司 | Circulating tumor cell capture device and preparation method thereof |
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