CN102465119A - Substrate for cell micropatterning growth as well as preparation method and application thereof - Google Patents
Substrate for cell micropatterning growth as well as preparation method and application thereof Download PDFInfo
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- CN102465119A CN102465119A CN2010105446063A CN201010544606A CN102465119A CN 102465119 A CN102465119 A CN 102465119A CN 2010105446063 A CN2010105446063 A CN 2010105446063A CN 201010544606 A CN201010544606 A CN 201010544606A CN 102465119 A CN102465119 A CN 102465119A
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Abstract
The invention provides a substrate for cell micropatterning growth. The substrate comprises an inert surface on which cells cannot be adhered and patterns on the inert surface, wherein the patterns are formed from substances which are allowable for cell adhesion, and the inert surface is not completely covered by the patterns. The invention further provides a preparation method of the substrate, wherein a one-step printing step is adopted in the method for preparing the substrate, so that the traditional method for preparing the substrate for the cell micropatterning growth is greatly simplified, and the range for using the substrate is broadened. The invention further provides application of the substrate to the cell micropatterning growth.
Description
Technical field
The present invention relates to cell culture technology, particularly, the present invention relates to a kind of substrate that can make the cell patterned growth.
Background technology
The patterning of cell is a kind of technology of vitro culture, and cell is confined to some region growing of base plane, and cell can not moved to the place outside the localized area.The method of cell patterning is different from commonly used directly carries out best cultivation with cell inoculation in petridish, the latter makes cell become the cell monolayer film of homogeneous in petridish middle berth spread.
The technology of cell patterning has very important use for the influence of cell migration and cell for the response of medicine irritation to the interaction between interaction, cell and the cell of research cell and substrate, shape.In addition, the technology of cell patterning can be the construct external model, in the behavior of tissue level research cell platform is provided.Relevant bibliographical information has, at article Jiang, X.Y., Bruzewicz, D.A.; Wong, A.P., Piel, M.; Whitesides, G.M., Proc.Natl.Acad.Sci.U.S.A., 102 (4); Among the 975-978 (2005), the author utilizes the method for self-assembled monolayer and micro-contact printing, and cell fixation is become the water droplet shape, then cell is discharged.Cell after the release more blunt end in its form moves.And for example, through with cell fixation in the patterns of different sizes, find cell Growth and Differentiation easily on than the pattern of big scale (＞10 microns), and in the pattern of less (＜10 microns) apoptosis (Chen C.S. easily; Mirksich, M., Huang, S.; Whitesides G.M., and Ingber, D.E.; Science, 276,1425-1428 (1997)).In these articles, the substrate of patterning has very big using value in the laboratory of some underlying issues that are used to study cytobiology.
At present more existing technology can be fixed on substrate surface with the cell patterning, for example self-assembled monolayer.Self-assembled monolayer is to utilize an end of molecule and substrate that very strong reactive force is arranged, and causes the molecule unitary film that chemisorption and regular arrangement form on substrate.For example thiol molecule just can be assembled the formation unitary film at gold surface, silane molecule at glass or silicon base surface.Thiol molecule can be imprinted on gold surface with method of printing.Just can obtain the thiol molecule of different functional groups like this in the different zones of gold surface.The thiol molecule of different functional groups is different for the interaction of cell, and have plenty of the cell of permission and attach in the above, the attaching of then resisting cell that has, the latter is called inactive surfaces.According to Search Results, the typical example of correlation technique has: one) through method of printing, cell can be printed onto on the metal base by adherent thiol molecule, other zone bubbles of substrate are gone up cell can not adherent another kind of thiol molecule; Perhaps cell can be printed onto on glass or the silicon base by adherent silane molecule, other zone bubbles of substrate are gone up cell can not adherent another kind of silane molecule; Two) form some closed systems with miniflow pipeline and substrate, in closed system, feed cell suspending liquid, after after a while, cell is grown in pipeline, removes pipeline, just can stay banded cell pattern; Three) through micro-machined method, film that contains the pattern hole of preparation is attached to film in the substrate earlier, cell suspending liquid is layered on above the substrate again, and cell will sink to film surface gradually.Open film, cell just forms pattern in the zone of hole; Four) can not carry out little pattern etch with modes such as laser, anodizing reduction in adherent surface to existing cell, thereby at the pattern of the regional supported cell adhesion of handling.
Though above method can well be controlled the patterning of cell in substrate; But need two kinds of molecules to assemble on the surface; And for the assembling substrate certain restriction (as first kind) is arranged; Improved the cost of substrate preparation like this, perhaps can not secular maintenance patterning (like second and third kind), perhaps depend on the equipment (as the 4th kind) of write-through.
Therefore; Exist at present the method that makes cell patterned growth in substrate and the demand of this substrate; Exploitation can keep the patterning of cell for a long time; Can reduce cost again, the method for the scope of application of expanded substrate, must promote the research of stechiology greatly, and the structure of external model.
Summary of the invention
The substrate that the objective of the invention is to overcome prior art is not generally used in the laboratory, needs specific installation, the defective that price is more expensive.The inventor has developed a series of substrates that are based on the general material prepn of biology laboratory through conscientiously research, and these substrates can be used in the patterning of cell after treatment, can address the deficiencies of the prior art like this, thereby accomplish the present invention.
Therefore, an object of the present invention is to provide a kind of substrate that is used for the cell patterned growth.Another object of the present invention provides the preparation method of said substrate.In addition, a further object of the present invention provides the application of said substrate.
The objective of the invention is to realize through following technical scheme:
On the one hand, the present invention provides a kind of substrate that is used for the cell patterned growth, and said substrate comprises:
A) cell can not adherent inactive surfaces; With
B) pattern on said inactive surfaces, this pattern is formed by the material that allows cell adhesion, and said inactive surfaces is not all by said pattern covers.
Preferably, the material of said permission cell adhesion is for gathering Dopamine HCL.
Wherein, said substrate can be the polystyrene plastic sheet, comprises the inactive surfaces that is formed by poly-lysine-polyethyleneglycol-graft copolymer (PLL-g-PEG) molecule on the said polystyrene sheet;
Perhaps, said substrate is sheet glass or silicon chip, comprises the inactive surfaces by poly-lysine-the polyethyleneglycol-graft copolymer molecule forms on said sheet glass or the silicon chip;
Perhaps, said substrate is metal titanium sheet or niobium sheet, on said titanium sheet or the niobium sheet by interior be titanium or niobium outward successively, titanium oxide or niobium oxides and by inactive surfaces that poly-lysine-the polyethyleneglycol-graft copolymer molecule forms;
Perhaps, said substrate is indium tin oxide conductive glass (ITO), on the said indium tin oxide conductive glass by interior be glass outward successively, indium tin oxide layer and by inactive surfaces that poly-lysine-the polyethyleneglycol-graft copolymer molecule forms;
Preferably, said poly-lysine-polyethyleneglycol-graft copolymer molecule is the PLL-g-PEG of different copolymer degree, the further preferred 20～375k of PLL wherein, and PEG is preferred 2～5k further, and the ratio of Methionin and PEG chain further preferred 3～5 among the PLL;
Perhaps, said substrate is by polyethylene glycol dimethacrylate illumination polymer formation;
Perhaps, said substrate is sheet glass or silicon chip, on said sheet glass or the silicon chip by the interior inactive surfaces that has metal level outward successively and form by thiol molecule with polyoxyethylene glycol ending;
Preferably, said metal level comprises the gold layer; Further preferably, said golden bed thickness 20～50nm;
Preferably, said metal level on sheet glass or the silicon chip by interior titanium adhesion layer and the gold layer of comprising successively outward; Further preferably, said titanium adheres to bed thickness 2～10nm, golden bed thickness 20～50nm;
Preferably, said thiol molecule is HS (CH
2) n (OCH
2CH
2) mOH (n＞3, m＞2), preferred HS (CH
2)
11(OCH
2CH
2)
6OH.
In addition, the pattern that on the substrate inactive surfaces, is formed by the material that allows cell adhesion is an arbitrary graphic pattern, includes but not limited to irregular pattern or the array be made up of point, line, bar, band, piece, irregularly shaped etc.;
Can be included but not limited to eukaryotic cell and bacterium by any cell of the adherent cell of substrate for surviving after can adhering to and adhere to.
On the other hand, the present invention provides the preparation method of above-mentioned substrate, and this method comprises, provide to have the substrate that cell can not adherent inactive surfaces, with the pattern that allows cell adhesion through micro-contact printing on the inactive surfaces of this substrate.
Preferably, the preparation method of said substrate may further comprise the steps:
1) inactive surfaces of preparation substrate:
Selection has polystyrene sheet, sheet glass, silicon chip, titanium sheet, niobium sheet or the indium tin oxide conductive glass of suitable size, is placed on to carry out oxide treatment in the air plasma oxidizer, and oxidization time is more than 1 minute.Hydroxyethyl piperazine second SULPHONIC ACID. (HEPES) solution (pH 7.4) of PLL-g-PEG that substrate after the oxidation is placed 0.5～10g/L is more than 0.5 hour, thus the substrate that obtains having inactive surfaces;
Alternatively; With the benzoin dimethylether light trigger that adds 1% (mass ratio) in the polyethylene glycol dimethacrylate liquid; The dissolving, under the UV-light of 250-400nm (＞60mW) irradiation more than 2 minutes, obtain polyethylene glycol dimethacrylate illumination polymkeric substance;
Alternatively, select sheet glass or silicon chip with suitable size, after the clean in surperficial evaporated metal layer (with reference to Pale-Grosdemange C.; Simon E.S., Prime K.L., and Whitesides G.M.; J.Am., Chem.Soc., 113; 12-20 (1991)), then with vapor deposition the substrate of metal level place 2～10mM concentration with the water of the thiol molecule of polyoxyethylene glycol ending or the spirituous solution soaking at room temperature of spending the night, thereby the substrate that obtains having inactive surfaces;
2) preparation seal:
The preparation seal makes on the surface of this seal and contains convexity, and said protruding composition will be formed on suprabasil pattern, and each protruding height is 1～40 micron, and protruding area is 10000 square nanometers～1 square millimeter; The material of preparation seal preferably adopts YSR 3286;
3) micro-contact printing:
It is 8-10 that seal is put into pH, and concentration is to spend the night in the dopamine solution of 0.1-100g/L, the Dopamine HCL spontaneous oxidation is gathered be incorporated in to form the one layer of polymeric film on the seal and promptly gather Dopamine HCL, takes out seal then, dries up; Wherein, the preferred pH of said dopamine solution is 8-9, concentration 0.5～10g/L;
The seal that dries up is lain on the inactive surfaces for preparing in the step 1), the one side that contains pattern is contacted with inactive surfaces, exert pressure make gather Dopamine HCL and transfer in the substrate after, take seal away, just obtained having the inactive surfaces of gathering the Dopamine HCL pattern.
In the above-mentioned method for preparing substrate, the said metal level in the step 1) comprises the gold layer of thick 20～50nm; Further preferably, said metal level comprises that direct vapor deposition is at the titanium adhesion layer of suprabasil 2～10nm and the gold layer of the 20～50nm of vapor deposition on the titanium adhesion layer;
Preferably, in step 1), said thiol molecule with the polyoxyethylene glycol ending is HS (CH
2) n (OCH
2CH
2) mOH (n＞3, m＞2), the HS (CH of preferred 1～5mM
2)
11(OCH
2CH
2)
6OH solution.
In the above-mentioned method for preparing substrate, said step 2) carry out through following steps:
At first, with the method processing mold of photoetching:, and print through film printer with the pattern of mapping software design demand; Adopt ordinary method that described pattern is carried out lithography process then; Select conventional silicon chip and photoresist material, after spin coating, preceding baking, exposure, back baking, developing, described pattern is just carried by the photoresist material on the silicon chip;
Then, using YSR 3286 to carry out pattern to the mould that obtains duplicates: the silicon chip that has photoresist material that will process is gone up YSR 3286 and is carried out duplicating molded; Then the baking oven at 80 degree toasted more than half a hour, made YSR 3286 form solid; Above silicon chip, take YSR 3286 off, pattern has just been transferred to above the YSR 3286; Cut the good YSR 3286 of polymerization as required, promptly can be used as the seal of micro-contact printing.
In the above-mentioned method for preparing substrate, the dopamine solution in the step 3) is the Tris-HCl buffer salt solution of the Dopamine HCL of pH8.5,2g/L; Preferably, the one side that seal is contained pattern was exerted pressure on seal 10～60 seconds with after inactive surfaces contacts, and took seal away, had just obtained having the inactive surfaces of gathering the Dopamine HCL pattern.
On the one hand, the present invention provides above-mentioned substrate at culturing cell, makes the application in the cell patterned growth again.In addition, the present invention also provides the application of said substrate in making the cell controlled release, and said substrate is preferably gold substrate.
Be detailed description of the present invention below: the present invention provides the device that cell is adhered to suprabasil method and realizes this method with the oldered array mode.
Said method is to use the elastomerics seal will allow the cell adhesion material to be printed onto inactive surfaces and to form specific pattern.
Said device comprises:
(1) substrate; Said substrate comprises cell can not adherent inactive surfaces.
Said substrate can be to be coated with the glass of metal level or silicon chip; Metal level up is followed successively by titanium adhesion layer and gold layer from sheet glass or silicon chip, on the gold layer, is assembled with thiol molecule with the polyoxyethylene glycol ending then, or contains the thiol molecule of phosphate group and/or uncle's nitrogen groups simultaneously.The structure of said metal level is that titanium adheres to bed thickness 2～10nm, golden bed thickness 20～50nm.
(2) elastomerics seal.There is one side to contain irregular micro-structure on the described seal.
Described elastomeric material is a YSR 3286.
Described micro-structure is a concaveconvex structure arbitrarily, can be array, also can be banded pattern.
Described micro-structure is certain planar pattern of protrusion seal, and its protrusion height is at 1～40 micron.
The area of described micro-structure is 10000 square nanometers to 1 square millimeter.
The cell that is used for patterning can be an eukaryotic cell, also can be bacterium, mikrobe.
According to an embodiment of the invention, the present invention provides cell is adhered to same suprabasil method with the oldered array mode, comprises following step:
1) preparation of surface metal-layer: vapor deposition titanium layer and gold layer successively in clean substrate;
2) preparation of the inadhesive inactive surfaces of cell: the substrate that will prepare metal level places the HS (CH of 1～5mM
2)
11(OCH
2CH
2)
6The immersion of spending the night in the OH solution can obtain inactive surfaces.
3) with the method processing mold of photoetching:
At first, with the described pattern of L-Edit software design, and print through film printer; Then described pattern is carried out lithography process; Because described pattern area is little, can be made on the silicon chip of 3 cun areas simultaneously; What photoresist material used is the negative glue of the thick type of SU-8 of MicroChem company.After through spin coating, preceding baking, exposure, back baking, development, described pattern is just carried by the photoresist material on the silicon chip;
4) using YSR 3286 to carry out pattern to the mould that obtains duplicates: the silicon chip that has photoresist material that will process is gone up YSR 3286 and is carried out duplicating molded; Then the baking oven at 80 degree toasted 1 hour, and YSR 3286 has just formed solid; This solid is elastic, can above silicon chip, take off; Take YSR 3286 off, pattern has just been transferred to above the YSR 3286.Cut the good YSR 3286 of polymerization as required, promptly can be used as the seal of micro-contact printing.
5) the Tris-HCl buffer salt solution of seal being put into the Dopamine HCL of 2g/L spends the night pH furnishing 8.5.Dopamine HCL is the spontaneous oxidation polymerization in above-mentioned condition, and on seal, forms polymeric film.Take out seal after spending the night, dry up.
6) micro-contact printing: the seal that dries up is lain on the inactive surfaces, and the one side that contains pattern contacts with gold surface, above seal, places a counterweight again.Behind 10～60s, take seal away, just obtained having the inactive surfaces of gathering the Dopamine HCL pattern.
7) patterning of cell: cell suspending liquid directly is added in has the inactive surfaces of gathering the Dopamine HCL pattern, through obtaining the cell of patterning after 2 hours.
The present invention is through can obtain once step printing can be with the method for the substrate of cell patterning.Compared with prior art, the present invention has following advantage at least:
One, a kind of substrate that is used for the patterning culturing cell provided by the invention need not carry out special processing and get final product inoculating cell, and pair cell is nontoxic; Employing gathers that Dopamine HCL forms pattern and the cell pattern that further obtains can be kept (seeing embodiment) about 3 days to 3 weeks;
Two, method steps of the present invention is simple; Only need directly provide the cell can not adherent surface; Method with micro-contact printing directly can be printed onto the surface by adherent material with cell then, for example directly sticks on the inactive surfaces with the physics contact form through gathering Dopamine HCL, and method is simple; Be adapted at operating in the ordinary laboratory, and can save reagent relatively;
Three, in the prior art, adopt the inadhesive inactive surfaces of cell of gold-plated sheet glass or silicon chip preparation usually, the inactive surfaces that perhaps obtains with the silane molecule assembling that contains polyoxyethylene glycol functional group on the silicon chip.Method of the present invention also is applicable to the polystyrene culture dish that biology laboratory is commonly used, and can extend to titanium, niobium, conductive glass and polyoxyethylene glycol illumination polymkeric substance.Therefore, this method has broken through the restriction for the gold-plated substrate of use that realizes the cell patterning, the selection of having widened the substrate kind;
Four, substrate of the present invention and method are applicable to that also the mode of analysing through electrolysis discharges the cell that fixes, thereby dynamically control the patterning of cell in substrate.
Description of drawings
Below, specify embodiment of the present invention in conjunction with accompanying drawing, wherein:
Fig. 1 is the synoptic diagram of preparation YSR 3286 seal;
Fig. 2 forms the synoptic diagram that gathers Dopamine HCL for Dopamine HCL at the seal surface aggregate;
Fig. 3 gathers Dopamine HCL to pass through the synoptic diagram that micro-contact printing forms pattern at the glass surface that PLL-g-PEG modifies;
To be mouse fibroblast cell (NIH 3T3) containing after the polystyrene surface that gathers the Dopamine HCL pattern cultivates 23 days the pattern of formation to Fig. 4;
Fig. 5 is containing after the polyethylene glycol dimethacrylate illumination polymer surfaces that gathers the Dopamine HCL pattern cultivates 2 days the pattern of formation for mouse fibroblast cell;
Fig. 6 is intestinal bacteria, cultivates the pattern that formed in 4 hours in gold surface;
Fig. 7 is a mouse fibroblast cell after gold surface is cultivated 6 hours, after gold surface is carried out electrolysis and analysed, and the result of the shape of per 2 h observation cells.
Embodiment
Below in conjunction with concrete embodiment, and comparable data describes in further detail the present invention.Should be understood that these embodiment just in order to demonstrate the invention, but not limit scope of invention by any way.
In following embodiment, various processes and the method do not described in detail are ordinary methods as known in the art.The source of agents useful for same, trade(brand)name and be necessary to list its moity person indicate when occurring first that all used thereafter identical reagent is like no specified otherwise, and is all identical with the content of indicating first.
The glass sheet surface printing that 1 couple of PLL-g-PEG of embodiment modifies gathers the Dopamine HCL pattern
1, sheet glass is carried out oxide treatment.
Sheet glass (25.4*76.2mm, sailing boat board slide glass, article No. 7101) is cut into 5 * 5mm size; Be positioned in acetone and the water ultrasonic cleaning successively 10 minutes; With the vitriol oil and the ydrogen peroxide 50 mixed with 3: 1, the cleaned slide of mixed liquid dipping 30 minutes dries up.Sheet glass is placed on oxidation 3min in the air plasma oxidizer.
2, sheet glass being carried out inertia modifies.
(pH 7.4 sheet glass after the oxidation to be put into hydroxyethyl piperazine second SULPHONIC ACID. (HEPES) solution of PLL-g-PEG solution of 0.1g/L; Soaked 2 hours 10mM); And the PLL-g-PEG that adopts is PLL (20)-g [3.5]-PEG (5); Be that PLL is 20k, PEG is 5k, and the ratio of Methionin and PEG chain is 3.5 among the PLL.
3, preparation YSR 3286 seal.
At first; With L-Edit software (The layout Editor Win 32 8.30, Tanner EDA, A division of Tanner Research; Inc.) the nail-like pattern formed by trilateral and rectangle of design, and be printed on (available from Suzhou U.S. precise and tiny photoelectricity ltd) above the chromium plate; Then described pattern is carried out lithography process; Lithography process can adopt usual method to carry out, the operating process (network address: http://www.microchem.com/products/pdf/SU-82000DataSheet2025thru 2075Ver4.pdf) that can provide referring to MicroChem company; What substrate was used is the silicon chip of 3 cun areas; What photoresist material used is the negative glue of the thick type of SU-8 of MicroChem company.After through spin coating, preceding baking, exposure, back baking, development, described pattern is just carried by the photoresist material on the silicon chip;
Then, using YSR 3286 to carry out pattern to the mould that obtains duplicates: the silicon chip that has photoresist material that will process is gone up YSR 3286 and is carried out duplicating molded; Then the baking oven at 80 degree toasted 1 hour, and YSR 3286 has just formed solid; This solid is elastic, can above silicon chip, take off; Take YSR 3286 off, pattern has just been transferred to above the YSR 3286.Cut the good YSR 3286 of polymerization as required, promptly can be used as the seal of micro-contact printing, as shown in Figure 1.
4, gather Dopamine HCL in the formation of seal surface.
With the polymerization of in concentration is the Tris-HCl solution of Dopamine HCL of 2g/L, spending the night of seal bubble, polymerizing condition is pH 8.0～8.5, and is as shown in Figure 2.
5, will gather Dopamine HCL and be printed on glass surface
Seal is taken out from solution, dry up, the glass surface that lies in the PLL-g-PEG modification took off seal through 30 seconds, was printed on glass surface thereby will gather Dopamine HCL, had formed the pattern that is used for the cell patterned growth, and is as shown in Figure 3.
The patterned growth of embodiment 2 cells in the PS substrate that PLL-g-PEG modifies
1, p-poly-phenyl ethene thin slice carries out oxide treatment.
PS (disposable use plastic culture dish likes to pursue progress medical plastic ltd) petridish is cut into the slice, thin piece of 5 * 5mm size, slice, thin piece is put into air plasma oxidizer oxidation 3min.
2, p-poly-phenyl ethene thin slice carries out the inertia modification; (pH 7.4 wherein to adopt hydroxyethyl piperazine second SULPHONIC ACID. (HEPES) solution of PLL-g-PEG solution of 10g/L; 10mM), and the PLL-g-PEG that adopts is PLL (375)-g [5]-PEG (5), and promptly PLL is 375k; PEG is 5k, and the ratio of Methionin and PEG chain is 5 among the PLL.
3, preparation YSR 3286 seal.
4, gather Dopamine HCL in the formation of seal surface.
5, will gather Dopamine HCL and be printed on the thin polystyrene sheet surface.
Wherein the step of 2-5 is consistent with corresponding steps among the embodiment 1.
6, be printed on the polystyrene surface inoculating cell that gathers Dopamine HCL.
To be printed on the thin polystyrene sheet of gathering Dopamine HCL and be placed in the petridish, with the cell suspending liquid under just digesting (cell is a mouse fibroblast cell, NIH 3T3, the concentration of cell suspending liquid is 10
5Individual/as mL) to place on the substrate, through 23 days cultivations (cell culture incubator, 37 ℃, 5%CO2, cell culture medium are that DMEM+10%FBS+1% is two anti-, during change substratum), obtained cell as shown in Figure 4 and in substrate, formed pattern.
The patterning of embodiment 3 cells on polyethylene glycol dimethacrylate illumination polymkeric substance given birth to
Long
1, the preparation of polyethylene glycol dimethacrylate illumination polymkeric substance
(sigma company buys with polyethylene glycol dimethacrylate liquid; CAS number is 25852-47-5) middle benzoin dimethylether light trigger (the sigma company purchase that adds 1% (mass ratio); CAS number is 24650-42-8), the vibration dissolving drips in polystyrene culture dish; Under the UV-light of 250-400nm (＞60mW) irradiation more than 2 minutes, obtain solidified polyethylene glycol dimethacrylate illumination polymkeric substance.In petridish, add entry, the immersion of spending the night.Polyethylene glycol dimethacrylate illumination polymkeric substance can break away from polystyrene surface automatically, forms film.Be placed on oven dry in the baking oven (40 degree, 30 minutes) to film.
2, preparation YSR 3286 seal.
3, gather Dopamine HCL in the formation of seal surface.
4, will gather Dopamine HCL and be printed on polyethylene glycol dimethacrylate illumination polymer surfaces.
Wherein the step of 2-4 is consistent with corresponding steps among the embodiment 1.
5, being printed on the polyethylene glycol dimethacrylate illumination polymer surfaces inoculating cell (identical) that gathers Dopamine HCL, cultivate and obtain cell as shown in Figure 5 pattern structure after 2 days at polyethylene glycol dimethacrylate illumination polymkeric substance with step 6 among the embodiment 2.
The patterned growth of embodiment 4 bacteriums on the gold plaque that thiol molecule is modified
1, the preparation of surface metal-layer
By this area routine techniques, vapor deposition titanium layer and gold layer successively at the bottom of the clean silicon wafer-based.Wherein, earlier at the titanium adhesion layer of substrate surface vapor deposition 10nm, then on titanium layer the gold layer of vapor deposition 40nm (with reference to Pale-Grosdemange C., Simon E.S.; Prime K.L., and Whitesides G.M., J.Am.; Chem.Soc., 113,12-20 (1991)).
2, the preparation of the YSR 3286 seal of nail-like array pattern.
3, the preparation of the inadhesive inactive surfaces of bacterium; (pH 7.4 wherein to adopt hydroxyethyl piperazine second SULPHONIC ACID. (HEPES) solution of PLL-g-PEG solution of 5g/L; 10mM), and the PLL-g-PEG that adopts is PLL (200)-g [2]-PEG (3), and promptly PLL is 200k; PEG is 3k, and the ratio of Methionin and PEG chain is 2 among the PLL.
Gold-plated sheet glass is placed on concentration soaked overnight in the HS of 2mM (CH2) 11 (OCH2CH2) 6OH solution, obtains the inactive surfaces of self-assembled monolayer.
4, gather Dopamine HCL in the formation of seal surface.
5, will gather Dopamine HCL and be printed on the gold plaque surface.
Wherein, 2,4 is identical with embodiment 1 corresponding steps with 5 operation steps.
6, the inoculation of bacterium.
To be printed on the gold plaque that gathers Dopamine HCL and be placed in the petridish, (intestinal bacteria, DSMZ of Institute of Micro-biology buys, ATCC11775, the OD of bacterium liquid with bacterium liquid
600Value is 0.4, is equivalent to 10
8CFU/mL, the culture soln of bacterium is a broth culture) being seeded in substrate surface, culture condition is 37 degree shaking table shaking culture.Through 4 hours, just can obtain bacterium and in substrate, form pattern, as shown in Figure 6, wherein Fig. 6 can see that for the bacterial growth situation of cultivation after 4 hours bacterium carries out patterned growth on the nail-like pattern of substrate surface.
The cell of 5 pairs of patternings of embodiment carries out controlled release
1, will have the substrate that gathers the Dopamine HCL pattern having of embodiment 4 step 1-5 acquisition and take out, and use step 6 inoculating cell of embodiment 2, then cell was cultivated in incubator 6 hours.
2, gold plaque is carried out electrolysis analyse: apply being placed on substrate in the substratum-the voltage 30s of 1.5V with voltage stabilized source, wash 3 times with substratum.
The substrate of electrolysis after analysing is reentered among the substratum, connects to clap with microscope and observe, can see that cell moves gradually from pattern, as shown in Figure 7.
Claims (12)
1. substrate that is used for the cell patterned growth, said substrate comprises:
A) cell can not adherent inactive surfaces; With
B) pattern on said inactive surfaces, this pattern is formed by the material that allows cell adhesion, and said inactive surfaces is not all by said pattern covers;
Preferably, the material of said permission cell adhesion is for gathering Dopamine HCL.
2. substrate as claimed in claim 1, wherein, said substrate is sheet glass or silicon chip, on said sheet glass or the silicon chip by the interior inactive surfaces that has metal level outward successively and form by thiol molecule with polyoxyethylene glycol ending;
Preferably, said metal level comprises the gold layer; Further preferably, said golden bed thickness 20～50nm;
Preferably, said metal level on sheet glass or the silicon chip by interior titanium adhesion layer and the gold layer of comprising successively outward; Further preferably, said titanium adheres to bed thickness 2～10nm, golden bed thickness 20～50nm;
Preferably, said thiol molecule is HS (CH
2) n (OCH
2CH
2) mOH (n＞3, m＞2), further preferred HS (CH
2)
11(OCH
2CH
2)
6OH.
3. substrate as claimed in claim 1, wherein, said substrate is the polystyrene plastic sheet, comprises the inactive surfaces by poly-lysine-the polyethyleneglycol-graft copolymer molecule forms on the said polystyrene sheet;
Alternatively, said substrate is sheet glass or silicon chip, comprises the inactive surfaces by poly-lysine-the polyethyleneglycol-graft copolymer molecule forms on said sheet glass or the silicon chip;
Alternatively, said substrate is metal titanium sheet or niobium sheet, on said titanium sheet or the niobium sheet by interior be titanium or niobium outward successively, titanium oxide or niobium oxides and by inactive surfaces that poly-lysine-the polyethyleneglycol-graft copolymer molecule forms;
Alternatively, said substrate is the indium tin oxide conductive glass, on the said indium tin oxide conductive glass by interior be glass outward successively, indium tin oxide and by inactive surfaces that poly-lysine-the polyethyleneglycol-graft copolymer molecule forms; Preferably, said poly-lysine-polyethyleneglycol-graft copolymer molecule is the PLL-g-PEG of different copolymer degree, the further preferred 20～375k of PLL wherein, and PEG is preferred 2～5k further, and the ratio of Methionin and PEG chain further preferred 3～5 among the PLL.
4. substrate as claimed in claim 1, wherein, said substrate is by polyethylene glycol dimethacrylate illumination polymer formation.
5. like each described substrate among the claim 1-4; Wherein, The pattern that on the substrate inactive surfaces, is formed by the material that allows cell adhesion is an arbitrary graphic pattern, includes but not limited to irregular pattern or the array be made up of point, line, bar, band, piece, irregularly shaped etc.;
Preferably, saidly can be included but not limited to eukaryotic cell and bacterium by any cell of the adherent cell of substrate for surviving after can adhering to and adhere to.
6. preparation method like each said substrate among the claim 1-5, wherein, said method comprises: provide to have the substrate that cell can not adherent inactive surfaces, with the pattern that allows cell adhesion through micro-contact printing on the inactive surfaces of this substrate.
7. method as claimed in claim 6 wherein, said method comprising the steps of:
1) inactive surfaces of preparation substrate:
Selection has polystyrene sheet, sheet glass, silicon chip, titanium sheet or the niobium sheet of suitable size, is placed on to carry out oxide treatment in the air plasma oxidizer, and oxidization time is more than 1 minute; It is the hydroxyethyl piperazine second SULPHONIC ACID. solution of PLL-g-PEG of 0.5～10g/L more than 0.5 hour that substrate after the oxidation is placed pH 7.4, concentration, thus the substrate that obtains having inactive surfaces;
Alternatively, with the benzoin dimethylether light trigger that adds 1% mass ratio in the polyethylene glycol dimethacrylate liquid, dissolving, 250-400nm,＞UV-light of 60mW shines more than 2 minutes down, obtains polyethylene glycol dimethacrylate illumination polymkeric substance;
Alternatively; Selection has the sheet glass or the silicon chip of suitable size; After the clean in surperficial evaporated metal layer; Then with vapor deposition the substrate of metal level place 2～10mM concentration with the water of the thiol molecule of polyoxyethylene glycol ending or the spirituous solution soaking at room temperature of spending the night, thereby the substrate that obtains having inactive surfaces;
2) preparation seal:
The preparation seal makes on the surface of this seal and contains convexity, and said protruding composition will be formed on suprabasil pattern, and each protruding height is 1～40 micron, and protruding area is 10000 square nanometers～1 square millimeter;
The material of preparation seal preferably adopts YSR 3286;
3) micro-contact printing:
It is 8～10 that seal is put into pH, and concentration is to spend the night in the dopamine solution of 0.1-100g/L, the Dopamine HCL spontaneous oxidation is gathered be incorporated in to form the one layer of polymeric film on the seal and promptly gather Dopamine HCL, takes out seal then, dries up; Wherein, the preferred pH of said dopamine solution is 8-9, and concentration is 0.5～10g/L;
The seal that dries up is lain on the inactive surfaces for preparing in the step 1), the one side that contains pattern is contacted with inactive surfaces, exert pressure make gather Dopamine HCL and transfer in the substrate after, take seal away, just obtained having the inactive surfaces of gathering the Dopamine HCL pattern.
8. method as claimed in claim 7, wherein, in step 1), the metal level of said vapor deposition comprises the gold layer of thick 20～50nm;
Preferably, said metal level comprises that direct vapor deposition is at the titanium adhesion layer of suprabasil 2～10nm and the gold layer of the 20～50nm of vapor deposition on the titanium adhesion layer;
Preferably, said thiol molecule solution with the polyoxyethylene glycol ending is HS (CH
2) n (OCH
2CH
2) mOH (n＞3, m＞2), the HS (CH of preferred 1～5mM
2)
11(OCH
2CH
2)
6OH solution.
9. like claim 7 or 8 described methods, wherein, said step 2) carry out through following steps:
At first, with the method processing mold of photoetching:, and print through film printer with the pattern of known mapping software design demand; Adopt ordinary method that described pattern is carried out lithography process then; Select conventional silicon chip and photoresist material, after spin coating, preceding baking, exposure, back baking, developing, described pattern is just carried by the photoresist material on the silicon chip;
Then, using YSR 3286 to carry out pattern to the mould that obtains duplicates: the silicon chip that has photoresist material that will process is gone up YSR 3286 and is carried out duplicating molded; Then the baking oven at 80 degree toasted more than half a hour, made YSR 3286 form solid; Above silicon chip, take YSR 3286 off, pattern has just been transferred to above the YSR 3286; Cut the good YSR 3286 of polymerization as required, promptly can be used as the seal of micro-contact printing.
10. like each described method among the claim 7-9, wherein, the dopamine solution in the said step 3) is the Tris-HCl buffer salt solution of the Dopamine HCL of pH8.5,2g/L;
Preferably, the one side that seal is contained pattern was exerted pressure on seal 10～60 seconds with after inactive surfaces contacts, and took seal away, had just obtained having the inactive surfaces of gathering the Dopamine HCL pattern.
11. make the application in the cell patterned growth at culturing cell like each said substrate among the claim 1-5.
12. like each said substrate application in making the cell controlled release among the claim 1-5;
Preferably, said substrate is a gold substrate.
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| CN103601181A (en) * | 2013-12-04 | 2014-02-26 | 南京大学扬州化学化工研究院 | Method for preparing nitrogen-doped graphene with polydopamine as raw material |
| CN103601181B (en) * | 2013-12-04 | 2015-07-22 | 南京大学扬州化学化工研究院 | Method for preparing nitrogen-doped graphene with polydopamine as raw material |
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| CN108641892B (en) * | 2018-04-17 | 2021-11-16 | 广州波奇亚标准及检测技术有限公司 | Micro-contact printing system for cell patterning |
| CN109776842A (en) * | 2019-01-11 | 2019-05-21 | 西南交通大学 | Regioselectivity controllably changes fixed bimolecular film and preparation method thereof |
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