CN108641892A - One kind being used for the patterned novel micro-contact printing system of cell - Google Patents
One kind being used for the patterned novel micro-contact printing system of cell Download PDFInfo
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- CN108641892A CN108641892A CN201810345302.0A CN201810345302A CN108641892A CN 108641892 A CN108641892 A CN 108641892A CN 201810345302 A CN201810345302 A CN 201810345302A CN 108641892 A CN108641892 A CN 108641892A
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- cell
- dopamine
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- oligomerization
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M33/00—Means for introduction, transport, positioning, extraction, harvesting, peeling or sampling of biological material in or from the apparatus
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
- C08G73/00—Macromolecular compounds obtained by reactions forming a linkage containing nitrogen with or without oxygen or carbon in the main chain of the macromolecule, not provided for in groups C08G12/00 - C08G71/00
- C08G73/06—Polycondensates having nitrogen-containing heterocyclic rings in the main chain of the macromolecule
- C08G73/0666—Polycondensates containing five-membered rings, condensed with other rings, with nitrogen atoms as the only ring hetero atoms
- C08G73/0672—Polycondensates containing five-membered rings, condensed with other rings, with nitrogen atoms as the only ring hetero atoms with only one nitrogen atom in the ring
Abstract
The present invention provides one kind being used for the patterned novel micro-contact printing system of cell.The system includes to form oligomerization dopamine by dopamine monomer polymerization, and for molecular weight between 1,000 10000 D, structural formula is as follows.Substrate is first chemically modified processing by the present invention, obtains the 'inertia' substrate of antibiont adherency；Then the aqueous solution for preparing oligomerization dopamine, prepares seal, is then dipped into seal in the aqueous solution of oligomerization dopamine, then the seal for being adsorbed with oligomerization dopamine is placed in 'inertia' substrate, to obtain the pattern on seal in substrate surface.The patterned substrate can be additionally used in the patterned growth of cell.The system is realized simply, is simplified existing for the patterned technology of cell.
Description
Technical field
The present invention relates to for the patterned novel micro-contact printing system of cell, it is used for more particularly, to one kind thin
The patterned novel micro-contact printing system of born of the same parents.
Background technology
Cell patterns, and is a kind of Cell culture invitro technology, and specifically, by preparing certain patterned substrate, induction is thin
The region that the response mechanism of born of the same parents itself is attached to design forms pattern.It is thin that this method is different from laboratory common directly plantation
It is cultivated in born of the same parents to culture dish, the latter is usually formed cell monolayer film.
Cell patterning techniques have become a kind of important tool of research and control cell behaviors, are widely used in research
Cell --- cell, cell --- interaction (including cell adhesion and apoptosis, polarization behavior, cell between substrate
Migration rate, cell differentiation behavior etc.)；External model can also be built, for organizational engineering, cell sensing, drug screening
With the fields such as trauma care.
Currently, cell patterning techniques can be roughly divided into two classes：(1) confinement method：One is formed by physically or chemically means
A little obstacles (such as micropore), by cell limitation, growth forms pattern wherein；(2) modification：Cell culture substrate is carried out
Modification forms close, the thin cell compartment of clear-cut so that cell selective grows to form pattern in close cell compartment.In crowd
In more cell patterning methods, microcontact printing techniques due to it is easy to operate, at low cost, can prepare large area pattern, use
Material (such as dimethyl silicone polymer, PDMS) it is nontoxic, it is transparent, it is easily molded the features such as, be widely applied.
In the research of early stage, micro-contact printing is used to mercaptan being transferred to gold surface, forms patterned self-assembled monolayer.It is logical
Normal process is that PDMS seals are dipped into thiol solution, is contacted with gold surface after drying, thiol molecule is just from the convex of seal
It plays part and is transferred to gold surface, form Iy self-assembled layer；The recessed part of seal is not contacted with substrate, so being exposed.It again will be golden
Substrate is dipped into another thiol molecule, and exposed part will be covered by second of thiol molecule, to form two types
The Iy self-assembled layer of type.For example the mercaptan with methyl can promote cell adhesion, the then anti-cellular adhesion of the mercaptan with polyethylene glycol, it will be thin
After born of the same parents are inoculated into gold surface, cell will form patterned growth.Later again developed to by silane molecule be printed onto glass or
In silicon base.But this printing process needs two kinds of molecules to carry out self assembly on surface, and has particular requirement to substrate.
For above-mentioned limitation, the Jiang Xing spaces seminar of State Nanometer Science Center has developed a kind of step print based on poly-dopamine
Brush method is used for the patterning of cell, and this method is suitable for a variety of substrates, such as gold surface, glass, silicon face, polystyrene (PS) table
Face etc., has the characteristics that easy to operate, at low cost, but also has the shortcomings that pattern is opaque, time-consuming.Therefore, it is necessary to a kind of systems
Make easier, cost is less expensive to realize the patterned means and method of cell.
Invention content
It being used for the patterned novel micro-contact printing system of cell it is a first objective of the present invention to provide a kind of.
The present invention uses oligomerization dopamine prepared by novel method, the easier operating process of use to realize the pattern of cell
Change.
Above-mentioned technical purpose that the invention is realized by the following technical scheme：
One kind being used for the patterned novel micro-contact printing system of cell, and the system includes to form widow by dopamine monomer polymerization
Poly-dopamine, for molecular weight between 1000-10000D, structural formula is as follows：
Preferably, realize that patterned operation is：Figuratum seal is immersed in the aqueous solution of oligomerization dopamine, is then turned
It prints in polyethylene glycols or the substrate of amphiphatic molecule substance modification, carries out cell culture, and obtain the pattern of observable.
Preferably, prepare the substrate of figuratum seal material be sheet glass, silicon chip, titanium sheet, gold plaque or thin polystyrene sheet,
Polyethylene glycol substances are methoxy poly (ethylene glycol) silane, mercapto-polyglycol or poly-D-lysine-polyethylene glycol；Antibiont is viscous
Attached chemical modification master substance to be used is polyethylene glycol substances or amphiphatic molecule.
Preferably, the polyethylene glycol substances are that methoxy poly (ethylene glycol) silane, mercapto-polyglycol or poly-D-lysine-are poly-
Ethylene glycol；Amphiphatic molecule is mainly carboxylic acid glycine betaine amphoteric ion polymer or sulphonic acid betaine type amphoteric ion polymer；
Preferably, realize that patterned the specific method is as follows for cell：
S1. the substrate of antibiont adherency modification is prepared：Simultaneously activated base is cleaned, polyethylene glycol substances or amphiphatic molecule are connect
Branch obtains the substrate of antibiont adherency modification in substrate；
S2. seal is prepared, the type siloxane performed polymer of liquid is positioned in the template with micron scale structures, after solidification,
It obtains with figuratum seal；
S3. it will be immersed in oligomerization dopamine solution with figuratum seal in S2, S1 moderate resistance bioadhesions be positioned over after dry
Substrate, obtain the surface with oligomerization dopamine pattern.
Wherein, primarily to obtaining the inert substrate that bio-resistant molecule sticks in S1；The inert substrate can be by poly-
Glycols silane or amphiphatic molecule form self assembled monolayer in substrate surface；The figure in inactive surfaces is obtained in S3
Case, the pattern are formed by the substance of permission albumen, cell adhesion, and the inactive surfaces are not covered by the pattern all
Lid；
In addition, the pattern formed by the substance of permission cell adherence in substrate inactive surfaces is arbitrary graphic pattern, including but it is unlimited
The irregular pattern or array of the compositions such as Yu Youdian, line, item, band, block, irregular shape.
Preferably, in S1 cleaning and activated base the specific steps are：Substrate is surpassed in acetone, ethanol solution respectively
Then sound boils 0.5~1.5h in the mixed liquor of the concentrated sulfuric acid and hydrogen peroxide, dry after washing with water, and using low temperature etc. from
It after daughter processing, is dipped into the solution of polyethylene glycols or amphiphatic molecule substance, triethylamine react certain time is added, clearly
It washes after drying to obtain the final product.
Preferably, the pH of oligomerization dopamine solution is 4.0~7.0 in S3, a concentration of 0.02~2mg/mL.
Preferably, the surface with oligomerization dopamine pattern is obtained in S3, the selectivity for cell is sticked, obtained patterned
Cell.
Compared with prior art, the invention has the advantages that and advantageous effect：
1. the present invention forms transparent pattern by oligomerization dopamine, substrate be not necessarily to by specially treated can inoculating cell, and
It is harmless to cytotoxic.
2. step of the present invention is simple, it only need to provide cell inadhesive substrate, then can be sticked cell by micro-contact printing
Attached print matter is attached to by physical contact method in substrate to surface, such as by oligomerization dopamine, and method is simple and practicable, no
Expensive instrument is needed, common laboratory is suitable for；Oligomerization dopamine is prepared simply, cheap；Widow used in the present invention
Poly-dopamine ink and the polyethylene glycol of substrate provide safeguard there are stronger active force to cell adhesion, are not in pattern
Obscission.
Description of the drawings
Fig. 1 is the schematic diagram for preparing dimethyl silicone polymer (PDMS) seal.
Fig. 2 is the schematic diagram that oligomerization dopamine forms monolayer in seal adsorption.
Fig. 3 be mouse fibroblast cell (NIH3T3) containing oligomerization dopamine pattern quartz plate surface cultivate 1 hour it
Afterwards, the pattern of formation.
Specific implementation mode
It is further illustrated the present invention below in conjunction with specific embodiments and the drawings, embodiment illustrates and is subject to later in the accompanying drawings
Illustrate, gives part detailed embodiment and specific operating process.Reagent that unless stated otherwise, the present invention uses,
Method and apparatus is the art conventional reagent, method and apparatus.
Embodiment 1：
1, the preparation of oligomerization dopamine
It among dopamine monomer molecule is put into the Tris-HCl buffer solutions of pH 8.5, places 8 hours, it is heavy with black to obtain
The substance in shallow lake.The centrifugation that 15000rpm is carried out to solution, obtains the yellow supernatant solution containing oligomerization dopamine.
2, the quartz plate surface printing oligomerization dopamine pattern hydride modified to polyethylene glycol
(1), oxidation processes are carried out to quartz plate
By quartz plate (8*8*0.5mm3, buy in quartz ware Co., Ltd of Donghai County Weida) successively as acetone and anhydrous second
It is cleaned by ultrasonic 10min in alcohol, then in the concentrated sulfuric acid and dioxygen water mixed liquid (volume ratio 3：1) 1h is boiled in, ultra-pure water cleans,
Drying.Quartz plate is placed in Low Temperature Plasma Treating instrument and aoxidizes 6min.
(2), inertia modification is carried out to quartz plate
The toluene solution that quartz plate after oxidation is put into the methoxy poly (ethylene glycol) silane (MW2000) of 6g/L impregnates for 24 hours, and
And the triethylamine of addition 1% is as catalyst.After the completion of reaction, with washes of absolute alcohol 2 times, ultra-pure water cleans 2 times, N2It blows
It is dry, and it is stored in N2In atmosphere.
3, dimethyl silicone polymer seal is prepared
First, with Auto-CAD softwares, round (50 μm of diameter, 100 μm of spacing), rectangle (50 μm of the length of side, 100 μ of spacing are designed
M), matrix (the 1*1 cm of triangle (50 μm of the length of side, 100 μm of spacing) etc.2), and be printed upon above chromium plate and (tie up light long in Shenzhen
Electric Co., Ltd)；Then lithography process being carried out to the pattern, lithography process can be used traditional method and carry out, reference can be made to
Operating process (the network address that MicroChem companies provide： http://microchem.com/pdf/SU-
82000DataSheet2025thru2075Ver4.pdf)；Substrate uses the silicon chip of 2 cun of areas；Photoresist uses
The SU-8 thicker negtive photoresists of MicroChem companies.By spin coating, front baking, exposure, after dry, development after, the pattern just by
Photoresist on silicon chip carries.
Then, pattern duplication is carried out using dimethyl silicone polymer to obtained mold：The silicon with photoresist that will be processed
Piece is placed in drier, and ten silicofluoroforms (sigma companies buy, and No. cas is 51851-37-7) of 20 μ L are added, vacuumize
1h；Then mixture (the mass ratio 10 of the performed polymer and curing agent of dimethyl silicone polymer is gone up：1), 1h is steeped in vacuum degassing,
Then the baking oven at 80 DEG C toasts 1h, and dimethyl silicone polymer is formed solid；This solid be it is elastic, can be from silicon chip
It takes off above；Take dimethyl silicone polymer off, pattern has been transferred to above dimethyl silicone polymer.It is cut into conjunction as needed
The dimethyl silicone polymer of suitable size, you can be used as the seal of micro-contact printing, as shown in Figure 1.
4, in seal adsorption oligomerization dopamine
By seal bubble in the aqueous solution of oligomerization dopamine, a concentration of 0.02~2mg/mL, pH are 4.0~7.0, and soaking time is
30min.As shown in Figure 2.
5, oligomerization dopamine is printed on quartz plate surface, and inoculating cell
Seal is taken out from solution, dries up, the hydride modified quartz plate surface of methoxy poly (ethylene glycol) is lain in, on seal
It puts the counterweight of 50g, after 1min, removes counterweight, take seal off, to which oligomerization dopamine is printed on quartz plate surface, formed
It is used for the pattern of cell patterned growth.The quartz plate for being printed on oligomerization dopamine is placed in 24 orifice plates, it will be under rigid digestion
Cell suspending liquid (cell is mouse fibroblast cell, NIH3T3, a concentration of the 10 of cell suspending liquid5A/mL) be placed in substrate it
On, by 1h culture (cell incubator, 37 DEG C, 5%CO2, cell culture medium DMEM+10%FBS+1% is dual anti-), it obtains
Cell as shown in Figure 3 forms pattern in substrate, which is by cell only in the selection of position that oligomerization dopamine print
Property is sticked, and to form preset pattern, and cell good sprawls form sticking region and have.
The above embodiment is only the preferred embodiment of the present invention, and the scope of protection of the present invention is not limited thereto, ability
The variation and replacement for any unsubstantiality that the technical staff in domain is done on the basis of the present invention belong to required by the present invention
The range of protection.
Claims (8)
1. one kind being used for the patterned novel micro-contact printing system of cell, which is characterized in that the system includes by dopamine
Monomer polymerization forms oligomerization dopamine, and for molecular weight between 1000-10000 D, structural formula is as follows：
。
2. being used for the patterned novel micro-contact printing system of cell according to claim 1, which is characterized in that will have pattern
Seal be immersed in the aqueous solution of oligomerization dopamine, be then transferred to antibiont adherency modification substrate on, carry out cell training
It supports, and obtains the pattern of observable.
3. being used for the patterned novel micro-contact printing system of cell according to claim 2, which is characterized in that preparation has figure
The material of the substrate of case is sheet glass, silicon chip, titanium sheet, gold plaque or thin polystyrene sheet；The chemical modification of antibiont adherency is main
The substance used is polyethylene glycol substances or amphiphatic molecule.
4. being used for the patterned novel micro-contact printing system of cell according to claim 3, which is characterized in that the poly- second
Di-alcohol is methoxy poly (ethylene glycol) silane, mercapto-polyglycol or poly-D-lysine-polyethylene glycol；Amphiphatic molecule is carboxylic
Sour glycine betaine amphoteric ion polymer or sulphonic acid betaine type amphoteric ion polymer.
5. being used for the patterned novel micro-contact printing system of cell according to claim 2, which is characterized in that realize cell
Patterned method is as follows：
S1. the substrate of antibiont coherent substance modification is prepared：Simultaneously activated base is cleaned, by polyethylene glycols or amphiphatic molecule
Substance is grafted in substrate, obtains the substrate of antibiont coherent substance modification；
S2. seal is prepared, the type siloxane performed polymer of liquid is positioned in the template with micron scale structures, after solidification,
It obtains with figuratum seal；
S3. it will be immersed in oligomerization dopamine solution with figuratum seal in S2, S1 moderate resistance bioadhesions be positioned over after dry
Substrate, obtain the surface with oligomerization dopamine pattern.
6. being used for the patterned novel micro-contact printing system of cell according to claim 5, which is characterized in that clear in S1
Wash and activated base the specific steps are：Substrate is carried out to ultrasound in acetone, ethanol solution respectively, then in the concentrated sulfuric acid and double
0.5 ~ 1.5 h is boiled in the mixed liquor of oxygen water, it is dry after washing with water, and using after Low Temperature Plasma Treating, be dipped into poly-
In the solution of glycol substance, triethylamine react certain time is added, after cleaning-drying to obtain the final product.
7. being used for the patterned novel micro-contact printing system of cell according to claim 5, which is characterized in that few in S3
The pH of poly-dopamine solution is 4.0 ~ 7.0, a concentration of 0.02 ~ 2 mg/mL.
8. being used for the patterned novel micro-contact printing system of cell according to claim 5, which is characterized in that obtained in S3
Surface with oligomerization dopamine pattern, the selectivity for cell are sticked, and patterned cell is obtained.
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| CN201810345302.0A CN108641892B (en) | 2018-04-17 | 2018-04-17 | Micro-contact printing system for cell patterning |
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| CN201810345302.0A CN108641892B (en) | 2018-04-17 | 2018-04-17 | Micro-contact printing system for cell patterning |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110628618A (en) * | 2019-09-26 | 2019-12-31 | 大连理工大学 | Method for accurately controlling shape of single cell by using PDMS stamp |
| CN111333759A (en) * | 2020-02-26 | 2020-06-26 | 青岛科技大学 | Preparation method of zwitterionic polymer pattern on surface of solid substrate |
| CN111424013A (en) * | 2019-01-10 | 2020-07-17 | 中国科学院苏州纳米技术与纳米仿生研究所 | CTC (CTC) capturing and separating substrate based on polystyrene nanospheres and preparation method of CTC capturing and separating substrate |
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| KR20130134080A (en) * | 2012-05-30 | 2013-12-10 | 한국과학기술원 | Method of manufacturing surfaces for controlling cell attachment using plasma-treated biopolymer |
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| KR20170029237A (en) * | 2015-09-07 | 2017-03-15 | 한양대학교 산학협력단 | Substrate with biopolymer pattern for culturing micro-sized cell sheet and method for delivering the same |
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| CN102465119A (en) * | 2010-11-12 | 2012-05-23 | 国家纳米科学中心 | Substrate for cell micropatterning growth as well as preparation method and application thereof |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111424013A (en) * | 2019-01-10 | 2020-07-17 | 中国科学院苏州纳米技术与纳米仿生研究所 | CTC (CTC) capturing and separating substrate based on polystyrene nanospheres and preparation method of CTC capturing and separating substrate |
| CN110628618A (en) * | 2019-09-26 | 2019-12-31 | 大连理工大学 | Method for accurately controlling shape of single cell by using PDMS stamp |
| CN111333759A (en) * | 2020-02-26 | 2020-06-26 | 青岛科技大学 | Preparation method of zwitterionic polymer pattern on surface of solid substrate |
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| CN108641892B (en) | 2021-11-16 |
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Inventor after: Wang Fen Inventor after: Sun Kang Inventor after: Wang Dongfang Inventor after: Wang Jian Inventor after: Pang Jiadong Inventor before: Wang Fen Inventor before: Sun Kang Inventor before: Wang Dongfang Inventor before: Wang Jian Inventor before: Pang Jiadong Inventor before: Zeng Pinqi |
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