CN109082155A - A kind of application of tannic acid as micro-contact printing ink in cell patterning - Google Patents
A kind of application of tannic acid as micro-contact printing ink in cell patterning Download PDFInfo
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- CN109082155A CN109082155A CN201810750720.8A CN201810750720A CN109082155A CN 109082155 A CN109082155 A CN 109082155A CN 201810750720 A CN201810750720 A CN 201810750720A CN 109082155 A CN109082155 A CN 109082155A
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- Prior art keywords
- tannic acid
- substrate
- pattern
- cell
- polyethylene glycol
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- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09D—COATING COMPOSITIONS, e.g. PAINTS, VARNISHES OR LACQUERS; FILLING PASTES; CHEMICAL PAINT OR INK REMOVERS; INKS; CORRECTING FLUIDS; WOODSTAINS; PASTES OR SOLIDS FOR COLOURING OR PRINTING; USE OF MATERIALS THEREFOR
- C09D11/00—Inks
- C09D11/02—Printing inks
- C09D11/03—Printing inks characterised by features other than the chemical nature of the binder
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/0068—General culture methods using substrates
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2533/00—Supports or coatings for cell culture, characterised by material
- C12N2533/10—Mineral substrates
- C12N2533/12—Glass
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2533/00—Supports or coatings for cell culture, characterised by material
- C12N2533/30—Synthetic polymers
Abstract
Substrate is first carried out the surface treatment of polyethylene glycol substances, obtains 'inertia' substrate by the application the present invention provides a kind of tannic acid as micro-contact printing ink in cell patterning, the present invention；Then the aqueous solution for matching preparing tannic acid, prepares seal, then seal is dipped into the aqueous solution of tannic acid, then the seal for being adsorbed with tannic acid is placed in 'inertia' substrate, to obtain the pattern on seal in substrate surface.The pattern has the characteristics that transparent, can chelate ferroferric oxide nano granules, visualize pattern；The patterned substrate can also be used in the patterned growth of cell.This method is easy to operate, simplifies existing for the patterned technology of cell.
Description
Technical field
The present invention relates to technical field of cell culture, more particularly, to a kind of tannic acid as micro-contact printing ink
Application in cell patterning.
Background technique
Cell patterning, is a kind of Cell culture invitro technology, specifically, by preparing certain patterned substrate, is lured
The region that the response mechanism of guided cell itself is attached to design forms pattern.This method is different from laboratory direct kind common
It plants cell to cultivate into culture dish, the latter is usually formed cell monolayer film.
Cell patterning techniques have become the important tool of a kind of research and control cell behaviors, are widely used in
Study cell --- cell, cell --- interaction between substrate (including cell adhesion and apoptosis, polarization behavior,
Cell migration rate, cell differentiation behavior etc.)；External model can also be constructed, for organizational engineering, cell sensing, drug
The fields such as screening and trauma care.
Currently, cell patterning techniques can be roughly divided into two classes: (1) confinement method: passing through physically or chemically means shape
At some obstacles (such as micropore), by cell limitation, growth forms pattern wherein；(2) modification: to cell culture substrate
It is modified, close, the thin cell compartment of clear-cut is formed, so that cell selective grows to form pattern in close cell compartment.
In numerous cell patterning methods, microcontact printing techniques due to it is easy to operate, at low cost, can prepare large area pattern,
The features such as material (such as dimethyl silicone polymer, PDMS) used is nontoxic, transparent, easily molded, is 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
Film.Common process is that PDMS seal is dipped into thiol solution, and after drying and golden watch face contact, thiol molecule is just from print
The convex portion of chapter is transferred to gold surface, forms Iy self-assembled layer；The recessed part of seal is not contacted with substrate, so being exposed.
Gold substrate is dipped into another thiol molecule again, exposed part will be covered by second of thiol molecule, to be formed
Two kinds of Iy self-assembled layer.For example the mercaptan with methyl can promote cell adhesion, then anti-cell is glutinous for the mercaptan with polyethylene glycol
Attached, after seeding cells into gold surface, cell just will form patterned growth.It develops to again later and is printed onto silane molecule
On glass or silicon base.But this printing process needs two kinds of molecules to carry out self assembly on surface, and has specific want to substrate
It asks.
For above-mentioned limitation, the Jiang Xing space seminar of State Nanometer Science Center has developed a kind of one based on poly-dopamine
The patterning that print process is used for cell is walked, this method is suitable for a variety of substrates, such as gold surface, glass, silicon face, polystyrene
(PS) surface 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 easier, the less expensive patterned means and method to realize cell of cost of production.
Summary of the invention
It is a first objective of the present invention to provide a kind of tannic acid as micro-contact printing ink cell patterning in
Application.
The present invention uses more cheap raw material (tannic acid), and is based on the basis of tannic acid, the easier operation of use
Process realizes the patterning of cell.
Above-mentioned technical purpose that the invention is realized by the following technical scheme:
A kind of application of tannic acid as micro-contact printing ink in cell patterning, the following institute of the structural formula of the tannic acid
Show:
。
Preferably, patterned operation is realized are as follows: figuratum seal is immersed in the aqueous solution of tannic acid, is then turned
It prints in the substrate of polyethylene glycol substances modification, after magnetic granular absorption, obtains the pattern of observable.
Preferably, the material for preparing the substrate of figuratum seal is sheet glass, silicon wafer, titanium sheet, gold plaque or polystyrene
Thin slice, polyethylene glycol substances are methoxy poly (ethylene glycol) silane, mercapto-polyglycol or poly-D-lysine-polyethylene glycol.
Preferably, realize that patterned the specific method is as follows for cell:
S1. it prepares the substrate of polyethylene glycol substances modification: cleaning simultaneously activated base, polyethylene glycol substances are grafted on substrate
On, obtain the substrate of polyethylene glycol substances modification；
S2. seal is prepared, type siloxane performed polymer is placed in template, after solidification, is obtained with figuratum seal；
S3. it will be immersed in tannic acid solution, be placed in after dry polyethyleneglycol modified in S1 with figuratum seal in S2
Substrate obtains the surface with tannic acid pattern.
Primarily to obtaining the inert substrate that bio-resistant molecule sticks in S1；The inert substrate can be through poly- second
Glycols silane substance forms self assembled monolayer on quartz plate surface；
The pattern in inactive surfaces is obtained in S3, which is formed by the substance of permission albumen, cell adhesion, and described lazy
Property surface is not covered by the pattern all；Particularly, it is described allow magnetic particle, cell adhesion substance be tannic acid.
The pattern in inactive surfaces is obtained in S3, the pattern have the characteristics that it is transparent, in visible-range, have about
100% translucency.
In addition, the pattern formed in substrate inactive surfaces by the substance of permission cell adherence is arbitrary graphic pattern, including but
It is not limited to the irregular pattern or array being made of point, line, item, band, block, irregular shape etc..
Preferably, the specific steps of cleaning and activated base in S1 are as follows: carry out substrate in acetone, ethanol solution respectively
Then ultrasound boils 0.5 ~ 1.5 h in the mixed liquor of the concentrated sulfuric acid and hydrogen peroxide, dry after washing with water, and using low temperature etc.
It after gas ions processing, is dipped into the solution of polyethylene glycol substances, triethylamine is added and reacts certain time, after cleaning-drying i.e.
?.
Preferably, the pH of tannic acid solution is 4.0 ~ 7.0 in S3, and concentration is 0.02 ~ 2 mg/mL.
Further, the surface with tannic acid pattern is obtained in S3, after carrying out magnetic-particle absorption, is obtained patterned
Cell.
Further, the surface with tannic acid pattern is obtained in S3, the selectivity for cell is sticked, patterned
Cell.
Preferably, the process of magnetic-particle absorption are as follows: magnetisable material is distributed in water, colloidal solution is added, room temperature is stirred
After mixing overnight, washed after being separated with magnet, obtain the magnetic particle of colloid modification, the magnetic particle that above-mentioned colloid is modified with single
The surface of peaceful acid pattern is mixed, and the substrate of magnetic particle pattern is obtained.
Compared with prior art, the invention has the following advantages and beneficial effects:
1. the present invention forms transparent pattern by tannic acid, substrate without pass through specially treated can inoculating cell, and to thin
Born of the same parents are nontoxic.
2. step of the present invention is simple, only it need to provide cell inadhesive substrate, it then can by cell by micro-contact printing
It is attached in substrate to surface, such as by tannic acid by physical contact method with the print matter sticked, method is simple and easy, no
Expensive instrument is needed, suitable for common laboratory；Tannic acid is from a wealth of sources, cheap；Tannic acid used in the present invention
Ink and the polyethylene glycol of substrate are provided safeguard there are stronger active force to cell adhesion, are not in pattern obscission.
Detailed description of the invention
Fig. 1 is the schematic diagram for preparing dimethyl silicone polymer (PDMS) seal.
Fig. 2 is the schematic diagram that tannic acid forms monolayer in seal adsorption.
Fig. 3 passes through micro-contact printing, (A) uv-spectrophotometric in the hydride modified quartz surfaces of polyethylene glycol for tannic acid
Test result is measured, (B) adsorbs the pattern after magnetic particle.
Fig. 4 is mouse fibroblast cell (NIH3T3) after the culture of quartz plate surface 1 hour containing tannic acid pattern,
The pattern of formation.
Specific embodiment
Further illustrate that the present invention, embodiment illustrate and later in the accompanying drawings below in conjunction with specific embodiments and the drawings
It is illustrated, gives part detailed embodiment and specific operating process.Unless stated otherwise, the examination that the present invention uses
Agent, method and apparatus are the art conventional reagent, method and apparatus.
Embodiment 1:
The quartz plate surface printing tannic acid pattern hydride modified to polyethylene glycol
1, oxidation processes are carried out to quartz plate
By quartz plate (8*8*0.5 mm3, buy in quartz ware Co., Ltd of Donghai County Weida) successively as acetone and anhydrous
It is cleaned by ultrasonic 10 min in ethyl alcohol, then boils 1 h in the concentrated sulfuric acid and dioxygen water mixed liquid (volume ratio 3:1), ultrapure water is clear
It washes, dries up.Quartz plate is placed in Low Temperature Plasma Treating instrument and aoxidizes 6 min.
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 6 g/L is impregnated into 24 h,
And the triethylamine of addition 1% is as catalyst.After the reaction was completed, with washes of absolute alcohol 2 times, ultrapure water is cleaned 2 times, N2It blows
It is dry, and it is stored in N2In atmosphere.
3, dimethyl silicone polymer seal is prepared
Firstly, with Auto-CAD software, design round (50 μm of diameter, 100 μm of spacing), rectangle (50 μm of side length, spacing 100
μm), matrix (the 1*1 cm of triangle (50 μm of side length, 100 μm of spacing) etc.2), and be printed upon above chromium plate and (tie up long Shenzhen
Photoelectric Co., Ltd.)；Then lithography process is carried out to the pattern, lithography process can be used traditional method and carry out, can join
See the operating process (network address: http://microchem.com/pdf/SU-82000DataShee that MicroChem company provides
T2025thru2075Ver4.pdf)；Substrate uses the silicon wafer of 2 cun of areas；Photoresist uses MicroChem company
SU-8 thicker negtive photoresist.By spin coating, front baking, exposure, after dry, development after, the pattern is just by the photoetching on silicon wafer
Glue carries.
Then, pattern duplication is carried out using dimethyl silicone polymer to obtained mold: will processed with photoresist
Silicon wafer, be placed in drier, ten silicofluoroforms (sigma company purchase, No. cas is 51851-37-7) of 20 μ L be added,
Vacuumize 1 h；Then the mixture (mass ratio 10:1) of the performed polymer of upper dimethyl silicone polymer and curing agent, vacuum are removed
1 h of bubble, then the baking oven at 80 DEG C toasts 1 h, and dimethyl silicone polymer is formed solid；This solid be it is elastic,
It can be taken off above silicon wafer；Take dimethyl silicone polymer off, pattern has been transferred to above dimethyl silicone polymer.According to need
It is cut into the dimethyl silicone polymer of suitable size, that is, can be used as the seal of micro-contact printing, as shown in Figure 1.
4, in seal adsorption tannic acid
By seal bubble in the aqueous solution of tannic acid (structural formula of tannic acid is as previously shown), concentration is 0.02 ~ 2 mg/mL, pH
It is 4.0 ~ 7.0, soaking time is 30 min.As shown in Figure 2.
5, tannic acid is printed on quartz plate surface
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 50 g, after 1 min, removes counterweight, take seal off, so that tannic acid is printed on quartz plate surface, form
For the patterning of magnetic particle and the pattern of cell patterned growth.The pattern has the characteristics that transparent, passes through ultraviolet spectrometry light
Degree meter (UV-Vis) is characterized, in visible-range, transmitance 100%, as shown in (A) figure of Fig. 3.
6, in the quartz plate adsorption magnetic particle for being printed on tannic acid
By Fe3O4For (100-300 nm, Aladdin) ultrasonic disperse into ultrapure water, gelatin is added in 1:5 in mass ratio
(gelatin), it after 2 h are stirred at room temperature, is separated with magnet, ultrapure washing 3 times removes extra gelatin and modifies to get to gelatin
Magnetic particle (Fe3O4@gelatin NPs).The quartz plate for being printed on tannic acid is placed in 24 orifice plates, 1 mL, 0.5 mg/ is added
The Fe of mL3O4It is taken out after@gelatin NPs, 1 h, ultrapure water, air blow drying, microscope light field is taken pictures, such as (B) of Fig. 3
Shown in figure.
7, in the quartz plate surface seeding cell for being printed on tannic acid
The quartz plate for being printed on tannic acid is placed in 24 orifice plates, by the cell suspending liquid under rigid digestion, (cell is mouse into fiber finer
Born of the same parents, NIH3T3, the concentration of cell suspending liquid are 105A/mL) be placed on substrate, by 1 h culture (cell incubator,
37 DEG C, 5%CO2, cell culture medium DMEM+10%FBS+1% is dual anti-), it has obtained cell as shown in Figure 4 and figure is formed on the substrate
Case, which only sticked in the regioselectivity of tannic acid printing by cell, to form preset pattern, and cell is glutinous
Attached region, which has, good sprawls form.
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,
The variation and replacement for any unsubstantiality that those skilled in the art is done on the basis of the present invention belong to institute of the present invention
Claimed range.
Claims (9)
1. a kind of application of tannic acid as micro-contact printing ink in cell patterning, which is characterized in that the tannic acid
Structural formula it is as follows:
。
2. application according to claim 1, which is characterized in that figuratum seal is immersed in the aqueous solution of tannic acid
In, it is then transferred in the substrate of polyethylene glycol substances modification, after magnetic granular absorption, obtains the pattern of observable.
3. application according to claim 1, which is characterized in that prepare figuratum substrate material be sheet glass, silicon wafer,
Titanium sheet, gold plaque or thin polystyrene sheet, polyethylene glycol substances are methoxy poly (ethylene glycol) silane, mercapto-polyglycol or poly
Lysine-polyethylene glycol.
4. application according to claim 3, which is characterized in that realize that the patterned method of cell is as follows:
S1. it prepares the substrate of polyethylene glycol substances modification: cleaning simultaneously activated base, polyethylene glycol substances are modified in substrate
On, obtain the substrate of polyethylene glycol substances modification；
S2. seal is prepared, type siloxane performed polymer is placed in template, after solidification, is obtained with figuratum seal；
S3. it will be immersed in tannic acid solution, be placed in after dry polyethyleneglycol modified in S1 with figuratum seal in S2
Substrate obtains the surface with tannic acid pattern.
5. application according to claim 4, which is characterized in that the specific steps of cleaning and activated base in S1 are as follows: by base
Bottom carries out ultrasound in acetone, ethanol solution respectively, and 0.5 ~ 1.5 h is then boiled in the mixed liquor of the concentrated sulfuric acid and hydrogen peroxide,
It is dry after washing with water, and using after Low Temperature Plasma Treating, it is dipped into the solution of polyethylene glycol substances, three second is added
Amine reacts certain time, after cleaning-drying to obtain the final product.
6. application according to claim 4, which is characterized in that the pH of tannic acid solution is 4.0 ~ 7.0 in S3, and concentration is
0.02~2 mg/mL。
7. application according to claim 4, which is characterized in that obtain the surface with tannic acid pattern in S3, carry out magnetic
After property granular absorption, visualization pattern is obtained.
8. application according to claim 4, which is characterized in that the surface with tannic acid pattern is obtained in S3, for thin
The selectivity of born of the same parents is sticked, and patterned cell is obtained.
9. application according to claim 7, which is characterized in that the process of magnetic-particle absorption are as follows: disperse magnetisable material
Into water, colloidal solution is added, after being stirred overnight at room temperature, is washed after being separated with magnet, obtains the magnetic particle of colloid modification, it will be upper
The magnetic particle for stating colloid modification is mixed with the surface with tannic acid pattern, obtains the substrate of magnetic particle pattern.
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| CN2017107375079 | 2017-08-24 | ||
| CN201710737507 | 2017-08-24 |
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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 |
| CN114114840A (en) * | 2021-11-26 | 2022-03-01 | 山东大学 | Positive photoetching material based on tannic acid, preparation method thereof, photoresist system and application in preparation of micro-nano circuit |
| CN114616260A (en) * | 2019-10-15 | 2022-06-10 | 拉迪瑟夫私人有限责任公司 | Polymer gels in polymer brush formation |
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| CN105218847A (en) * | 2015-09-14 | 2016-01-06 | 北京化工大学 | A kind of bill of material interface function method of modifying based on polyphenolic compound |
| CN106644622A (en) * | 2016-09-12 | 2017-05-10 | 大连理工大学 | Method for realizing platelet patterning by polyphenols on material surface |
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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 |
| CN105218847A (en) * | 2015-09-14 | 2016-01-06 | 北京化工大学 | A kind of bill of material interface function method of modifying based on polyphenolic compound |
| CN106644622A (en) * | 2016-09-12 | 2017-05-10 | 大连理工大学 | Method for realizing platelet patterning by polyphenols on material surface |
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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 |
| CN114616260A (en) * | 2019-10-15 | 2022-06-10 | 拉迪瑟夫私人有限责任公司 | Polymer gels in polymer brush formation |
| CN114114840A (en) * | 2021-11-26 | 2022-03-01 | 山东大学 | Positive photoetching material based on tannic acid, preparation method thereof, photoresist system and application in preparation of micro-nano circuit |
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