CN106544269A - A kind of preparation method with collagen nano-wire array layer cuvette - Google Patents
A kind of preparation method with collagen nano-wire array layer cuvette Download PDFInfo
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- CN106544269A CN106544269A CN201610975025.2A CN201610975025A CN106544269A CN 106544269 A CN106544269 A CN 106544269A CN 201610975025 A CN201610975025 A CN 201610975025A CN 106544269 A CN106544269 A CN 106544269A
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- 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
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- 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
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Abstract
The invention discloses a kind of preparation method with collagen nano-wire array layer cuvette.Methods described utilizes phosphate buffered saline collagen monomer solution, collagen monomer solution covers in the drop of micarex cuvette surface, and buffer solution for cleaning simultaneously cultivates a period of time, obtains collagen nano-wire array layer cuvette.The collagen that the cuvette of the present invention has good hydrophilicity, surface has excellent biocompatibility, can be used for cell culture.Simultaneously cuvette of the present invention relative to two mica sheets be into unidirectional nano-wire array, with good polarization characteristic, as the biology sensor of mask combined with electrochemical method synthesizing new, or the bioprobe of high specific, or synthesizing new micro-nano photoelectric material can be prepared into.
Description
Technical field
The present invention relates to the improvement of cuvette, and in particular to a kind of system with collagen nano-wire array layer cuvette
Preparation Method.
Background technology
Epitaxial growth (epitaxial growth) often refers to that a kind of mineral crystals are given birth on another kind of mineral crystals surface
It is long, the phenomenon that there is two kinds of mineral matters same crystal structure to be orientated on interface, using epitaxial growth principle in crystal substrate table
Face has become a popular domain come the film layer structure and growth kinetics for controlling and studying plating membrane molecule.Recent two decades come,
With reference to size and the adjustable molecule synthesis technology of function and the matrix of microcosmic crystal structure, under ultrahigh vacuum background, utilize
Molecular beam deposition technology controls the growth of organic molecular film layer crystal body in stromal surface, is that preparation function is predictable new organic
Micro-nano photoelectric material provides new method with device.For example, (hot wall epitaxy) technology is grown by hot-wall epitaxial, it is right
The blue light small molecule material of the high-quantum efficiencies such as six biphenyl (para-sexiphenyl) is in mica, TiO2, the stromal surface such as ITO
Orderly nano-wire array film can be generated, film shows very strong optical anisotropy due to structure long-range order, this
Outward, film can produce optical gain, have in the application of electroluminescent device of polarization characteristic Random Laser and preparation is obtained
Great market potential.
During the biomineralization of nature generally existing, on meso-scale, the mineral matter with regular repeating structures is brilliant
Body (hydroxyapatite etc.) can recognize mutually that with collagen fabric skeleton collagen fabric beam can provide support to mineralising,
And effectively regulate and control the startup of mineral crystals growth and block, so as to the organ such as specification bone, tooth in size with orientation
Growth.But anti-phase controlled in liquid phase buffer solution brilliant using mineral crystals and the recognition reaction of large biological molecule
There is not been reported for the large biological molecule self assembly in body surface face.If can by large biological molecule stromal surface epitaxial growth, for
Further investigation large biological molecule function, synthesizing new micro-nano biomaterial, and prepare that function is special and efficient bioprobe
All it is significant.
The content of the invention
Present invention aim at providing a kind of preparation method with collagen nano-wire array layer cuvette.
Realize that the technical scheme of above-mentioned purpose is as follows：
A kind of preparation method with collagen nano-wire array layer cuvette, comprises the following steps that：
Mica sheet is bonded to cuvette by step 1, sterilizes, dry after cleaning；
Step 2, prepares the collagen monomer solution of 5-10 μ g/mL with the sodium phosphate buffer that pH is 6.8～8.0；
Collagen monomer solution is added dropwise the sodium ascorbyl phosphate cushioning liquid behind cuvette surface, 15-20 minutes by step 3
Cleaning, removes absorption albumen loosely；
Step 4, covers cuvette surface with sodium phosphate buffer that pH is 6.8～8.0, carry out collagen from group
Dress, after self assembly terminates, washes, is dried, obtain the cuvette with collagen nano-wire array layer.
In step 4, the time of described self assembly is more than 12h.
In the present invention, protein monomers receive mica crystal surface, that is, (001) crystal face is advised leads, in " anti-phase biological ore deposit
Under change ", standard is epitaxially-formed collagen nano-wire array.Mica sheet structure cell is the structure that bilayer is mutually 120 °.In each list
In layer, the Al at center is combined with octahedral structure with O and is sandwiched by the Si/O layers on surface.The Si/O on top layer is connected with tetrahedral structure
Connect and formed the electronegative oxonium ion cavity of hexagon to accommodate the K ions of interlayer.Due to the deformation of Si/O tetrahedral structures,
So that a part of oxonium ion depression is gone down so as to form edge on whole crystal faceThe long and narrow electronegative groove in direction.Cloud
Master slice can be torn layer by layer and expose with preceding layer in 120 ° of brand-new crystal face.Collagen monomer is mutual with mica crystal surface
Identification, has effectively regulated and controled the direction of growth of azelon, and two albumen for being mutually 120 ° are formed in defective plane of crystal
Fiber array.
When pH is 6.8～8.0, the collagen monomer in sodium salt solution can be in micarex cuvette inner surface certainly
Assembling, forms the consistent azelon nano-wire array of orientation, and the nano wire in array is with the increase of collagen monomer concentration
Become finer and close, wall scroll nano wire is longer, and the crosslinking between nano wire is gradually notable.Measurement result shows, dense by difference
The width of the wall scroll nano wire that the collagen monomer solution of degree is cultivated with it is highly relatively stable, width is substantially left in 60nm
The right side, height is substantially in 1.5nm or so.
The present invention is easy to operate, is capable of the collagen nanowire array structure of precise control generation, and cuvette surface connects
Feeler average reaches 9.5 °, with good hydrophilicity.The present invention be with collagen nano-wire array layer cuvette, can
For cell culture.Additionally, cuvette of the present invention relative to two mica sheets on protein nano linear array into unified direction, tool
There is good polarization characteristic, can be used as mask, the biology sensor of combined with electrochemical method synthesizing new.
Description of the drawings
Fig. 1 is the structural representation with collagen nano-wire array layer cuvette.
Fig. 2 is the AFM height map with collagen nano-wire array layer cuvette obtained by embodiment 1.
Fig. 3 is the AFM height map with collagen nano-wire array layer cuvette obtained by embodiment 2.
Fig. 4 is the static contact angle on mica crystal face, and wherein A is brand-new (001) crystal face, and B is to receive with collagen
The crystal face that rice noodles film layer is covered.
Fig. 5 is the TEM/SAED figures of the mica crystal face that collagen nanofiber array is covered, wherein, A is unfocused
Constituency TEM photos, B are the diffraction pattern at the round dot occupied without fiber in figure A.
Fig. 6 is perspective view of the negative oxygen ion of mica crystal surface Si/O layers on (001) crystal face, wherein, A is 1 brilliant
Born of the same parents' structure, B are 9 cell configurations.
Fig. 7 is the EBSD result figures of the mica crystal face that collagen nanofiber array is covered, wherein, A is shone for constituency SEM
Piece, B are the diffraction pattern in A at the round dot that occupies without fiber, and C is nanofiber along micaDirection rotates 120 ° of simulation
Figure.
Specific embodiment
With reference to embodiment and accompanying drawing, the invention will be further described.
Embodiment 1
A kind of preparation method of the cuvette with collagen nano-wire array layer, comprises the following steps that：
Mica sheet is made cuvette structure by step 1, with one jiao of tape-stripping mica sheet tear off above which floor, expose light
Sliding mica sheet；
Step 2, prepares the Na of 0.2mol/L2HPO4With 0.3mol/L NaH2PO4Solution, by Na2HPO4Solution and NaH2PO4
Solution is hybridly prepared into the cushioning liquid of PH=6.8, and the collagen monomer for preparing 5 μ g/ml using sodium phosphate buffer is molten
Liquid；
Step 3, collagen solution is uniformly titrated on mica sheet, buffer solution for cleaning mica sheet is used after 15-20min；
Step 4, Titration Buffer on mica sheet after cleaning cover mica sheet, cultivate 12h, and washing is dried naturally
Afterwards, obtain the cuvette with collagen nano-wire array layer.
AFM observes self-assembled structures of the collagen on mica, as a result as shown in Figure 2.In fig. 2, it can be seen that battle array
Than sparse, the crosslinking between nano wire is less, the wall scroll nano wire that collagen monomer solution is cultivated for nano wire in row
Width with it is highly relatively stable, substantially in 60nm or so, height substantially has collagen nano wire in 1.5nm or so to width
The cuvette of array layer has good self-assembled structures, linear array characteristic.
Embodiment 2
A kind of preparation method of the cuvette with collagen nano-wire array layer, comprises the following steps that：
Mica sheet is made cuvette structure by step 1, with one jiao of tape-stripping mica sheet tear off above which floor, expose light
Sliding mica sheet；
Step 2, prepares the Na of 0.2mol/L2HPO4With 0.3mol/L NaH2PO4Solution, by Na2HPO4Solution and NaH2PO4
Solution is hybridly prepared into the cushioning liquid of PH=7, and the collagen monomer for preparing 10 μ g/ml using sodium phosphate buffer is molten
Liquid；
Step 3, collagen solution is uniformly titrated on mica sheet, buffer solution for cleaning mica sheet is used after 15-20min；
Step 4, Titration Buffer on mica sheet after cleaning cover mica sheet, cultivate 12h, and washing is dried naturally
Afterwards, obtain the cuvette with collagen nano-wire array layer.
AFM observes self-assembled structures of the collagen on mica, as a result as shown in Figure 3.In fig. 2, it is seen that battle array
Nano wire in row becomes finer and close with the increase of collagen monomer concentration, and wall scroll nano wire is longer, and between nano wire
Crosslinking it is gradually notable.But measurement result shows, the wall scroll nanometer cultivated by the collagen monomer solution of variable concentrations
The width of line with it is highly relatively stable, width and height not with change in concentration.
Static Water is carried out to brand-new mica (001) the crystal face sample covered with existing collagen film with nano lines respectively
The baseline circule method test of drop contact angle, is to reduce measure error, has taken to same sample that to take which after 3 points are measured average
Value.As a result it is as shown in Figure 4.Mica crystal face Contact-angle measurement average brand-new in Figure 4 A is 25.8 °, and Fig. 4 B have protein nano
It is only 9.5 ° that line covers the crystal face contact angle average of (10 μ g/ml).It will be apparent that collagen film with nano lines is significantly enhanced
The hydrophily of mica crystal face.
Performance is carried out on AFM and contact angle tester to the obtained cuvette with collagen nano-wire array layer
Characterization result shows the technological parameter according to example 2, and the cuvette of obtained collagen nano-wire array layer has well
Self-assembled structures, linear array characteristic, little contact angle, good hydrophilic property, cell culture characteristic, biometric characteristic sensor.
Embodiment 3
A kind of preparation method of the cuvette with collagen nano-wire array layer, comprises the following steps that：
Mica sheet is made cuvette structure by step 1, with one jiao of tape-stripping mica sheet tear off above which floor, expose light
Sliding mica sheet；
Step 2, prepares the Na of 0.2mol/L2HPO4With 0.3mol/L NaH2PO4Solution, Na2HPO4Solution and NaH2PO4It is molten
Liquid is hybridly prepared into the cushioning liquid of PH=8, prepares the collagen monomer solution of 10 μ g/ml using sodium phosphate buffer；
Step 3, collagen solution is uniformly titrated on mica sheet, buffer solution for cleaning mica sheet is used after 15-20min；
Step 4, Titration Buffer on mica sheet after cleaning cover mica sheet, cultivate 12h, and washing is dried naturally
Afterwards, obtain the cuvette with collagen nano-wire array layer.
Mica substrate to covering nanofiber film layer carries out the selected diffraction (SAED) of TEM, as a result as shown in figure 5, figure
Constituency sem images of the 5A for TEM, nanofiber are visible.Fig. 5 B are the electronic diffraction decorative pattern figure of certain point mica crystal, by right
The demarcation of diffraction pattern and coordinate analysis, the orientation of nano wire is substantially with mica [001] crystallographic axis into 60 °.
Gold-plated process is carried out to protein self-assembled structures mica sheet cuvette, and EBSD experiments is carried out with SEM, observe which
Crystal structure, as a result as shown in Figure 6.
In Fig. 6 A, it can be seen that in each individual layer, the Al at center is combined with octahedral structure with O and by the Si/O on surface
Layer is sandwiched.(001) Si/O of crystal face connected with tetrahedral structure to form the oxonium ion cavity of hexagon accommodate the K of interlayer from
Son.In the solution, the potassium ion of plane of crystal can be replaced by other ions or charged group.Due to the change of Si/O tetrahedral structures
Shape causes a part of oxonium ion depression to be gone downSo that oxonium ion cavity is into irregular hexagon, and on whole crystal face
Form edge as shown in Figure 6BDirection, with [001] crystallographic axis into 60 ° of negatively charged elongated slot, this is also electricity on (001) crystal face
The diadaxis of lotus distribution.The symmetry axis causes to have edge onlyDirection or perpendicular toThe distribution of charges in direction is
Uniquely.According to the unicity that the symmetry and collagen nano-wire array are orientated, may infer that the orientation of nano wire will
Parallel to the long and narrow negative oxygen ion groove, or being perpendicularly to the direction.
Fig. 7 A are certain specific sample constituency SEM image, and Fig. 7 B are area's EBSD decorative pattern figure.Based on to the area
Multiple spot diffraction decorative pattern is demarcated and coordinate analysis, and the orientation of nano wire is but substantially with mica [001] crystallographic axis into 0 °.This may system
Mica crystal face is damaged during sample, caused by exposing the crystal face that lower floor reverses 120 (see Fig. 7 C).This and nanometer in Fig. 5
Line is orientated edgeDirection meets.If being perpendicular toDirection, nanofiber then can not possibly be flat with mica [001] crystallographic axis
OK.Therefore nano wire is oriented parallel to the long and narrow negative oxygen ion groove, it is impossible to be perpendicularly to the direction.
Claims (3)
1. a kind of preparation method with collagen nano-wire array layer cuvette, it is characterised in that comprise the following steps that：
Mica sheet is bonded to cuvette by step 1, sterilizes, dry after cleaning；
Step 2, prepares the collagen monomer solution of 5-10 μ g/mL with the sodium phosphate buffer that pH is 6.8～8.0；
Collagen monomer solution is added dropwise the sodium ascorbyl phosphate cushioning liquid cleaning behind cuvette surface, 15-20 minutes by step 3,
Remove absorption albumen loosely；
Step 4, covers cuvette surface with the sodium phosphate buffer that pH is 6.8～8.0, carries out the self assembly of collagen,
After self assembly terminates, wash, be dried, obtain the cuvette with collagen nano-wire array layer.
2. preparation method according to claim 1, it is characterised in that in step 4, the time of described self assembly is 12h
More than.
3. cuvette obtained in preparation method according to claim 1 and 2.
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Cited By (2)
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| CN108120830A (en) * | 2017-12-13 | 2018-06-05 | 合肥安为康医学检验有限公司 | A kind of new microplate reader |
| CN109001240A (en) * | 2018-08-17 | 2018-12-14 | 胜科纳米（苏州）有限公司 | The method for preparing non-conductive material sample |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108120830A (en) * | 2017-12-13 | 2018-06-05 | 合肥安为康医学检验有限公司 | A kind of new microplate reader |
| CN109001240A (en) * | 2018-08-17 | 2018-12-14 | 胜科纳米（苏州）有限公司 | The method for preparing non-conductive material sample |
| CN109001240B (en) * | 2018-08-17 | 2021-04-13 | 胜科纳米（苏州）有限公司 | Method for preparing non-conductive material sample |
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Application publication date: 20170329 |