CN102643446A - Process for preparing protein micropattern on surface of PET (polyethylene glycol terephthalate) base material - Google Patents

Process for preparing protein micropattern on surface of PET (polyethylene glycol terephthalate) base material Download PDF

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CN102643446A
CN102643446A CN2012101194310A CN201210119431A CN102643446A CN 102643446 A CN102643446 A CN 102643446A CN 2012101194310 A CN2012101194310 A CN 2012101194310A CN 201210119431 A CN201210119431 A CN 201210119431A CN 102643446 A CN102643446 A CN 102643446A
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base material
pet
little pattern
solution
substrate surface
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CN102643446B (en
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吴仲岿
唐红肖
李少英
杨军
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Wuhan University of Technology WUT
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Abstract

The invention relates to a process for preparing protein micropattern on the surface of a PET (polyethylene glycol terephthalate) base material, which includes steps of firstly, preparing metal silver micropattern on the surface of the PET base material, soaking the PET base materials with the metal silver micropattern into SHPEG water solution so that a silver-plated area adsorbs SHPEG repelling protein and a non-silver-plated area reserves the property of the base material adsorbing protein, and accordingly, preparing the protein micropattern on the PET base material, and controlling selective adsorption of protein on the surface of the PET base material. The protein micropattern on the surface of the PET base material can be applied to surface biological diagnosis and manufacture of biosensors and biomedical devices and the like and creates a certain foundation for research of tissue engineering. Meanwhile, the protein micropattern on the surface of the PET base material is simple in preparation process, easy in control of preparation steps, low in cost and fine in fidelity and has excellent repeatability.

Description

A kind of technology for preparing the little pattern of albumen at the PET substrate surface
Technical field
The present invention relates to the field of inorganic chemistry, biological chemistry and organic polymer material surface and interface modification technology, particularly relate to a kind of technology for preparing the little pattern of albumen at PET (polyethylene terephthalate) substrate surface.
Background technology
The present material hot research fields is transferred in the research to the material surface micro-property from the research to macroscopical fields such as mechanical property of material; The fine setting control of material surface submicron-scale upper surface property and structure and little processing become the focus of people's research gradually, and the development of many modern technologies all derives from constructing of new microstructures and microminiaturization.In present life science; Can through to material surface modifying with realize that the material surface micro-patterning obtains they and the information of biomolecules and surrounding environment interphase interaction, this is significant to the detection that realizes various molecules in medical science, the industrial and environment; In addition, at the bottom of the substratum of micro-patterning on the specific region can be used to start protein adsorption and cell and attach, other zone is then played anti-albumen and is suppressed the effect that cell attaches, thereby can organize and control cell growth from the teeth outwards.This shows; The little pattern of nano-micro level albumen can be realized the regulation and control of film micro area character; This makes the little pattern of albumen have important application in a lot of fields; For example can be used to make biochip, biosensor, biomedical articles, microelectromechanical systems and be used for surface biological diagnosis etc. that [Science 293 (5537), 2001:2101 ~ 2105; Nano Lett 4 (10), 2004:1869 ~ 1872].Biomolecules optionally is fixed in the surface of solid material with a kind of mode that can control and still keeps its biological activity is to make the significant challenge that the biological device of microstructure is faced.
At present, about the report that on hard substrate such as silicon chip, glass and gold surface, prepares the albumen pattern has a lot, but because the preparation cost of this type of biomaterial is high, shortcomings such as toughness is low, light transmission difference make its application receive very big restriction.Thereby, people sight has been turned to have excellent biological compatibility, light transmission is good, toughness is high, chemicalstability is high, the organic polymer material of light weight, advantage such as with low cost.
Preparing the little method of patterning of albumen at material surface has a variety of; Micro-contact printing [Appl. Phys. Lett 63 (14) for example; 1993:2002 ~ 2004], little moulding of capillary [Annu Rev Mater Sci 281,998:153 ~ 184], electron beam lithography [Sci. Technol. B 16,1998:3526 ~ 3534] etc.; But all there is the weak point of self in these technology, like the particular requirement of the control of process, selection, fidelity of reproduction, repeatable relatively poor etc.A kind of suitable can to prepare the technology of the little pattern of albumen at the organic polymer material surface selectivity significant so seek.
Summary of the invention
Technical problem to be solved by this invention is to propose a kind of direct, easy process method for preparing the little pattern of albumen at the PET substrate surface to above-mentioned prior art; The preparation technology of the little pattern of albumen of this PET substrate surface is simple; The preparation process is easy to control; With low cost, the little pattern fidelity of albumen is good, and has good repeatability.
The present invention solves the problems of the technologies described above the technical scheme that is adopted: a kind ofly prepare the technology of the little pattern of albumen at the PET substrate surface, it is characterized in that including following steps:
1) absorption of sulfydryl polyoxyethylene glycol:
PET base material immersion 10 ~ 20 ml concentration that the surface had the little pattern of argent are in SHPEG (sulfydryl polyoxyethylene glycol) aqueous solution of 0.01 ~ 0.1 g/ml; Shake 10 ~ 20 min with have gentle hands, take out the PET base material that the surface has the little pattern of argent, with zero(ppm) water ultrasonic cleaning 3 ~ 6 times; Each 5 ~ 10 min; Nitrogen dries up the PET base material that the surface that has obtained adsorbing SHPEG has the little pattern of argent, is kept in the vacuum drying oven, and is subsequent use;
2) absorption of FITC (fluorescein isothiocyanate) mark BSA (bovine serum albumin):
Get BSA and be dissolved in NaHCO 3Obtain BSA solution in the buffered soln; The FITC of 5 ~ 10 mg is dissolved among 5 ~ 15 ml DMSO (anhydrous dimethyl sulfoxide); Getting the FITC/DMSO solution that 120 ~ 180 μ l obtain dropwise slowly joins in the BSA solution of 1 ~ 2 ml gained; Slightly shake while adding, reaction 6 ~ 10 h under-4 ~ 8 ℃ environment add 0.00545 ~ 0.00595 g NH afterwards 4Cl finishes reaction, in above-mentioned solution, adds the USP Kosher of 0.1 ~ 0.5 ml afterwards again, and concussion 5 ~ 10 min to isolate the not FITC of absorption, again with the solution ultrafiltration, thereby obtain the BSA solution of spissated FITC mark;
The PET base material that the surface of having adsorbed SHPEG is had the little pattern of argent is put into 24 orifice plates, and the little pattern of argent simultaneously makes progress the BSA solution of adding 0.15 ~ 0.30 ml FITC mark; On shaking table, shake lightly under the room temperature; Absorption 2 ~ 3 h, the BSA solution of sucking-off FITC mark shakes cleaning 3 ~ 5 times with the PBS buffered soln of 0.15 ~ 0.30 ml on shaking table; Each 10 ~ 15 min can prepare the little pattern of albumen at the PET substrate surface.
Press such scheme, the preparation method that the surface has the PET base material of the little pattern of argent is: photomask is fixed on the PET substrate surface that cleans up, places under the ultra-violet lamp of 165 ~ 195 nm; Light application time is 10 ~ 20 min; PET base material after the illumination is placed silver plating solution, treat that the silvered film of light appears in the PET substrate surface after, take out the PET base material; With distilled water flushing 3 ~ 5 times; And dry up with nitrogen and to obtain the PET base material that the surface has the little pattern of argent, be kept in the vacuum drier, subsequent use.
Press such scheme, silver plating solution be that reductive agent and the oxygenant of 1:1 formed by volume ratio, wherein reductive agent is made up of glucose (10 ~ 20 g/l) and Seignette salt (3 ~ 8 g/l), oxygenant is silver ammino solution (20 ~ 30 g/l).
Press such scheme, described NaHCO 3The pH of buffered soln is 7 ~ 9, and concentration is 5 ~ 15 mg/ml.
Press such scheme, the compound method of described PBS buffered soln is: take by weighing the NaCl of 15 ~ 20 g respectively, the NaH of 0.6 ~ 1.0 g 2PO 4, the Na of 2 ~ 5 g 2HPO 4, be dissolved in 1.5 ~ 2.5 L deionized waters, promptly get PBS buffered soln.
Ultimate principle of the present invention is: adopt the high energy UV-light that the PET base material is carried out the film micro area radiation; Utilize radiation areas and the selective deposition that is not realized argent in the chemical silvering process by the difference of radiation areas PET substrate surface chemical property; The PET base material that will have the little pattern of argent is immersed in the SHPEG aqueous solution; The proteinic SHPEG of repulsion is gone up in silver-plated zone just absorption, and not silver-plated zone still keeps the base material character of adhesion protein easily itself, then in follow-up protein adsorption process; Protein molecular will be adsorbed on not silver-plated zone, promptly can form and silver-plated little pattern complementary albumen pattern at substrate surface.Thereby on the PET base material, prepare the little pattern of albumen, realize albumen on the PET base material selective adsorption.
The present invention diagnoses at surface biological, and make aspects such as biosensor and biomedical articles and have purposes very widely, and the little pattern of the albumen of gained, also established certain basis for next step research of carrying out organizational project.In addition, the argent pattern of PET substrate surface and albumen pattern also can be realized docking of microelectronics and biosensor, have solved the signal transmission issues of interface bio-molecular interaction.
The present invention compares with other method of prior art; Its beneficial effect is: the present invention need not in material surface grafting unimolecular film, thereby also low or in the grafting process, introduce the fidelity that other impurity, pollutent reduce the albumen pattern because of the unimolecular film grafting density with regard to not existing in the protein adsorption process; Secondly, have little pattern of albumen and the little pattern of argent on the PET base material among the present invention simultaneously, because argent has electroconductibility, this just is easy to realize the mutual conversion between bio signal and the electrical signal that the thing preparation of devices of making a living provides maybe.At last, directly to the PET substrate surface functionalized after, tend to make the stability of surface functional group to reduce because of the rearrangement of macromolecular chain; Influence constructing of generate subsequent article patternization; Through to PET substrate surface argent micro-patterning,, can form the rigidity network structure at the PET substrate surface because argent is deposited on modification area; Thereby stop the rearrangement of macromolecular chain, improve the stability of function of surface group.
Description of drawings
Fig. 1 is a preparation technology's of the present invention synoptic diagram;
Among Fig. 2, Fig. 2 a is the silver-plated little pattern that utilizes fluorescent microscope observed PET substrate surface under natural light, and Fig. 2 b is the little pattern of bovine serum albumin that utilizes fluorescent microscope observed PET substrate surface under the wavelength of 495 nm.
Embodiment
In order to understand the present invention better, further illustrate content of the present invention below in conjunction with embodiment, but content of the present invention not only is confined to following embodiment.
Embodiment 1:
As shown in Figure 1, a kind ofly prepare the little method of patterning of bovine serum albumin at the PET substrate surface, it comprises the steps:
1) preparation of the little pattern of argent:
Photomask (copper mesh, the about 20 μ m of live width) is fixed on the PET substrate surface of 1 cm * 1 cm that cleans up, places under the ultra-violet lamp of 165 nm, light application time is 10 min.Base material after the illumination is placed silver plating solution, and (solution composition is V (reductive agent): V (oxygenant)=1:1, wherein reductive agent is made up of glucose (12 g/l) and Seignette salt (3 g/l), oxygenant is silver ammino solution (21 g/l)) in; After treating that the silvered film of light appears in substrate surface; Take out base material,, and dry up with nitrogen with distilled water flushing 3 times; Be kept in the vacuum drier, subsequent use.
2) absorption of sulfydryl polyoxyethylene glycol (SHPEG):
The PET base material that the surface is had the little pattern of argent immerses in the SHPEG solution that 10 ml concentration are 0.04 g/ml, shakes 10 min with have gentle hands.Afterwards, base material is taken out, with sheet material ultrasonic cleaning 3 times, each 5 min will wash attached to the SHPEG in silver-plated zone not with zero(ppm) water, and nitrogen dries up, and is kept in the vacuum drying oven, and is subsequent use.
3) absorption of fluorescein isothiocyanate (FITC) mark bovine serum albumin (BSA):
BSA is dissolved in NaHCO 3In the buffered soln (pH is 7), concentration is 5 mg/ml.The FITC of 5 mg is dissolved in the 5 ml anhydrous dimethyl sulfoxides (DMSO), and the FITC/DMSO solution of getting 120 μ l dropwise slowly joins in the above-mentioned BSA solution of 1 ml, slightly shakes while adding, afterwards reaction 6 h under-2 ℃ environment.Add 0.00545 g NH 4Cl finishes reaction, in above-mentioned solution, adds the USP Kosher of 0.2 ml afterwards again, shakes 5 min, to isolate the not FITC of absorption, again with solution ultrafiltration (2000 rpm), thereby obtains the BSA solution (being BSA-FITC solution) of spissated FITC mark.
Take by weighing the NaCl of 15 g respectively, the NaH of 0.6 g 2PO 4, the Na of 2 g 2HPO 4, be dissolved in the 1.5 L deionized waters, promptly get PBS buffered soln.
The PET base material that has silver-plated little pattern that has adsorbed SHPEG is put into 24 orifice plates; Patterned surfaces upwards adds 0.15 ml BSA-FITC solution, on shaking table, shakes (40 rpm) under the room temperature lightly and makes unmodified zone absorption 2 hs of BSA-FITC at PET; Absorption finishes; With suction pipe sucking-off BSA-FITC solution carefully, shake on shaking table with the PBS buffered soln of 0.15 ml and to clean each 10 min 3 times.Whole experiment will be carried out under the lucifuge condition.Through above-mentioned steps, can on the PET substrate surface, prepare the fidelity little pattern of bovine serum albumin preferably.
In Fig. 2 a, darker grid-like zone is the argent that is deposited on the PET substrate surface, then is base material part than bright area.In Fig. 2 b; The net region demonstrates green because adsorbed bovine serum albumin under fluorescent microscope; Darker grid-like silver-plated zone then fluorescence can not occur because of having adsorbed the proteic SHPEG of row; These two patterns are complementary obviously, and the pattern corner angle are clearly demarcated, explain that the fidelity of pattern is good.
Embodiment 2:
A kind ofly prepare the little method of patterning of bovine serum albumin at the PET substrate surface, it comprises the steps:
(1) preparation of the little pattern of argent:
Photomask (copper mesh, the about 20 μ m of live width) is fixed on the PET substrate surface of 1 cm * 1 cm that cleans up, places under the ultra-violet lamp of 170 nm, light application time is 15 min.Base material after the illumination is placed silver plating solution, and (solution composition is V (reductive agent): V (oxygenant)=1:1, wherein reductive agent is made up of glucose (15 g/l) and Seignette salt (6 g/l), oxygenant is silver ammino solution (24 g/l)) in; After treating that the silvered film of light appears in substrate surface; Take out base material,, and dry up with nitrogen with distilled water flushing 4 times; Be kept in the vacuum drier, subsequent use.
(2) absorption of sulfydryl polyoxyethylene glycol (SHPEG):
The PET base material that the surface is had the little pattern of argent immerses in the SHPEG solution that 15 ml concentration are 0.06 g/ml, shakes 15 min with have gentle hands.Afterwards, base material is taken out, with sheet material ultrasonic cleaning 5 times, each 8 min will wash attached to the SHPEG in silver-plated zone not with zero(ppm) water, and nitrogen dries up, and is kept in the vacuum drying oven, and is subsequent use.
(3) absorption of fluorescein isothiocyanate (FITC) mark bovine serum albumin (BSA):
BSA is dissolved in NaHCO 3In the buffered soln (pH is 8), concentration is 10 mg/ml.The FITC of 8 mg is dissolved in the 8 ml anhydrous dimethyl sulfoxides (DMSO), and the FITC/DMSO solution of getting 150 μ l dropwise slowly joins in the above-mentioned BSA solution of 1.5 ml, slightly shakes while adding, afterwards reaction 8 h under 0 ℃ environment.Add 0.00565 g NH 4Cl finishes reaction, in above-mentioned solution, adds the USP Kosher of 0.4 ml afterwards again, shakes 8 min, to isolate the not FITC of absorption, again with solution ultrafiltration (2500 rpm), thereby obtains the BSA solution (being BSA-FITC solution) of spissated FITC mark.
Take by weighing the NaCl of 18 g respectively, the NaH of 0.8 g 2PO 4, the Na of 3 g 2HPO 4, be dissolved in the 2 L deionized waters, promptly get PBS buffered soln.
The PET base material that has silver-plated little pattern that has adsorbed SHPEG is put into 24 orifice plates, and patterned surfaces upwards adds 0.2 ml BSA-FITC solution; On shaking table, shake (100 rpm) under the room temperature lightly; Make the unmodified zone absorption 2 .5 hs of BSA-FITC at PET, absorption finishes, with suction pipe sucking-off BSA-FITC solution carefully; PBS buffered soln with 0.25 ml shakes cleaning 4 times, each 12 min on shaking table.Whole experiment will be carried out under the lucifuge condition.
Adopt present embodiment identical with embodiment 1 at the little pattern of bovine serum albumin of PET substrate surface preparation.
Embodiment 3:
A kind ofly prepare the little method of patterning of bovine serum albumin at the PET substrate surface, it comprises the steps:
(1) preparation of the little pattern of argent:
Photomask (copper mesh, the about 20 μ m of live width) is fixed on the PET substrate surface of 1 cm * 1 cm that cleans up, places under the ultra-violet lamp of 185 nm, light application time is 20 min.Base material after the illumination is placed silver plating solution, and (solution composition is V (reductive agent): V (oxygenant)=1:1, wherein reductive agent is made up of glucose (19 g/l) and Seignette salt (8 g/l), oxygenant is silver ammino solution (27 g/l)) in; After treating that the silvered film of light appears in substrate surface; Take out base material,, and dry up with nitrogen with distilled water flushing 5 times; Be kept in the vacuum drier, subsequent use.
(2) absorption of sulfydryl polyoxyethylene glycol (SHPEG):
The PET base material that the surface is had the little pattern of argent immerses in the SHPEG solution that 20 ml concentration are 0.1 g/ml, shakes 20 min with have gentle hands.Afterwards, base material is taken out, with sheet material ultrasonic cleaning 6 times, each 10 min will wash attached to the SHPEG in silver-plated zone not with zero(ppm) water, and nitrogen dries up, and is kept in the vacuum drying oven, and is subsequent use.
(3) absorption of fluorescein isothiocyanate (FITC) mark bovine serum albumin (BSA):
BSA is dissolved in NaHCO 3In the buffered soln (pH is 9), concentration is 15 mg/ml.The FITC of 10 mg is dissolved in the 15 ml anhydrous dimethyl sulfoxides (DMSO), and the FITC/DMSO solution of getting 180 μ l dropwise slowly joins in the above-mentioned BSA solution of 2 ml, slightly shakes while adding, afterwards reaction 9 h under 6 ℃ environment.Add 0.00595 g NH 4Cl finishes reaction, in above-mentioned solution, adds the USP Kosher of 0.5 ml afterwards again, shakes 10 min, to isolate the not FITC of absorption, again with solution ultrafiltration (3000 rpm), thereby obtains the BSA solution (being BSA-FITC solution) of spissated FITC mark.
Take by weighing the NaCl of 20 g respectively, the NaH of 1.0 g 2PO 4, the Na of 5 g 2HPO 4, be dissolved in the 2.5 L deionized waters, promptly get PBS buffered soln.
The PET base material that has silver-plated little pattern that has adsorbed SHPEG is put into 24 orifice plates, and patterned surfaces upwards adds 0.30 ml BSA-FITC solution; On shaking table, shake (240 rpm) under the room temperature lightly; Make unmodified zone absorption 3 hs of BSA-FITC at PET, absorption finishes, with suction pipe sucking-off BSA-FITC solution carefully; PBS buffered soln with 0.3 ml shakes cleaning 5 times, each 15 min on shaking table.Whole experiment will be carried out under the lucifuge condition.
Adopt present embodiment identical with embodiment 1 at the little pattern of bovine serum albumin of PET substrate surface preparation.

Claims (5)

1. one kind prepares the technology of the little pattern of albumen at the PET substrate surface, it is characterized in that including following steps:
1) absorption of sulfydryl polyoxyethylene glycol:
PET base material immersion 10 ~ 20 ml concentration that the surface had the little pattern of argent are in the SHPEG solution of 0.01 ~ 0.1 g/ml; Shake 10 ~ 20 min with have gentle hands, take out the PET base material that the surface has the little pattern of argent, with zero(ppm) water ultrasonic cleaning 3 ~ 6 times; Each 5 ~ 10 min; Nitrogen dries up the PET base material that the surface that has obtained adsorbing SHPEG has the little pattern of argent, is kept in the vacuum drying oven, and is subsequent use;
2) absorption of FITC mark BSA:
Get BSA and be dissolved in NaHCO 3Obtain BSA solution in the buffered soln; The FITC of 5 ~ 10 mg is dissolved among 5 ~ 15 ml DMSO; Getting the FITC/DMSO solution that 120 ~ 180 μ l obtain dropwise slowly joins in the BSA solution of 1 ~ 2 ml gained; Slightly shake while adding, reaction 6 ~ 10 h under-4 ~ 8 ℃ environment add 0.00545 ~ 0.00595 g NH afterwards 4Cl finishes reaction, in above-mentioned solution, adds the USP Kosher of 0.1 ~ 0.5 ml afterwards again, and concussion 5 ~ 10 min to isolate the not FITC of absorption, again with the solution ultrafiltration, thereby obtain the BSA solution of spissated FITC mark;
The PET base material that the surface of having adsorbed SHPEG is had the little pattern of argent is put into 24 orifice plates, and the little pattern of argent simultaneously makes progress the BSA solution of adding 0.15 ~ 0.30 ml FITC mark; On shaking table, shake lightly under the room temperature; Absorption 2 ~ 3 h, the BSA solution of sucking-off FITC mark shakes cleaning 3 ~ 5 times with the PBS buffered soln of 0.15 ~ 0.30 ml on shaking table; Each 10 ~ 15 min can prepare the little pattern of albumen at the PET substrate surface.
2. prepare the technology of the little pattern of albumen by claim 1 is described at the PET substrate surface, it is characterized in that the preparation method that the surface has a PET base material of the little pattern of argent is: photomask is fixed on the PET substrate surface that cleans up, places under the ultra-violet lamp of 165 ~ 195 nm; Light application time is 10 ~ 20 min; PET base material after the illumination is placed silver plating solution, treat that the silvered film of light appears in the PET substrate surface after, take out the PET base material; With distilled water flushing 3 ~ 5 times; And dry up with nitrogen and to obtain the PET base material that the surface has the little pattern of argent, be kept in the vacuum drier, subsequent use.
3. by the described technology for preparing the little pattern of albumen at the PET substrate surface of claim 2; What it is characterized in that silver plating solution is that reductive agent and the oxygenant of 1:1 formed by volume ratio; Wherein reductive agent is made up of glucose and Seignette salt, and oxygenant is a silver ammino solution.
4. describedly prepare the technology of the little pattern of albumen by claim 1 or 3, it is characterized in that step 2 at the PET substrate surface) described NaHCO 3The pH of buffered soln is 7 ~ 9, and concentration is 5 ~ 15 mg/ml.
5. describedly preparing the technology of the little pattern of albumen by claim 1 or 3, it is characterized in that step 2 at the PET substrate surface) compound method of described PBS buffered soln is: take by weighing the NaCl of 15 ~ 20 g respectively, the NaH of 0.6 ~ 1.0 g 2PO 4, the Na of 2 ~ 5 g 2HPO 4, be dissolved in 1.5 ~ 2.5 L deionized waters, promptly get PBS buffered soln.
CN2012101194310A 2012-04-23 2012-04-23 Process for preparing protein micropattern on surface of PET (polyethylene glycol terephthalate) base material Expired - Fee Related CN102643446B (en)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103951280A (en) * 2014-04-22 2014-07-30 西南交通大学 Method for preparing fibrinogen graphics on surface of target material by using titanium oxide seal
CN106349660A (en) * 2016-08-29 2017-01-25 张德轩 Decoration material added with fluorescein and preparation method of decoration material
CN110194847A (en) * 2019-05-24 2019-09-03 陕西师范大学 A kind of preparation method of pet sheet face polymer pattern

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103951280A (en) * 2014-04-22 2014-07-30 西南交通大学 Method for preparing fibrinogen graphics on surface of target material by using titanium oxide seal
CN103951280B (en) * 2014-04-22 2016-05-18 西南交通大学 A kind of method of utilizing titanium oxide seal to prepare fibrinogen figure on target material surface
CN106349660A (en) * 2016-08-29 2017-01-25 张德轩 Decoration material added with fluorescein and preparation method of decoration material
CN110194847A (en) * 2019-05-24 2019-09-03 陕西师范大学 A kind of preparation method of pet sheet face polymer pattern

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