CN102085125A - Method for fixing polypeptide aptamers on surface of cardiovascular implanting device - Google Patents
Method for fixing polypeptide aptamers on surface of cardiovascular implanting device Download PDFInfo
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- CN102085125A CN102085125A CN 201010589076 CN201010589076A CN102085125A CN 102085125 A CN102085125 A CN 102085125A CN 201010589076 CN201010589076 CN 201010589076 CN 201010589076 A CN201010589076 A CN 201010589076A CN 102085125 A CN102085125 A CN 102085125A
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Abstract
The invention discloses a method for fixing polypeptide aptamers on the surface of a cardiovascular implanting device, which ensures that endothelial progenitor cells in the blood can be captured after the ardiovascular implanting device is implanted into a human body. The method comprises the steps of: surface activation, silanization treatment, biotin fixation, avidin fixation, albumin fixation, biotinylated aptamers fixation and the like. The method for fixing specific polypeptide aptamers for capturing the endothelial progenitor cells on the surface of the cardiovascular implanting device ensure that the cardiovascular implanting device can be used for capturing the endothelial progenitor cells in the blood after being implanted into a human body, and is favorable for inducing the rapid endothelium transition of the surface of the implanting device and improving the blood compatibility of a material, and thus, the implanting device has favorable functions of blood anticoagulation and restenosis inhibition.
Description
Technical field
The present invention relates to the modification technology of inorganic or metal material surface, particularly the modification technology of artificial organ material, bio-sensing modulator material, anticorrosive and bioreediation material surface.
Background technology
[0002] cardiovascular system diseases is one of important diseases of harm humans health, and at the beginning of 21 century, about 1,700 ten thousand people in the whole world die from cardiovascular and cerebrovascular disease, expect death toll in 2020 and will reach 25,000,000 people.The whole world is annual now approximately need accept percutaneous transluminal coronary angioplasty (PTCA) above 2,000,000 patients with coronary heart disease, but acute or subacute arteria coronaria has taken place and has blocked in the patient who has implemented PTCA nearly 10% people, the incidence rate of restenosis is up to 20%～30% after 6 months, restenosis has become the key issue of PTCA, becomes the topmost factor of restriction PTCA development.
Research about medicine coating blood vessel support is study at most in the present prevention of restenosis method a kind of.At the stent surface coated medicine, form medication coat and stop the medicine of hamartoplasia to have a lot, what study often at present is rapamycin, paclitaxel and actinomycin D etc.Though coating stent of medicine can reduce the restenosis incidence rate, there is high relatively advanced thrombus risk in it, this be since its surperficial endothelialization not exclusively due to.
The endotheliocyte that one deck merges can improve the anti-thrombogenic capacity of implant, and can prevent false neointimal hyperplasia.Therefore make up a kind of finishing means of the coating of the quick endothelialization of inner membrance that promote to damage, be expected to solve support and implant long-term blood coagulation in back and restenosis problem as intravascular stent.Endothelial progenitor cells plays a crucial role in impaired cardiovascular endothelialization, and the endothelial progenitor cells in the blood circulation can be by chemotactic to cardiovascular damage location, and its differentiation, multiplication potentiality can make the structure function of damage endothelium repair fast.The quick endothelialization of damage inner membrance is important measures that do not solve neointimal hyperplasia and restenosis, has a good application prospect.
Summary of the invention
Shortcoming and defect in view of prior art, the surface that the purpose of this invention is to provide a kind of blood vessel implanting device is the method for the adaptive son of endothelial progenitor cells specific polypeptide fixedly, make it adaptive sub-orientation is fixed on the surface of cardiovascular implantation device, can catch endothelial progenitor cells in the blood behind the implant into body, induce the quick endothelialization of implanting device, thereby make implanting device have the function of good anticoagulation function and the generation of inhibition restenosis.
The objective of the invention is to realize by following means.
A kind of method of the adaptive son of surperficial immobilized polypeptide of cardiovascular implantation device can be caught endothelial progenitor cells in the blood after making the cardiovascular implantation device implant into body, comprises following preparation process:
A, surface active: sample inserted in the activated solution activate;
B, silanization handle: the implanting device after the A step is handled places 3-aminopropyl triethoxysilane (APTES) backflow system 1～24 h that refluxes;
C, fixed biologically element: implanting device is immersed in the biotin covalent coupling solution system that concentration is 0.005mg/mL～0.5mg/mL after the B step handles, reaction 1～36h;
D, fixing Avidin: implanting device is handled 1～12 h in the normal saline that back immersion concentration is 0.1～5 mg/mL Avidin through the C step;
E, albuminous fixing: D is gone on foot in the albumin solution of the implanting device immersion of handling 0.1%～5% earlier, hatch 0.2-36 h;
Fixing of F, the adaptive son of biotinylation: 1～36 h in the normal saline of the adaptive son of biotinylation polypeptide of implanting device taking-up immersion 1 ng～1 mg/mL that the E step handles.
Chemical reaction process, mechanism in the preparation process of the present invention are:
By the immersion of NaOH or orthophosphoric acid solution under the A step condition, make the surface of cardiovascular implantation device form loose structure and have a large amount of hydrophilic hydroxyls.
Pass through silane coupler at B in step again---the ethoxy group hydrolysis in the 3-aminopropyl three oxygen ethylsilane generates the hydroxyl generation covalency condensation on Si-OH and implanting device surface; Form the silane coupler monolayer at apparatus surface; Again in the 1-(3-dimethylaminopropyl)-3-ethyl carbodiimide catalyst action under, the amino of silane coupling agent layer and the carboxyl of Avidin react, and the fixing from the teeth outwards Avidin layer that forms.
The non-specific site of fixedly having sealed Avidin of E albumin (BSA) in the step makes the biotin end of adaptive son combine with the Avidin specificity of material surface, in conjunction with firm, thereby guarantees that the grafted adaptive sub-density of material surface is uniform and stable.
The biotin terminal specific of the adaptive son of biotinylation in F step in the fixed Avidin in implanting device surface and the soaking solution combines, thereby the ordered sequence of adaptive son is outwards arranged away from the surface of solids, realize that the orientation of adaptive son fixes, in order to catch the endothelial progenitor cells in the blood circulation.
Compared with prior art, the invention has the beneficial effects as follows:
C-terminus at the adaptive son of polypeptide that can the specificity capturing endothelial ancestral cell when design is synthetic has added the flexible arm of a GGGC, and has added a biotin end.By the activation processing material surface, make material surface obtain can the grafting Avidin group, behind the material surface grafting Avidin, thereby the biotin of adaptive sub-c-terminus combine fixing of the adaptive son of realization polypeptide with the Avidin of material surface.The method has realized that not only the orientation of adaptive son fixes, and has guaranteed the activity of peptide sequence, and the adaptive son of polypeptide that can the specificity capturing endothelial ancestral cell is fixed on the cardiovascular implantation device surface, has promptly constructed endothelial progenitor cells specific recognition surface.Because efficient, the adaptive son of specific binding polypeptide of the energy of the endothelial progenitor cells surface of cell membrane in the blood circulation, therefore, the present invention adopts on the device materials surface the fixedly adaptive son of EPC specific polypeptide, improved the efficient of catching endothelial progenitor cells in the blood, induce the quick endothelialization on implanting device surface, thereby make implanting device have the function of good anticoagulation function and the generation of inhibition restenosis.
Experimental results show that effectively capturing endothelial ancestral cell is induced implanting device surface endothelialization fast, has excellent anticoagulation function with the fixing material surface behind the adaptive son of endothelial progenitor cells polypeptide of the inventive method.
Description of drawings
The femoral artery that the unmodified implanting device of Fig. 1 is implanted adult dog takes out sem observation figure after 20 days.
The femoral artery that implanting device behind the adaptive son of Fig. 2 immobilized polypeptide of the present invention is implanted adult dog takes out sem observation figure after 20 days.
The specific embodiment
The invention will be further described below in conjunction with embodiment.Each chemical reagent that adopts among the embodiment except that dated especially, is analytical pure.Wherein, normal saline phosphoric acid buffer substitutes.
Embodiment 1
A, activation processing
Sample is inserted 83% phosphoric acid solution, activation 0.5 h in 120 ℃ of baking ovens, deionized water for ultrasonic is cleaned, and dries up.Again sample is immersed 10 min in 8.3% the phosphoric acid solution, behind 120 ℃ of insulation 12 h, deionized water ultrasonic cleaning 3 times, each 10 min, drying;
B, silanization are handled:
With A go on foot implanting device after the activation processing place concentration be 5% APTES/ anhydrous tetrahydrofuran solution (
V/
V) in, 5 h that reflux take out sample, continue backflow 5h in anhydrous tetrahydro furan.Ultrasonic cleaning, vacuum drying;
C, fixed biologically element
The biotin of 0.1 mg is joined in 20 mL, 0.001 mol/L 2-morpholino b acid (MES) aqueous solution, add 1 * 10 successively
-5Mol N-hydroxy-succinamide (NHS), 1 * 10
-5Mol 1-(3-dimethylaminopropyl)-and 3-ethyl carbodiimide (EDC), with the carboxyl on the activation biotin.The sample of silanization is immersed in the above-mentioned solution, and behind room temperature reaction 4 h, deionized water for ultrasonic is cleaned, vacuum drying.
D, fixing Avidin
Immerse 12 h in the normal saline that Avidin concentration is 0.1 mg/mL, take out the back with cleaning drying;
Fixing of E, albumin (BSA):
Implanting device after B step handled immerses in 0.1% the albumin solution, and incubated at room 40 min contain its immersion 12 h in the normal saline of BSA of 0.01 mg/mL again, take out the back and clean drying;
Fixing of F, the adaptive son of biotinylation:
The absorption that E step is obtained albuminous implanting device immerse 12 h in the normal saline of the adaptive sub-TPSLEQRTVYAK-GGGC-K of biotinylation polypeptide (BIOTIN)-COOH of 0.1 mg/mL.By the recognition reaction of biotin-avidin, the adaptive son of immobilized polypeptide takes out the back and cleans drying.
Table 1. acid activation (soaking for the first time) conditional parameter
Orthophosphoric acid activation experiment L
9
(3
3
) orthogonal experiment
| ? | Phosphoric acid concentration (%) | Reaction temperature (℃) | Soak time (h) |
| 1 | 1(83%) | 1 (room temperature) | 1(0.5h) |
| 2 | 1(83%) | 2(60℃) | 2(12h) |
| 3 | 1(83%) | 3(120℃) | 3(24h) |
| 4 | 2(85%) | 1 (room temperature) | 1(0.5h) |
| 5 | 2(85%) | 2(60℃) | 2(12h) |
| 6 | 2(85%) | 3(120℃) | 3(24h) |
| 7 | 3(98%) | 1 (room temperature) | 1(0.5h) |
| 8 | 3(98%) | 2(60℃) | 2(12h) |
| 9 | 3(98%) | 3(120℃) | 3(24h) |
Table 2. acid activation (soaking for the second time) conditional parameter
Orthophosphoric acid activation experiment L
9
(3
3
) orthogonal experiment
| ? | Phosphoric acid concentration (%) | Reaction temperature (℃) | Soak time (h) |
| 1 | 1(8.3%) | 1(60℃) | 1(0.5h) |
| 2 | 1(8.3%) | 2(90℃) | 2(12h) |
| 3 | 1(8.3%) | 3(120℃) | 3(24h) |
| 4 | 2(8.5%) | 1(60℃) | 1(0.5h) |
| 5 | 2(8.5%) | 2(90℃) | 2(12h) |
| 6 | 2(8.5%) | 3(120℃) | 3(24h) |
| 7 | 3(9.8%) | 1(60℃) | 1(0.5h) |
| 8 | 3(9.8%) | 2(90℃) | 2(12h) |
| 9 | 3(9.8%) | 3(120℃) | 3(24h) |
Embodiment 2
A, activation processing
Cardiovascular implantation device is soaked 24h in 90 ℃ 4mol/L NaOH solution, taking-up is placed in 60 ℃ of deionized waters and is incubated 0.5h, drying;
B, silanization are handled:
With A go on foot implanting device after the activation processing place concentration be 5% APTES/ anhydrous tetrahydrofuran solution (
V/
V) in, 5 h that reflux take out sample, continue backflow 5h in anhydrous tetrahydro furan.Ultrasonic cleaning, vacuum drying;
C, fixed biologically element
The biotin of 0.1 mg is joined in 20 mL, 0.001 mol/L 2-morpholino b acid (MES) aqueous solution, add 1 * 10 successively
-5Mol N-hydroxy-succinamide (NHS), 1 * 10
-5Mol 1-(3-dimethylaminopropyl)-and 3-ethyl carbodiimide (EDC), with the carboxyl on the activation biotin.The sample of silanization is immersed in the above-mentioned solution, and behind room temperature reaction 4 h, deionized water for ultrasonic is cleaned, vacuum drying.
D, fixing Avidin
Immersed in the normal saline that Avidin concentration is 0.1 mg/mL 12 hours, and took out the back with cleaning drying;
Fixing of E, albumin (BSA):
Implanting device after B step handled immerses in 0.1% the albumin solution, and incubated at room 40 min contain its immersion 12 h in the normal saline of BSA of 0.01 mg/mL again, take out the back and clean drying;
Fixing of F, the adaptive son of biotinylation:
The absorption that E step is obtained albuminous implanting device immerse 12 h in the normal saline of the adaptive sub-TPSLEQRTVYAK-GGGC-K of biotinylation polypeptide (BIOTIN)-COOH of 0.1 mg/mL.By the recognition reaction of biotin-avidin, the adaptive son of immobilized polypeptide takes out the back and cleans drying.
Table 3. alkali activation condition parameter
NaOH activation experiment L
9
(3
3
) orthogonal experiment
| ? | NaOH concentration (mol/L) | Reaction temperature (℃) | Soak time (h) |
| 1 | 1(0.5mol/L) | 1 (room temperature) | 1(0.5h) |
| 2 | 1(0.5mol/L) | 2(60℃) | 2(12h) |
| 3 | 1(0.5mol/L) | 3(120℃) | 3(24h) |
| 4 | 2(3mol/L) | 1 (room temperature) | 1(0.5h) |
| 5 | 2(3mol/L) | 2(60℃) | 2(12h) |
| 6 | 2(3mol/L) | 3(120℃) | 3(24h) |
| 7 | 3(5mol/L) | 1 (room temperature) | 1(0.5h) |
| 8 | 3(5mol/L) | 2(60℃) | 2(12h) |
| 9 | 3(5mol/L) | 3(120℃) | 3(24h) |
Embodiment 3
A, activation processing
Cardiovascular implantation device is soaked 24h in 90 ℃ 4mol/L NaOH solution, taking-up is placed in 120 ℃ of deionized waters and is incubated 0.5h, drying;
B, silanization are handled:
With A go on foot implanting device after the activation processing place concentration be 5% APTES/ anhydrous tetrahydrofuran solution (
V/
V) in, 5 h that reflux take out sample, continue backflow 5h in anhydrous tetrahydro furan.Ultrasonic cleaning, vacuum drying;
C, fixed biologically element
The biotin of 0.1 mg is joined in 20 mL, 0.001 mol/L 2-morpholino b acid (MES) aqueous solution, add 1 * 10 successively
-5Mol N-hydroxy-succinamide (NHS), 1 * 10
-5Mol 1-(3-dimethylaminopropyl)-and 3-ethyl carbodiimide (EDC), with the carboxyl on the activation biotin.The sample of silanization is immersed in the above-mentioned solution, and behind room temperature reaction 4 h, deionized water for ultrasonic is cleaned, vacuum drying.
D, fixing Avidin
Immersed in the normal saline that Avidin concentration is 0.1 mg/mL 12 hours, and took out the back with cleaning drying;
Fixing of E, albumin (BSA):
Implanting device after B step handled immerses in 0.1% the albumin solution, and incubated at room 40 min contain its immersion 12 h in the normal saline of BSA of 0.01 mg/mL again, take out the back and clean drying;
Fixing of F, the adaptive son of biotinylation:
The absorption that E step is obtained albuminous implanting device immerse 12 h in the normal saline of the adaptive sub-TPSLEQRTVYAK-GGGC-K of biotinylation polypeptide (BIOTIN)-COOH of 0.1 mg/mL.By the recognition reaction of biotin-avidin, the adaptive son of immobilized polypeptide takes out the back and cleans drying.
Embodiment 4
A, activation processing
Cardiovascular implantation device is soaked 24h in 90 ℃ 4mol/L NaOH solution, taking-up is placed in 60 ℃ of deionized waters and is incubated 24h, drying;
B, silanization are handled:
With A go on foot implanting device after the activation processing place concentration be 5% APTES/ anhydrous tetrahydrofuran solution (
V/
V) in, 5 h that reflux take out sample, continue backflow 5h in anhydrous tetrahydro furan.Ultrasonic cleaning, vacuum drying;
C, fixed biologically element
The biotin of 0.1 mg is joined in 20 mL, 0.001 mol/L 2-morpholino b acid (MES) aqueous solution, add 1 * 10 successively
-5Mol N-hydroxy-succinamide (NHS), 1 * 10
-5Mol 1-(3-dimethylaminopropyl)-and 3-ethyl carbodiimide (EDC), with the carboxyl on the activation biotin.The sample of silanization is immersed in the above-mentioned solution, and behind room temperature reaction 4 h, deionized water for ultrasonic is cleaned, vacuum drying.
D, fixing Avidin
Immersed in the normal saline that Avidin concentration is 0.1 mg/mL 12 hours, and took out the back with cleaning drying;
Fixing of E, albumin (BSA):
Implanting device after B step handled immerses in 0.1% the albumin solution, and incubated at room 40 min contain its immersion 12 h in the normal saline of BSA of 0.01 mg/mL again, take out the back and clean drying;
Fixing of F, the adaptive son of biotinylation:
The absorption that E step is obtained albuminous implanting device immerse 12 h in the normal saline of the adaptive sub-TPSLEQRTVYAK-GGGC-K of biotinylation polypeptide (BIOTIN)-COOH of 0.1 mg/mL.By the recognition reaction of biotin-avidin, the adaptive son of immobilized polypeptide takes out the back and cleans drying.
Embodiment 5
A, activation processing
Cardiovascular implantation device is soaked 24h in 90 ℃ 4mol/L NaOH solution, taking-up is placed in 120 ℃ of deionized waters and is incubated 24h, drying;
B, silanization are handled:
With A go on foot implanting device after the activation processing place concentration be 5% APTES/ anhydrous tetrahydrofuran solution (
V/
V) in, 5 h that reflux take out sample, continue backflow 5h in anhydrous tetrahydro furan.Ultrasonic cleaning, vacuum drying;
C, fixed biologically element
The biotin of 0.1 mg is joined in 20 mL, 0.001 mol/L 2-morpholino b acid (MES) aqueous solution, add 1 * 10 successively
-5Mol N-hydroxy-succinamide (NHS), 1 * 10
-5Mol 1-(3-dimethylaminopropyl)-and 3-ethyl carbodiimide (EDC), with the carboxyl on the activation biotin.The sample of silanization is immersed in the above-mentioned solution, and behind room temperature reaction 4 h, deionized water for ultrasonic is cleaned, vacuum drying.
D, fixing Avidin
Immersed in the normal saline that Avidin concentration is 0.1 mg/mL 12 hours, and took out the back with cleaning drying;
Fixing of E, albumin (BSA):
Implanting device after B step handled immerses in 0.1% the albumin solution, and incubated at room 40 min contain its immersion 12 h in the normal saline of BSA of 0.01 mg/mL again, take out the back and clean drying;
Fixing of F, the adaptive son of biotinylation:
The absorption that E step is obtained albuminous implanting device immerse 12 h in the normal saline of the adaptive sub-TPSLEQRTVYAK-GGGC-K of biotinylation polypeptide (BIOTIN)-COOH of 0.1 mg/mL.By the recognition reaction of biotin-avidin, the adaptive son of immobilized polypeptide takes out the back and cleans drying.
Embodiment 6
A, activation processing
Sample is inserted 83% phosphoric acid solution, activation 0.5 h in 120 ℃ of baking ovens, deionized water for ultrasonic is cleaned, and dries up.Again sample is immersed 10 min in 8.3% the phosphoric acid solution, behind 120 ℃ of insulation 12 h, deionized water ultrasonic cleaning 3 times, each 10 min, drying;
B, silanization are handled:
With A go on foot implanting device after the activation processing place concentration be 0.01% APTES/ anhydrous tetrahydrofuran solution (
V/
V) in, 5 h that reflux take out sample, continue backflow 5h in anhydrous tetrahydro furan.Ultrasonic cleaning, vacuum drying;
C, fixed biologically element
The biotin of 0.1 mg is joined in 20 mL, 0.001 mol/L 2-morpholino b acid (MES) aqueous solution, add 1 * 10 successively
-5Mol N-hydroxy-succinamide (NHS), 1 * 10
-5Mol 1-(3-dimethylaminopropyl)-and 3-ethyl carbodiimide (EDC), with the carboxyl on the activation biotin.The sample of silanization is immersed in the above-mentioned solution, and behind room temperature reaction 4 h, deionized water for ultrasonic is cleaned, vacuum drying.
D, fixing Avidin
Immerse 12 h in the normal saline that Avidin concentration is 0.1 mg/mL, take out the back with cleaning drying;
Fixing of E, albumin (BSA):
Implanting device after B step handled immerses in 0.1% the albumin solution, and incubated at room 40 min contain its immersion 12 h in the normal saline of BSA of 0.01 mg/mL again, take out the back and clean drying;
Fixing of F, the adaptive son of biotinylation:
The absorption that E step is obtained albuminous implanting device immerse 12 h in the normal saline of the adaptive sub-TPSLEQRTVYAK-GGGC-K of biotinylation polypeptide (BIOTIN)-COOH of 0.1 mg/mL.By the recognition reaction of biotin-avidin, the adaptive son of immobilized polypeptide takes out the back and cleans drying.
Embodiment 7
A, activation processing
Sample is inserted 83% phosphoric acid solution, activation 0.5 h in 120 ℃ of baking ovens, deionized water for ultrasonic is cleaned, and dries up.Again sample is immersed 10 min in 8.3% the phosphoric acid solution, behind 120 ℃ of insulation 12 h, deionized water ultrasonic cleaning 3 times, each 10 min, drying;
B, silanization are handled:
With A go on foot implanting device after the activation processing place concentration be APTES/ethanol solution of 0.01% (
V/
V) in, 5 h that reflux take out sample, continue backflow 5h in dehydrated alcohol.Ultrasonic cleaning, vacuum drying;
C, fixed biologically element
The biotin of 0.1 mg is joined in 20 mL, 0.001 mol/L 2-morpholino b acid (MES) aqueous solution, add 1 * 10 successively
-5Mol N-hydroxy-succinamide (NHS), 1 * 10
-5Mol 1-(3-dimethylaminopropyl)-and 3-ethyl carbodiimide (EDC), with the carboxyl on the activation biotin.The sample of silanization is immersed in the above-mentioned solution, and behind room temperature reaction 4 h, deionized water for ultrasonic is cleaned, vacuum drying.
D, fixing Avidin
Immerse 12 h in the normal saline that Avidin concentration is 0.1 mg/mL, take out the back with cleaning drying;
Fixing of E, albumin (BSA):
Implanting device after B step handled immerses in 0.1% the albumin solution, and incubated at room 40 min contain its immersion 12 h in the normal saline of BSA of 0.01 mg/mL again, take out the back and clean drying;
Fixing of F, the adaptive son of biotinylation:
The absorption that E step is obtained albuminous implanting device immerse 12 h in the normal saline of the adaptive sub-TPSLEQRTVYAK-GGGC-K of biotinylation polypeptide (BIOTIN)-COOH of 0.1 mg/mL.By the recognition reaction of biotin-avidin, the adaptive son of immobilized polypeptide takes out the back and cleans drying.
Embodiment 8
A, activation processing
Sample is inserted 83% phosphoric acid solution, activation 0.5 h in 120 ℃ of baking ovens, deionized water for ultrasonic is cleaned, and dries up.Again sample is immersed 10 min in 8.3% the phosphoric acid solution, behind 120 ℃ of insulation 12 h, deionized water ultrasonic cleaning 3 times, each 10 min, drying;
B, silanization are handled:
With A go on foot implanting device after the activation processing place concentration be APTES/ethanol solution of 5% (
V/
V) in, 5 h that reflux take out sample, continue backflow 5h in dehydrated alcohol.Ultrasonic cleaning, vacuum drying;
C, fixed biologically element
The biotin of 0.1 mg is joined in 20 mL, 0.001 mol/L 2-morpholino b acid (MES) aqueous solution, add 1 * 10 successively
-5Mol N-hydroxy-succinamide (NHS), 1 * 10
-5Mol 1-(3-dimethylaminopropyl)-and 3-ethyl carbodiimide (EDC), with the carboxyl on the activation biotin.The sample of silanization is immersed in the above-mentioned solution, and behind room temperature reaction 4 h, deionized water for ultrasonic is cleaned, vacuum drying.
D, fixing Avidin
Immerse 12 h in the normal saline that Avidin concentration is 0.1 mg/mL, take out the back with cleaning drying;
Fixing of E, albumin (BSA):
Implanting device after B step handled immerses in 0.1% the albumin solution, and incubated at room 40 min contain its immersion 12 h in the normal saline of BSA of 0.01 mg/mL again, take out the back and clean drying;
Fixing of F, the adaptive son of biotinylation:
The absorption that E step is obtained albuminous implanting device immerse 12 h in the normal saline of the adaptive sub-TPSLEQRTVYAK-GGGC-K of biotinylation polypeptide (BIOTIN)-COOH of 0.1 mg/mL.By the recognition reaction of biotin-avidin, the adaptive son of immobilized polypeptide takes out the back and cleans drying.
Embodiment 9
A, activation processing
Sample is inserted 83% phosphoric acid solution, activation 0.5 h in 120 ℃ of baking ovens, deionized water for ultrasonic is cleaned, and dries up.Again sample is immersed 10 min in 8.3% the phosphoric acid solution, behind 120 ℃ of insulation 12 h, deionized water ultrasonic cleaning 3 times, each 10 min, drying;
B, silanization are handled:
With A go on foot implanting device after the activation processing place concentration be APTES/anhydrous toluene solution of 0.1% (
V/
V) in, 5 h that reflux take out sample, continue backflow 5h in dry toluene.Ultrasonic cleaning, vacuum drying;
C, fixed biologically element
The biotin of 0.1 mg is joined in 20 mL, 0.001 mol/L 2-morpholino b acid (MES) aqueous solution, add 1 * 10 successively
-5Mol N-hydroxy-succinamide (NHS), 1 * 10
-5Mol 1-(3-dimethylaminopropyl)-and 3-ethyl carbodiimide (EDC), with the carboxyl on the activation biotin.The sample of silanization is immersed in the above-mentioned solution, and behind room temperature reaction 4 h, deionized water for ultrasonic is cleaned, vacuum drying.
D, fixing Avidin
Immerse 12 h in the normal saline that Avidin concentration is 0.1 mg/mL, take out the back with cleaning drying;
Fixing of E, albumin (BSA):
Implanting device after B step handled immerses in 0.1% the albumin solution, and incubated at room 40 min contain its immersion 12 h in the normal saline of BSA of 0.01 mg/mL again, take out the back and clean drying;
Fixing of F, the adaptive son of biotinylation:
The absorption that E step is obtained albuminous implanting device immerse 12 h in the normal saline of the adaptive sub-TPSLEQRTVYAK-GGGC-K of biotinylation polypeptide (BIOTIN)-COOH of 0.1 mg/mL.By the recognition reaction of biotin-avidin, the adaptive son of immobilized polypeptide takes out the back and cleans drying.
Embodiment 10
A, activation processing
Sample is inserted 83% phosphoric acid solution, activation 0.5 h in 120 ℃ of baking ovens, deionized water for ultrasonic is cleaned, and dries up.Again sample is immersed 10 min in 8.3% the phosphoric acid solution, behind 120 ℃ of insulation 12 h, deionized water ultrasonic cleaning 3 times, each 10 min, drying;
B, silanization are handled:
With A go on foot implanting device after the activation processing place concentration be 5% APTES/ anhydrous tetrahydrofuran solution (
V/
V) in, 1 h that refluxes takes out sample, continues backflow 1h in anhydrous tetrahydro furan.Ultrasonic cleaning, vacuum drying;
C, fixed biologically element
The biotin of 0.1 mg is joined in 20 mL, 0.001 mol/L 2-morpholino b acid (MES) aqueous solution, add 1 * 10 successively
-5Mol N-hydroxy-succinamide (NHS), 1 * 10
-5Mol 1-(3-dimethylaminopropyl)-and 3-ethyl carbodiimide (EDC), with the carboxyl on the activation biotin.The sample of silanization is immersed in the above-mentioned solution, and behind room temperature reaction 4 h, deionized water for ultrasonic is cleaned, vacuum drying.
D, fixing Avidin
Immerse 12 h in the normal saline that Avidin concentration is 0.1 mg/mL, take out the back with cleaning drying;
Fixing of E, albumin (BSA):
Implanting device after B step handled immerses in 0.1% the albumin solution, and incubated at room 40 min contain its immersion 12 h in the normal saline of BSA of 0.01 mg/mL again, take out the back and clean drying;
Fixing of F, the adaptive son of biotinylation:
The absorption that E step is obtained albuminous implanting device immerse 12 h in the normal saline of the adaptive sub-TPSLEQRTVYAK-GGGC-K of biotinylation polypeptide (BIOTIN)-COOH of 0.1 mg/mL.By the recognition reaction of biotin-avidin, the adaptive son of immobilized polypeptide takes out the back and cleans drying.
Embodiment 11
A, activation processing
Sample is inserted 83% phosphoric acid solution, activation 0.5 h in 120 ℃ of baking ovens, deionized water for ultrasonic is cleaned, and dries up.Again sample is immersed 10 min in 8.3% the phosphoric acid solution, behind 120 ℃ of insulation 12 h, deionized water ultrasonic cleaning 3 times, each 10 min, drying;
B, silanization are handled:
With A go on foot implanting device after the activation processing place concentration be 5% APTES/ anhydrous tetrahydrofuran solution (
V/
V) in, 24 h that reflux take out sample, continue backflow 24h in anhydrous tetrahydro furan.Ultrasonic cleaning, vacuum drying;
C, fixed biologically element
The biotin of 0.1 mg is joined in 20 mL, 0.001 mol/L 2-morpholino b acid (MES) aqueous solution, add 1 * 10 successively
-5Mol N-hydroxy-succinamide (NHS), 1 * 10
-5Mol 1-(3-dimethylaminopropyl)-and 3-ethyl carbodiimide (EDC), with the carboxyl on the activation biotin.The sample of silanization is immersed in the above-mentioned solution, and behind room temperature reaction 4 h, deionized water for ultrasonic is cleaned, vacuum drying.
D, fixing Avidin
Immerse 12 h in the normal saline that Avidin concentration is 0.1 mg/mL, take out the back with cleaning drying;
Fixing of E, albumin (BSA):
Implanting device after B step handled immerses in 0.1% the albumin solution, and incubated at room 40 min contain its immersion 12 h in the normal saline of BSA of 0.01 mg/mL again, take out the back and clean drying;
Fixing of F, the adaptive son of biotinylation:
The absorption that E step is obtained albuminous implanting device immerse 12 h in the normal saline of the adaptive sub-TPSLEQRTVYAK-GGGC-K of biotinylation polypeptide (BIOTIN)-COOH of 0.1 mg/mL.By the recognition reaction of biotin-avidin, the adaptive son of immobilized polypeptide takes out the back and cleans drying.
Embodiment 12
A, activation processing
Sample is inserted 83% phosphoric acid solution, activation 0.5 h in 120 ℃ of baking ovens, deionized water for ultrasonic is cleaned, and dries up.Again sample is immersed 10 min in 8.3% the phosphoric acid solution, behind 120 ℃ of insulation 12 h, deionized water ultrasonic cleaning 3 times, each 10 min, drying;
B, silanization are handled:
With A go on foot implanting device after the activation processing place concentration be 5% APTES/ anhydrous tetrahydrofuran solution (
V/
V) in, 5 h that reflux take out sample, continue backflow 5h in anhydrous tetrahydro furan.Ultrasonic cleaning, vacuum drying;
C, fixed biologically element
The biotin of 10 mg is joined in 20 mL, 0.001 mol/L 2-morpholino b acid (MES) aqueous solution, add 1 * 10 successively
-5Mol N-hydroxy-succinamide (NHS), 1 * 10
-5Mol 1-(3-dimethylaminopropyl)-and 3-ethyl carbodiimide (EDC), with the carboxyl on the activation biotin.The sample of silanization is immersed in the above-mentioned solution, and behind room temperature reaction 4 h, deionized water for ultrasonic is cleaned, vacuum drying.
D, fixing Avidin
Immerse 12 h in the normal saline that Avidin concentration is 0.1 mg/mL, take out the back with cleaning drying;
Fixing of E, albumin (BSA):
Implanting device after B step handled immerses in 0.1% the albumin solution, and incubated at room 40 min contain its immersion 12 h in the normal saline of BSA of 0.01 mg/mL again, take out the back and clean drying;
Fixing of F, the adaptive son of biotinylation:
The absorption that E step is obtained albuminous implanting device immerse 12 h in the normal saline of the adaptive sub-TPSLEQRTVYAK-GGGC-K of biotinylation polypeptide (BIOTIN)-COOH of 0.1 mg/mL.By the recognition reaction of biotin-avidin, the adaptive son of immobilized polypeptide takes out the back and cleans drying.
Embodiment 13
A, activation processing
Sample is inserted 83% phosphoric acid solution, activation 0.5 h in 120 ℃ of baking ovens, deionized water for ultrasonic is cleaned, and dries up.Again sample is immersed 10 min in 8.3% the phosphoric acid solution, behind 120 ℃ of insulation 12 h, deionized water ultrasonic cleaning 3 times, each 10 min, drying;
B, silanization are handled:
With A go on foot implanting device after the activation processing place concentration be 5% APTES/ anhydrous tetrahydrofuran solution (
V/
V) in, 5 h that reflux take out sample, continue backflow 5h in anhydrous tetrahydro furan.Ultrasonic cleaning, vacuum drying;
C, fixed biologically element
The biotin of 10 mg is joined in 20 mL, 0.1 mol/L 2-morpholino b acid (MES) aqueous solution, add 1 * 10 successively
-5Mol N-hydroxy-succinamide (NHS), 1 * 10
-5Mol 1-(3-dimethylaminopropyl)-and 3-ethyl carbodiimide (EDC), with the carboxyl on the activation biotin.The sample of silanization is immersed in the above-mentioned solution, and behind room temperature reaction 4 h, deionized water for ultrasonic is cleaned, vacuum drying.
D, fixing Avidin
Immerse 12 h in the normal saline that Avidin concentration is 0.1 mg/mL, take out the back with cleaning drying;
Fixing of E, albumin (BSA):
Implanting device after B step handled immerses in 0.1% the albumin solution, and incubated at room 40 min contain its immersion 12 h in the normal saline of BSA of 0.01 mg/mL again, take out the back and clean drying;
Fixing of F, the adaptive son of biotinylation:
The absorption that E step is obtained albuminous implanting device immerse 12 h in the normal saline of the adaptive sub-TPSLEQRTVYAK-GGGC-K of biotinylation polypeptide (BIOTIN)-COOH of 0.1 mg/mL.By the recognition reaction of biotin-avidin, the adaptive son of immobilized polypeptide takes out the back and cleans drying.
Embodiment 14
A, activation processing
Sample is inserted 83% phosphoric acid solution, activation 0.5 h in 120 ℃ of baking ovens, deionized water for ultrasonic is cleaned, and dries up.Again sample is immersed 10 min in 8.3% the phosphoric acid solution, behind 120 ℃ of insulation 12 h, deionized water ultrasonic cleaning 3 times, each 10 min, drying;
B, silanization are handled:
With A go on foot implanting device after the activation processing place concentration be 5% APTES/ anhydrous tetrahydrofuran solution (
V/
V) in, 5 h that reflux take out sample, continue backflow 5h in anhydrous tetrahydro furan.Ultrasonic cleaning, vacuum drying;
C, fixed biologically element
The biotin of 10 mg is joined in 20 mL, 0.1 mol/L 2-morpholino b acid (MES) aqueous solution, add 9 * 10 successively
-5Mol N-hydroxy-succinamide (NHS), 9 * 10
-5Mol 1-(3-dimethylaminopropyl)-and 3-ethyl carbodiimide (EDC), with the carboxyl on the activation biotin.The sample of silanization is immersed in the above-mentioned solution, and behind room temperature reaction 1 h, deionized water for ultrasonic is cleaned, vacuum drying.
D, fixing Avidin
Immerse 12 h in the normal saline that Avidin concentration is 0.1 mg/mL, take out the back with cleaning drying;
Fixing of E, albumin (BSA):
Implanting device after B step handled immerses in 0.1% the albumin solution, and incubated at room 40 min contain its immersion 12 h in the normal saline of BSA of 0.01 mg/mL again, take out the back and clean drying;
Fixing of F, the adaptive son of biotinylation:
The absorption that E step is obtained albuminous implanting device immerse 12 h in the normal saline of the adaptive sub-TPSLEQRTVYAK-GGGC-K of biotinylation polypeptide (BIOTIN)-COOH of 0.1 mg/mL.By the recognition reaction of biotin-avidin, the adaptive son of immobilized polypeptide takes out the back and cleans drying.
Embodiment 15
A, activation processing
Sample is inserted 83% phosphoric acid solution, activation 0.5 h in 120 ℃ of baking ovens, deionized water for ultrasonic is cleaned, and dries up.Again sample is immersed 10 min in 8.3% the phosphoric acid solution, behind 120 ℃ of insulation 12 h, deionized water ultrasonic cleaning 3 times, each 10 min, drying;
B, silanization are handled:
With A go on foot implanting device after the activation processing place concentration be 5% APTES/ anhydrous tetrahydrofuran solution (
V/
V) in, 5 h that reflux take out sample, continue backflow 5h in anhydrous tetrahydro furan.Ultrasonic cleaning, vacuum drying;
C, fixed biologically element
The biotin of 10 mg is joined in 20 mL, 0.1 mol/L 2-morpholino b acid (MES) aqueous solution, add 9 * 10 successively
-5Mol N-hydroxy-succinamide (NHS), 9 * 10
-5Mol 1-(3-dimethylaminopropyl)-and 3-ethyl carbodiimide (EDC), with the carboxyl on the activation biotin.The sample of silanization is immersed in the above-mentioned solution, and behind room temperature reaction 36 h, deionized water for ultrasonic is cleaned, vacuum drying.
D, fixing Avidin
Immerse 12 h in the normal saline that Avidin concentration is 0.1 mg/mL, take out the back with cleaning drying;
Fixing of E, albumin (BSA):
Implanting device after B step handled immerses in 0.1% the albumin solution, and incubated at room 40 min contain its immersion 12 h in the normal saline of BSA of 0.01 mg/mL again, take out the back and clean drying;
Fixing of F, the adaptive son of biotinylation:
The absorption that E step is obtained albuminous implanting device immerse 12 h in the normal saline of the adaptive sub-TPSLEQRTVYAK-GGGC-K of biotinylation polypeptide (BIOTIN)-COOH of 0.1 mg/mL.By the recognition reaction of biotin-avidin, the adaptive son of immobilized polypeptide takes out the back and cleans drying.
Embodiment 16
A, activation processing
Sample is inserted 83% phosphoric acid solution, activation 0.5 h in 120 ℃ of baking ovens, deionized water for ultrasonic is cleaned, and dries up.Again sample is immersed 10 min in 8.3% the phosphoric acid solution, behind 120 ℃ of insulation 12 h, deionized water ultrasonic cleaning 3 times, each 10 min, drying;
B, silanization are handled:
With A go on foot implanting device after the activation processing place concentration be 5% APTES/ anhydrous tetrahydrofuran solution (
V/
V) in, 5 h that reflux take out sample, continue backflow 5h in anhydrous tetrahydro furan.Ultrasonic cleaning, vacuum drying;
C, fixed biologically element
The biotin of 10 mg is joined in 20 mL, 0.1 mol/L 2-morpholino b acid (MES) aqueous solution, add 9 * 10 successively
-5Mol N-hydroxy-succinamide (NHS), 9 * 10
-5Mol 1-(3-dimethylaminopropyl)-and 3-ethyl carbodiimide (EDC), with the carboxyl on the activation biotin.The sample of silanization is immersed in the above-mentioned solution, and behind room temperature reaction 36 h, deionized water for ultrasonic is cleaned, vacuum drying.
D, fixing Avidin
Immerse 12 h in the normal saline that Avidin concentration is 5 mg/mL, take out the back with cleaning drying;
Fixing of E, albumin (BSA):
Implanting device after B step handled immerses in 0.1% the albumin solution, and incubated at room 40 min contain its immersion 12 h in the normal saline of BSA of 0.01 mg/mL again, take out the back and clean drying;
Fixing of F, the adaptive son of biotinylation:
The absorption that E step is obtained albuminous implanting device immerse 12 h in the normal saline of the adaptive sub-TPSLEQRTVYAK-GGGC-K of biotinylation polypeptide (BIOTIN)-COOH of 0.1 mg/mL.By the recognition reaction of biotin-avidin, the adaptive son of immobilized polypeptide takes out the back and cleans drying.
Embodiment 17
A, activation processing
Sample is inserted 83% phosphoric acid solution, activation 0.5 h in 120 ℃ of baking ovens, deionized water for ultrasonic is cleaned, and dries up.Again sample is immersed 10 min in 8.3% the phosphoric acid solution, behind 120 ℃ of insulation 12 h, deionized water ultrasonic cleaning 3 times, each 10 min, drying;
B, silanization are handled:
With A go on foot implanting device after the activation processing place concentration be 5% APTES/ anhydrous tetrahydrofuran solution (
V/
V) in, 5 h that reflux take out sample, continue backflow 5h in anhydrous tetrahydro furan.Ultrasonic cleaning, vacuum drying;
C, fixed biologically element
The biotin of 10 mg is joined in 20 mL, 0.1 mol/L 2-morpholino b acid (MES) aqueous solution, add 9 * 10 successively
-5Mol N-hydroxy-succinamide (NHS), 9 * 10
-5Mol 1-(3-dimethylaminopropyl)-and 3-ethyl carbodiimide (EDC), with the carboxyl on the activation biotin.The sample of silanization is immersed in the above-mentioned solution, and behind room temperature reaction 36 h, deionized water for ultrasonic is cleaned, vacuum drying.
D, fixing Avidin
Immerse 1 h in the normal saline that Avidin concentration is 5 mg/mL, take out the back with cleaning drying;
Fixing of E, albumin (BSA):
Implanting device after B step handled immerses in 0.1% the albumin solution, and incubated at room 40 min contain its immersion 12 h in the normal saline of BSA of 0.01 mg/mL again, take out the back and clean drying;
Fixing of F, the adaptive son of biotinylation:
The absorption that E step is obtained albuminous implanting device immerse 12 h in the normal saline of the adaptive sub-TPSLEQRTVYAK-GGGC-K of biotinylation polypeptide (BIOTIN)-COOH of 0.1 mg/mL.By the recognition reaction of biotin-avidin, the adaptive son of immobilized polypeptide takes out the back and cleans drying.
Embodiment 18
A, activation processing
Sample is inserted 83% phosphoric acid solution, activation 0.5 h in 120 ℃ of baking ovens, deionized water for ultrasonic is cleaned, and dries up.Again sample is immersed 10 min in 8.3% the phosphoric acid solution, behind 120 ℃ of insulation 12 h, deionized water ultrasonic cleaning 3 times, each 10 min, drying;
B, silanization are handled:
With A go on foot implanting device after the activation processing place concentration be 5% APTES/ anhydrous tetrahydrofuran solution (
V/
V) in, 5 h that reflux take out sample, continue backflow 5h in anhydrous tetrahydro furan.Ultrasonic cleaning, vacuum drying;
C, fixed biologically element
The biotin of 10 mg is joined in 20 mL, 0.1 mol/L 2-morpholino b acid (MES) aqueous solution, add 9 * 10 successively
-5Mol N-hydroxy-succinamide (NHS), 9 * 10
-5Mol 1-(3-dimethylaminopropyl)-and 3-ethyl carbodiimide (EDC), with the carboxyl on the activation biotin.The sample of silanization is immersed in the above-mentioned solution, and behind room temperature reaction 36 h, deionized water for ultrasonic is cleaned, vacuum drying.
D, fixing Avidin
Immerse 1 h in the normal saline that Avidin concentration is 5 mg/mL, take out the back with cleaning drying;
Fixing of E, albumin (BSA):
Implanting device after B step handled immerses in 1% the albumin solution, and incubated at room 40 min contain its immersion 12 h in the normal saline of BSA of 0.1 mg/mL again, take out the back and clean drying;
Fixing of F, the adaptive son of biotinylation:
The absorption that E step is obtained albuminous implanting device immerse 12 h in the normal saline of the adaptive sub-TPSLEQRTVYAK-GGGC-K of biotinylation polypeptide (BIOTIN)-COOH of 0.1 mg/mL.By the recognition reaction of biotin-avidin, the adaptive son of immobilized polypeptide takes out the back and cleans drying.
Embodiment 19
A, activation processing
Sample is inserted 83% phosphoric acid solution, activation 0.5 h in 120 ℃ of baking ovens, deionized water for ultrasonic is cleaned, and dries up.Again sample is immersed 10 min in 8.3% the phosphoric acid solution, behind 120 ℃ of insulation 12 h, deionized water ultrasonic cleaning 3 times, each 10 min, drying;
B, silanization are handled:
With A go on foot implanting device after the activation processing place concentration be 5% APTES/ anhydrous tetrahydrofuran solution (
V/
V) in, 5 h that reflux take out sample, continue backflow 5h in anhydrous tetrahydro furan.Ultrasonic cleaning, vacuum drying;
C, fixed biologically element
The biotin of 10 mg is joined in 20 mL, 0.1 mol/L 2-morpholino b acid (MES) aqueous solution, add 9 * 10 successively
-5Mol N-hydroxy-succinamide (NHS), 9 * 10
-5Mol 1-(3-dimethylaminopropyl)-and 3-ethyl carbodiimide (EDC), with the carboxyl on the activation biotin.The sample of silanization is immersed in the above-mentioned solution, and behind room temperature reaction 36 h, deionized water for ultrasonic is cleaned, vacuum drying.
D, fixing Avidin
Immerse 1 h in the normal saline that Avidin concentration is 5 mg/mL, take out the back with cleaning drying;
Fixing of E, albumin (BSA):
Implanting device after B step handled immerses in 5% the albumin solution, and incubated at room 40 min contain its immersion 12 h in the normal saline of BSA of 0.5 mg/mL again, take out the back and clean drying;
Fixing of F, the adaptive son of biotinylation:
The absorption that E step is obtained albuminous implanting device immerse 12 h in the normal saline of the adaptive sub-TPSLEQRTVYAK-GGGC-K of biotinylation polypeptide (BIOTIN)-COOH of 0.1 mg/mL.By the recognition reaction of biotin-avidin, the adaptive son of immobilized polypeptide takes out the back and cleans drying.
Embodiment 20
A, activation processing
Sample is inserted 83% phosphoric acid solution, activation 0.5 h in 120 ℃ of baking ovens, deionized water for ultrasonic is cleaned, and dries up.Again sample is immersed 10 min in 8.3% the phosphoric acid solution, behind 120 ℃ of insulation 12 h, deionized water ultrasonic cleaning 3 times, each 10 min, drying;
B, silanization are handled:
With A go on foot implanting device after the activation processing place concentration be 5% APTES/ anhydrous tetrahydrofuran solution (
V/
V) in, 5 h that reflux take out sample, continue backflow 5h in anhydrous tetrahydro furan.Ultrasonic cleaning, vacuum drying;
C, fixed biologically element
The biotin of 10 mg is joined in 20 mL, 0.1 mol/L 2-morpholino b acid (MES) aqueous solution, add 9 * 10 successively
-5Mol N-hydroxy-succinamide (NHS), 9 * 10
-5Mol 1-(3-dimethylaminopropyl)-and 3-ethyl carbodiimide (EDC), with the carboxyl on the activation biotin.The sample of silanization is immersed in the above-mentioned solution, and behind room temperature reaction 36 h, deionized water for ultrasonic is cleaned, vacuum drying.
D, fixing Avidin
Immerse 1 h in the normal saline that Avidin concentration is 5 mg/mL, take out the back with cleaning drying;
Fixing of E, albumin (BSA):
Implanting device after B step handled immerses in 1% the albumin solution, and incubated at room 10 min contain its immersion 12 h in the normal saline of BSA of 0.1 mg/mL again, take out the back and clean drying;
Fixing of F, the adaptive son of biotinylation:
The absorption that E step is obtained albuminous implanting device immerse 12 h in the normal saline of the adaptive sub-TPSLEQRTVYAK-GGGC-K of biotinylation polypeptide (BIOTIN)-COOH of 0.1 mg/mL.By the recognition reaction of biotin-avidin, the adaptive son of immobilized polypeptide takes out the back and cleans drying.
Embodiment 21
A, activation processing
Sample is inserted 83% phosphoric acid solution, activation 0.5 h in 120 ℃ of baking ovens, deionized water for ultrasonic is cleaned, and dries up.Again sample is immersed 10 min in 8.3% the phosphoric acid solution, behind 120 ℃ of insulation 12 h, deionized water ultrasonic cleaning 3 times, each 10 min, drying;
B, silanization are handled:
With A go on foot implanting device after the activation processing place concentration be 5% APTES/ anhydrous tetrahydrofuran solution (
V/
V) in, 5 h that reflux take out sample, continue backflow 5h in anhydrous tetrahydro furan.Ultrasonic cleaning, vacuum drying;
C, fixed biologically element
The biotin of 10 mg is joined in 20 mL, 0.1 mol/L 2-morpholino b acid (MES) aqueous solution, add 9 * 10 successively
-5Mol N-hydroxy-succinamide (NHS), 9 * 10
-5Mol 1-(3-dimethylaminopropyl)-and 3-ethyl carbodiimide (EDC), with the carboxyl on the activation biotin.The sample of silanization is immersed in the above-mentioned solution, and behind room temperature reaction 36 h, deionized water for ultrasonic is cleaned, vacuum drying.
D, fixing Avidin
Immerse 1 h in the normal saline that Avidin concentration is 5 mg/mL, take out the back with cleaning drying;
Fixing of E, albumin (BSA):
Implanting device after B step handled immerses in 1% the albumin solution, and incubated at room 36 h contain its immersion 12 h in the normal saline of BSA of 0.1 mg/mL again, take out the back and clean drying;
Fixing of F, the adaptive son of biotinylation:
The absorption that E step is obtained albuminous implanting device immerse 12 h in the normal saline of the adaptive sub-TPSLEQRTVYAK-GGGC-K of biotinylation polypeptide (BIOTIN)-COOH of 0.1 mg/mL.By the recognition reaction of biotin-avidin, the adaptive son of immobilized polypeptide takes out the back and cleans drying.
Embodiment 22
A, activation processing
Sample is inserted 83% phosphoric acid solution, activation 0.5 h in 120 ℃ of baking ovens, deionized water for ultrasonic is cleaned, and dries up.Again sample is immersed 10 min in 8.3% the phosphoric acid solution, behind 120 ℃ of insulation 12 h, deionized water ultrasonic cleaning 3 times, each 10 min, drying;
B, silanization are handled:
With A go on foot implanting device after the activation processing place concentration be 5% APTES/ anhydrous tetrahydrofuran solution (
V/
V) in, 5 h that reflux take out sample, continue backflow 5h in anhydrous tetrahydro furan.Ultrasonic cleaning, vacuum drying;
C, fixed biologically element
The biotin of 10 mg is joined in 20 mL, 0.1 mol/L 2-morpholino b acid (MES) aqueous solution, add 9 * 10 successively
-5Mol N-hydroxy-succinamide (NHS), 9 * 10
-5Mol 1-(3-dimethylaminopropyl)-and 3-ethyl carbodiimide (EDC), with the carboxyl on the activation biotin.The sample of silanization is immersed in the above-mentioned solution, and behind room temperature reaction 36 h, deionized water for ultrasonic is cleaned, vacuum drying.
D, fixing Avidin
Immerse 1 h in the normal saline that Avidin concentration is 5 mg/mL, take out the back with cleaning drying;
Fixing of E, albumin (BSA):
Implanting device after B step handled immerses in 1% the albumin solution, and incubated at room 40 min contain its immersion 12 h in the normal saline of BSA of 0.1 mg/mL again, take out the back and clean drying;
Fixing of F, the adaptive son of biotinylation:
The absorption that E step is obtained albuminous implanting device immerse 12 h in the normal saline of the adaptive sub-TPSLEQRTVYAK-GGGC-K of biotinylation polypeptide (BIOTIN)-COOH of 1 ng/mL.By the recognition reaction of biotin-avidin, the adaptive son of immobilized polypeptide takes out the back and cleans drying.
Embodiment 23
A, activation processing
Sample is inserted 83% phosphoric acid solution, activation 0.5 h in 120 ℃ of baking ovens, deionized water for ultrasonic is cleaned, and dries up.Again sample is immersed 10 min in 8.3% the phosphoric acid solution, behind 120 ℃ of insulation 12 h, deionized water ultrasonic cleaning 3 times, each 10 min, drying;
B, silanization are handled:
With A go on foot implanting device after the activation processing place concentration be 5% APTES/ anhydrous tetrahydrofuran solution (
V/
V) in, 5 h that reflux take out sample, continue backflow 5h in anhydrous tetrahydro furan.Ultrasonic cleaning, vacuum drying;
C, fixed biologically element
The biotin of 10 mg is joined in 20 mL, 0.1 mol/L 2-morpholino b acid (MES) aqueous solution, add 9 * 10 successively
-5Mol N-hydroxy-succinamide (NHS), 9 * 10
-5Mol 1-(3-dimethylaminopropyl)-and 3-ethyl carbodiimide (EDC), with the carboxyl on the activation biotin.The sample of silanization is immersed in the above-mentioned solution, and behind room temperature reaction 36 h, deionized water for ultrasonic is cleaned, vacuum drying.
D, fixing Avidin
Immerse 1 h in the normal saline that Avidin concentration is 5 mg/mL, take out the back with cleaning drying;
Fixing of E, albumin (BSA):
Implanting device after B step handled immerses in 1% the albumin solution, and incubated at room 40 min contain its immersion 12 h in the normal saline of BSA of 0.1 mg/mL again, take out the back and clean drying;
Fixing of F, the adaptive son of biotinylation:
The absorption that E step is obtained albuminous implanting device immerse 12 h in the normal saline of the adaptive sub-TPSLEQRTVYAK-GGGC-K of biotinylation polypeptide (BIOTIN)-COOH of 1 mg/mL.By the recognition reaction of biotin-avidin, the adaptive son of immobilized polypeptide takes out the back and cleans drying.
Embodiment 24
A, activation processing
Sample is inserted 83% phosphoric acid solution, activation 0.5 h in 120 ℃ of baking ovens, deionized water for ultrasonic is cleaned, and dries up.Again sample is immersed 10 min in 8.3% the phosphoric acid solution, behind 120 ℃ of insulation 12 h, deionized water ultrasonic cleaning 3 times, each 10 min, drying;
B, silanization are handled:
With A go on foot implanting device after the activation processing place concentration be 5% APTES/ anhydrous tetrahydrofuran solution (
V/
V) in, 5 h that reflux take out sample, continue backflow 5h in anhydrous tetrahydro furan.Ultrasonic cleaning, vacuum drying;
C, fixed biologically element
The biotin of 10 mg is joined in 20 mL, 0.1 mol/L 2-morpholino b acid (MES) aqueous solution, add 9 * 10 successively
-5Mol N-hydroxy-succinamide (NHS), 9 * 10
-5Mol 1-(3-dimethylaminopropyl)-and 3-ethyl carbodiimide (EDC), with the carboxyl on the activation biotin.The sample of silanization is immersed in the above-mentioned solution, and behind room temperature reaction 36 h, deionized water for ultrasonic is cleaned, vacuum drying.
D, fixing Avidin
Immerse 1 h in the normal saline that Avidin concentration is 5 mg/mL, take out the back with cleaning drying;
Fixing of E, albumin (BSA):
Implanting device after B step handled immerses in 1% the albumin solution, and incubated at room 40 min contain its immersion 12 h in the normal saline of BSA of 0.1 mg/mL again, take out the back and clean drying;
Fixing of F, the adaptive son of biotinylation:
The absorption that E step is obtained albuminous implanting device immerse 1 h in the normal saline of the adaptive sub-TPSLEQRTVYAK-GGGC-K of biotinylation polypeptide (BIOTIN)-COOH of 1 ng/mL.By the recognition reaction of biotin-avidin, the adaptive son of immobilized polypeptide takes out the back and cleans drying.
Embodiment 25
A, activation processing
Sample is inserted 83% phosphoric acid solution, activation 0.5 h in 120 ℃ of baking ovens, deionized water for ultrasonic is cleaned, and dries up.Again sample is immersed 10 min in 8.3% the phosphoric acid solution, behind 120 ℃ of insulation 12 h, deionized water ultrasonic cleaning 3 times, each 10 min, drying;
B, silanization are handled:
With A go on foot implanting device after the activation processing place concentration be 5% APTES/ anhydrous tetrahydrofuran solution (
V/
V) in, 5 h that reflux take out sample, continue backflow 5h in anhydrous tetrahydro furan.Ultrasonic cleaning, vacuum drying;
C, fixed biologically element
The biotin of 10 mg is joined in 20 mL, 0.1 mol/L 2-morpholino b acid (MES) aqueous solution, add 9 * 10 successively
-5Mol N-hydroxy-succinamide (NHS), 9 * 10
-5Mol 1-(3-dimethylaminopropyl)-and 3-ethyl carbodiimide (EDC), with the carboxyl on the activation biotin.The sample of silanization is immersed in the above-mentioned solution, and behind room temperature reaction 36 h, deionized water for ultrasonic is cleaned, vacuum drying.
D, fixing Avidin
Immerse 1 h in the normal saline that Avidin concentration is 5 mg/mL, take out the back with cleaning drying;
Fixing of E, albumin (BSA):
Implanting device after B step handled immerses in 1% the albumin solution, and incubated at room 40 min contain its immersion 12 h in the normal saline of BSA of 0.1 mg/mL again, take out the back and clean drying;
Fixing of F, the adaptive son of biotinylation:
The absorption that E step is obtained albuminous implanting device immerse 36 h in the normal saline of the adaptive sub-TPSLEQRTVYAK-GGGC-K of biotinylation polypeptide (BIOTIN)-COOH of 1 ng/mL.By the recognition reaction of biotin-avidin, the adaptive son of immobilized polypeptide takes out the back and cleans drying.
Embodiment 26
A, activation processing
Sample is inserted 83% phosphoric acid solution, activation 0.5 h in 120 ℃ of baking ovens, deionized water for ultrasonic is cleaned, and dries up.Again sample is immersed 10 min in 8.3% the phosphoric acid solution, behind 120 ℃ of insulation 12 h, deionized water ultrasonic cleaning 3 times, each 10 min, drying;
B, silanization are handled:
With A go on foot implanting device after the activation processing place concentration be 5% APTES/ anhydrous tetrahydrofuran solution (
V/
V) in, 5 h that reflux take out sample, continue backflow 5h in anhydrous tetrahydro furan.Ultrasonic cleaning, vacuum drying;
C, fixed biologically element
The biotin of 10 mg is joined in 20 mL, 0.1 mol/L 2-morpholino b acid (MES) aqueous solution, add 9 * 10 successively
-5Mol N-hydroxy-succinamide (NHS), 9 * 10
-5Mol 1-(3-dimethylaminopropyl)-and 3-ethyl carbodiimide (EDC), with the carboxyl on the activation biotin.The sample of silanization is immersed in the above-mentioned solution, and behind room temperature reaction 36 h, deionized water for ultrasonic is cleaned, vacuum drying.
D, fixing Avidin
Immerse 1 h in the normal saline that Avidin concentration is 5 mg/mL, take out the back with cleaning drying;
Fixing of E, albumin (BSA):
Implanting device after B step handled immerses in 1% the albumin solution, and incubated at room 40 min contain its immersion 12 h in the normal saline of BSA of 0.1 mg/mL again, take out the back and clean drying;
Fixing of F, the adaptive son of biotinylation:
The absorption that E step is obtained albuminous implanting device immerse 36 h in the normal saline of the adaptive sub-SYQTLKQHLPYG-GGGC-K of biotinylation polypeptide (BIOTIN)-COOH of 1 ng/mL.By the recognition reaction of biotin-avidin, the adaptive son of immobilized polypeptide takes out the back and cleans drying.
Embodiment 27
A, activation processing
Sample is inserted 83% phosphoric acid solution, activation 0.5 h in 120 ℃ of baking ovens, deionized water for ultrasonic is cleaned, and dries up.Again sample is immersed 10 min in 8.3% the phosphoric acid solution, behind 120 ℃ of insulation 12 h, deionized water ultrasonic cleaning 3 times, each 10 min, drying;
B, silanization are handled:
With A go on foot implanting device after the activation processing place concentration be 5% APTES/ anhydrous tetrahydrofuran solution (
V/
V) in, 5 h that reflux take out sample, continue backflow 5h in anhydrous tetrahydro furan.Ultrasonic cleaning, vacuum drying;
C, fixed biologically element
The biotin of 10 mg is joined in 20 mL, 0.1 mol/L 2-morpholino b acid (MES) aqueous solution, add 9 * 10 successively
-5Mol N-hydroxy-succinamide (NHS), 9 * 10
-5Mol 1-(3-dimethylaminopropyl)-and 3-ethyl carbodiimide (EDC), with the carboxyl on the activation biotin.The sample of silanization is immersed in the above-mentioned solution, and behind room temperature reaction 36 h, deionized water for ultrasonic is cleaned, vacuum drying.
D, fixing Avidin
Immerse 1 h in the normal saline that Avidin concentration is 5 mg/mL, take out the back with cleaning drying;
Fixing of E, albumin (BSA):
Implanting device after B step handled immerses in 1% the albumin solution, and incubated at room 40 min contain its immersion 12 h in the normal saline of BSA of 0.1 mg/mL again, take out the back and clean drying;
Fixing of F, the adaptive son of biotinylation:
The absorption that E step is obtained albuminous implanting device immerse 36 h in the normal saline of the adaptive sub-SWDILKPNPQPL-GGGC-K of biotinylation polypeptide (BIOTIN)-COOH of 1 ng/mL.By the recognition reaction of biotin-avidin, the adaptive son of immobilized polypeptide takes out the back and cleans drying.
Cardiovascular implantation device after the inventive method modification and unmodified implanting device are implanted respectively in the femoral artery of adult dog, after 20 days, taken out sem observation.It contrasts as depicted in figs. 1 and 2: unmodified implanting device surface deposition fibrin forms thrombosis.Implanting device surface after the modification is covered by oval or flat cell, and cell is sprawled at material surface.As seen, the cardiovascular implantation device after the inventive method modification is capturing endothelial ancestral cell effectively, and the induced material surface is endothelialization fast, has excellent anticoagulation function.
The used cardiovascular implantation device of the present invention is conventional vascular stent material, such as titanium, titanium alloy, rustless steel, platinumiridio, magnesium alloy, Nitinol, ferrum, cochrome.
Claims (8)
1. the method for the adaptive son of cardiovascular implantation device surface immobilized polypeptide can be caught endothelial progenitor cells in the blood after making the cardiovascular implantation device implant into body, comprises following preparation process:
A, surface active: sample inserted in the activated solution activate;
B, silanization handle: the implanting device after the A step is handled places APTES backflow system 1～24 h that refluxes;
C, fixed biologically element: implanting device is immersed in the biotin covalent coupling solution system that concentration is 0.005mg/mL～0.5mg/mL after the B step handles, reaction 1～36h;
D, fixing Avidin: implanting device is handled 1～12 h in the normal saline that back immersion concentration is 0.1～5 mg/mL Avidin through the C step;
E, albuminous fixing: D is gone on foot in the albumin solution of the implanting device immersion of handling 0.1%～5% earlier, hatch 0.2-36 h;
Fixing of F, the adaptive son of biotinylation: 1～36 h in the normal saline of the adaptive son of biotinylation polypeptide of implanting device taking-up immersion 1 ng～1 mg/mL that the E step handles.
2. according to the method for the adaptive son of the described cardiovascular implantation device surface immobilized polypeptide of claim 1, it is characterized in that described activated solution is 83%～98% phosphoric acid solution, activates 0.5～24 h in ℃ baking oven of room temperature～120.
3.
?
The method of the adaptive son of cardiovascular implantation device surface immobilized polypeptide according to claim 1, it is characterized in that described activated solution is 0.5～5mol/L NaOH solution, in room temperature～120 ℃, activate 0.5～24 h in the baking oven, take out to be placed in 60 ℃～120 ℃ deionized waters and be incubated 0.5～24 h.
4. the method for the adaptive son of cardiovascular implantation device surface immobilized polypeptide according to claim 1 is characterized in that described cardiovascular implantation device is made by a kind of of following material: titanium, titanium alloy, rustless steel, platinumiridio, magnesium alloy, Nitinol, ferrum, cochrome.
5. the method for the adaptive son of cardiovascular implantation device according to claim 1 surface immobilized polypeptide, it is characterized in that: APTES backflow system adopts one of following system: APTES/anhydrous tetrahydrofuran solution of 0.01%～5%, APTES/ethanol solution of 0.01%～5%, APTES/anhydrous toluene solution of 0.01%～5%.
6. the method for the adaptive son of cardiovascular implantation device surface immobilized polypeptide according to claim 1, it is characterized in that: biotin covalent coupling solution system adopts following formation: the biotin of 0.1～20 mg is joined in 20 mL, 0.001～0.1 mol/L 2-morpholino b acid aqueous solution, add 1～9 * 10 successively
-5The mol N-hydroxy-succinamide, 1～9 * 10
-5Mol 1-(3-dimethylaminopropyl)-the 3-ethyl carbodiimide.
7. the method for the adaptive son of cardiovascular implantation device surface immobilized polypeptide according to claim 1 is characterized in that: the adaptive son of the fixed biotinylation polypeptide of institute can be one of following material:
TPSLEQRTVYAK-GGGC-K(BIOTIN)-COOH、SYQTLKQHLPYG-GGGC-K(BIOTIN)-COOH?SWDILKPNPQPL-GGGC-K(BIOTIN)-COOH。
8. the method for the adaptive son of cardiovascular implantation device surface immobilized polypeptide according to claim 1 is characterized in that used normal saline phosphoric acid buffer substitutes.
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| US20080288057A1 (en) * | 2004-04-05 | 2008-11-20 | Carpenter Kenneth W | Bioactive Stents For Type II Diabetics and Methods for Use Thereof |
| CN101370444A (en) * | 2006-01-11 | 2009-02-18 | 阿费内基有限公司 | Compositions and methods for promoting attachment of cells of endothelial cell lineage to medical devices |
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| CN115518194B (en) * | 2022-09-19 | 2024-02-27 | 西南交通大学 | Preparation method of metal-based implant material for combined loading of exosomes, product and application thereof |
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