CN106754860A - A kind of method to interface Rapid Modification Avidin based on liposome - Google Patents
A kind of method to interface Rapid Modification Avidin based on liposome Download PDFInfo
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- CN106754860A CN106754860A CN201611029323.9A CN201611029323A CN106754860A CN 106754860 A CN106754860 A CN 106754860A CN 201611029323 A CN201611029323 A CN 201611029323A CN 106754860 A CN106754860 A CN 106754860A
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- liposome
- avidin
- interface
- modification
- pure water
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
- C12N11/14—Enzymes or microbial cells immobilised on or in an inorganic carrier
Abstract
The present invention relates to a kind of method to interface Rapid Modification Avidin based on liposome, phosphatide is mixed with biotinylated phosphatidyl ethanol, add chloroform stirring ultrasound, nitrogen drying, vacuum drying is removed；Adding neutral solution, squeezer can form the liposome that size is not waited after being equipped with the extruding repeatedly of the film in different size aperture；After glass slide cleaning is clean, it is put into 1% hydrofluoric acid solution after soaking tens of seconds, is cleaned with pure water, nitrogen drying；Slide is put in clean vessel, and the liposome of a certain amount of synthesis is added dropwise, and after being incubated some minutes higher than phase transition temperature, excessive liposome is cleaned with ultra-pure water；Then, after adding Avidin to be incubated some minutes, excessive Avidin is washed away with ultra-pure water or solution, you can obtaining surface modification has the glass interface of Avidin.The method modification step is easy, quick, and modification is uniform, closing is complete, and due to not introducing the reagents such as silanization in modification, ambient interferences are smaller.Prepared interface can meet the requirement of unimolecule enzymology.
Description
Technical field
The present invention relates to one kind using liposome as medium, the method that Avidin modification is quickly carried out to interface.The present invention
Belong to nanoscale interfacial materials field.
Background technology
Unimolecule zymetology field with single enzyme molecule as research object is focus and the forward position of scientific research.Normal conditions
Under, unimolecule enzymology takes some way to be fixed unimolecule enzyme, and is remembered in real time using single molecular fluorescence microscope
The Strength Changes of the fluorescence signal that record enzyme is produced in catalytic process, so as to reconstruct the uniqueness in single enzyme molecule catalytic process
Dynamic process.Unimolecule zymetology is disclosed and is hidden under ensemble average level with microcosmic visual angle, single enzyme molecule and substrate
Dynamic fluctuation during effect, has deepened understanding of the people to differential responses system, thus has been subject to field of biology since the birth
Extensive concern.
, it is necessary to carry out observing for a long time the catalysis letter for being enriched enough to institute's research object in unimolecule enzymology
Breath, thus fixation of the enzyme on interface is particularly significant.Under normal circumstances, most easy method is to use for interface to carry out Avidin
Modification（streptavidin）Afterwards, biotinylated enzyme molecule is fixed.Such as by the bovine serum albumin with biotin
（biotin-BSA）Or polyethylene glycol（biotin-PEG）After being incubated with interface, fixed Avidin（avidin）, Jin Ergu
Fixed biotinylated enzyme molecule.Above method is widely applied in single molecule study, however, the method is time-consuming numerous
It is trivial, and because BSA and PEG are easy to reunite, easily appearance is modified uneven, interface and closes incomplete existing during modification
As.Simultaneously when being modified with biotinylation polyethylene glycol, need to carry out silylation modification to glass in advance, easily cause glass table
The impurity in face is reunited, and influences the observation to single molecular fluorescence.So that a kind of easy, uniform and clean background boundary of exploitation
Surface modification method.
The content of the invention
The present invention in view of the shortcomings of the prior art, proposes a kind of side to interface Rapid Modification Avidin based on liposome
Method, using liposome as medium, in the method that surface of glass slide carries out Avidin modification.The method has quick, easy, modification equal
The characteristics of even and smooth clean interfaces.In the inventive solutions, with 1,2- dioleyl phosphatidyl cholines（DOPC）For
It is main, biotinylated phosphatidyl-ethanolamine is added, synthesis carries the liposome of biotin, with this liposome incubation slide, and with
It is incubated Avidin again afterwards, that is, obtains having modified the slide of Avidin.
A kind of method to interface Rapid Modification Avidin based on liposome, it is characterised in that comprise the following steps：
（1）A certain amount of phosphatide is mixed with biotinylated phosphatidyl ethanol, ultrasound after adding chloroform to stir is passed through stream
Logical nitrogen drying, and it is vacuum dried the organic solvent for removing remnants；It is subsequently added neutral solution and promotes immobilized artificial membrane aquation again,
Being equipped with after the extruding repeatedly of the film in different size aperture using squeezer can form the liposome that size is not waited；
（2）After slide is cleaned up with lotion, ultra-pure water, acetone, sodium hydroxide solution and ultra-pure water respectively, 1% hydrofluoric acid is put into
After tens of seconds are soaked in solution, cleaned with pure water, nitrogen drying；Slide is put in clean vessel, and a certain amount of synthesis is added dropwise
Liposome, after being incubated some minutes higher than phase transition temperature, excessive liposome is cleaned with ultra-pure water；Then, Avidin is added
After being incubated some minutes, excessive Avidin is washed away with ultra-pure water or solution, you can obtaining surface modification has the glass of Avidin
Interface.
Phosphatide used is 1,2- dioleyl phosphatidyl cholines（DOPC）, DPPC (DPPC), two
Dimyristoylphosphatidycholine（DMPC）, dilinoleoylphosphatidylcholine（DLPC）, two peanut phosphatidyl cholines（DAPC）
One kind or its combination in phosphatid ylcholine.
The liposomal particle size size of synthesis is 50 ~ 200 nm.
Avidin used is Streptavidin.
The ratio of phosphatide and biotinylated phosphatidyl ethanol is 80:1-120:1.
According to the glass size modified, the volume of the general liposome that synthesis is added dropwise is at 50-300 microlitres.
Liposome is incubated 5-10 min higher than phase transition temperature with slide, and incubation period liposome can not be dried.
The method can prepare the various sizes of liposome with biotin by control condition, be incubated Avidin
Afterwards, can obtain being modified with the interface of Avidin.The method modification step is easy, quick, and modification is uniform, closing is complete, and by
The reagents such as silanization are not introduced in modification, ambient interferences are smaller.Prepared interface can meet unimolecule enzymology
Requirement.
The method have the characteristics that：The present invention modifies Avidin, material therefor as medium using liposome in glass interface
Good biocompatibility, and one layer of liposome is only only spread in interface surface, modifying interface is uniform, and closing is complete.Modification side of the invention
Method is easy, and simple to operate, time-consuming, clean background reduces interference of the background signal to target single molecular fluorescence.
Brief description of the drawings
Fig. 1 is the grain-size graph of the liposome prepared by embodiment 1.
Fig. 2 is the AFM image of the liposome prepared by embodiment 1.
Fig. 3 is the fluorescence microscope image of the Avidin glass interface prepared by embodiment 1.
Fig. 4 is that the interface of the Avidin modification prepared by embodiment 1 is incubated the fluorescence after biotin labeling Cy5 fluorescence molecules
Microscope imaging figure.
Fig. 5 is the grain-size graph of the liposome prepared by embodiment 2.
Fig. 6 is that the interface of the Avidin modification prepared by Fig. 4 case study on implementation 2 of embodiment 2 is incubated mark green fluorescence and life
Fluorescence microscope image after thing element nanometer bead.
Fig. 7 is the grain-size graph of the liposome prepared by embodiment 3.
Fig. 8 is the interface incubation atto-488 fluorescence and the DNA of biotin labeling of the Avidin modification prepared by embodiment 1
Fluorescence microscope image afterwards.
Specific embodiment
Technology of the invention is further described by case study on implementation in detail below.Following embodiment is to this hair
Bright further illustrates, and does not limit the scope of the invention.
Embodiment 1：
By DOPC with biotinylated phosphatidyl ethanol with 100:1 ratio is dissolved in 5 mL chloroforms, after being well mixed, plus
Enter the nitrogen drying of flowing, then 3 hr of vacuum drying remove remaining organic solvent.1 mL adds PBS in the phosphatide of gained
（pH=7.4）Make individual layer phosphatide aquation, final concentration reaches 5 mg/mL.Using the squeezer extruding 20 with 50 nm apertures filter membranes
It is secondary, you can to obtain the liposome with biotin.Slide is ultrasonic 10 minutes with lotion, ultra-pure water respectively, subsequent acetone, hydroxide
Sodium solution ultrasound after ultrapure water is clean, is put into 1% hydrofluoric acid solution after soaking about 30 seconds after 30 minutes, uses ultrapure washing
Only, nitrogen drying is stand-by.Glass surface sticks fence, the liposome prepared by 100 μ L is added in fence, then at 45 DEG C
It is incubated 10 minutes.Then, replaced 20 times using PBS, clean excessive liposome.0.2 mg/mL Avidins are added in fence
After being incubated 10 minutes, replaced 20 times with PBS, wash away excessive Avidin, you can obtaining surface modification has glass circle of Avidin
Face.After the biotinylated fluorescence molecule Cy5 of 100 pM are incubated 10 minutes with the glass for having modified Avidin, replaced using PBS
The fluorescence molecule dissociated in solution is washed away for 20 times, using fluorescence microscopy.Fig. 1 is the particle diameter distribution of prepared liposome
Figure, as seen from the figure, liposomal particle size is about 75 nm.Fig. 2 shows that liposome can form even uniform on interface simultaneously
Film.As seen from Figure 3, under the irradiation of 561 nm laser, the glass interface background that Avidin has only been modified merely is very clean.While by
Fig. 4 is visible, and Avidin glass interface can observe the cy3 fluorescence molecules of modifying interface, thus can be utilized and carry out to list
Molecule enzyme or substrate are fixed and carry out single molecule study.
Embodiment 2：
By DOPC with biotinylated phosphatidyl-ethanolamine with 100:1 ratio is dissolved in 5 mL chloroforms, after being well mixed,
The nitrogen drying of flowing is added, then 3 hr of vacuum drying remove remaining organic solvent.1 mL is added in the phosphatide of gained
PBS（pH=7.4）Make individual layer phosphatide aquation, final concentration reaches 5 mg/mL.Squeezed using the squeezer with 100 nm apertures filter membranes
Pressure 20 times, you can obtain the liposome with biotin.Slide is ultrasonic 10 minutes with lotion, ultra-pure water respectively, subsequent acetone, hydrogen
Sodium hydroxide solution ultrasound after ultrapure water is clean, is put into 1% hydrofluoric acid solution after soaking about 30 seconds after 30 minutes, with ultrapure
Water is cleaned, and nitrogen drying is stand-by.Glass surface sticks fence, and the liposome prepared by 100 μ L is added in fence, then exists
45 DEG C are incubated 10 minutes.Then, replaced 20 times using PBS, clean excessive liposome.0.2 mg/mL parents are added in fence
After being incubated 10 minutes with element, replaced 20 times with PBS, wash away excessive Avidin, you can obtaining surface modification has the glass of Avidin
Glass interface.10 will be diluted6After the green fluorescence bead of times biotin labeling is incubated 3 minutes with the glass for having modified Avidin, adopt
The fluorescence beads dissociated in solution are washed away for 20 times with PBS displacements, using fluorescence microscopy.Fig. 5 is prepared by case study on implementation 2
Liposome grain size distribution, as seen from the figure, liposomal particle size is about 100 nm.As seen from Figure 6, affine element glass simultaneously
Interface further combined with the green fluorescence bead of biotin modification, thus can be utilized and carry out to unimolecule enzyme or substrate
Fix and carry out single molecule study.
Embodiment 3：
By DOPC with biotinylated phosphatidyl-ethanolamine with 100:1 ratio is dissolved in 5 mL chloroforms, after being well mixed,
The nitrogen drying of flowing is added, then 3 hr of vacuum drying remove remaining organic solvent.PBS is added in the phosphatide of gained
（pH=7.4）Make individual layer phosphatide aquation, final concentration reaches 5 mg/mL.Extruded using the squeezer with 200 nm apertures filter membranes
20 times, you can obtain the liposome with biotin.Slide is ultrasonic 10 minutes with lotion, ultra-pure water respectively, subsequent acetone, hydrogen-oxygen
Change sodium solution ultrasound after 30 minutes, after ultrapure water is clean, be put into 1% hydrofluoric acid solution after soaking about 30 seconds, use ultra-pure water
Clean, nitrogen drying is stand-by.Glass surface sticks fence, the liposome prepared by 100 μ L is added in fence, then 45
DEG C be incubated 10 minutes.Then, replaced 20 times using PBS, clean excessive liposome.Add 0.2 mg/mL affine in fence
After element is incubated 10 minutes, replaced 20 times with PBS, wash away excessive Avidin, you can obtaining surface modification has the glass of Avidin
Interface.10 pM are marked simultaneously atto-488 fluorescence molecules be incubated 10 with the glass for having modified Avidin with the DNA of biotin
After minute, the fluorescence molecule dissociated in solution is washed away for 20 times using PBS displacements, using fluorescence microscopy.Fig. 7 is implementation case
The grain size distribution of the liposome prepared by example 3, as seen from the figure, liposomal particle size is about 200 nm.As seen from Figure 8, it is affine
The DNA that element glass interface can be modified further combined with biotin and atto-488, thus can be utilized and carry out to unimolecule
Enzyme or substrate are fixed and carry out single molecule study.
Claims (7)
1. a kind of method to interface Rapid Modification Avidin based on liposome, it is characterised in that comprise the following steps：
（1）A certain amount of phosphatide is mixed with biotinylated phosphatidyl ethanol, ultrasound after adding chloroform to stir is passed through stream
Logical nitrogen drying, and it is vacuum dried the organic solvent for removing remnants；It is subsequently added neutral solution and promotes immobilized artificial membrane aquation again,
Being equipped with after the extruding repeatedly of the film in different size aperture using squeezer can form the liposome that size is not waited；
（2）After slide is cleaned up with lotion, ultra-pure water, acetone, sodium hydroxide solution and ultra-pure water respectively, 1% hydrofluoric acid is put into
After tens of seconds are soaked in solution, cleaned with pure water, nitrogen drying；Slide is put in clean vessel, and a certain amount of synthesis is added dropwise
Liposome, after being incubated some minutes higher than phase transition temperature, excessive liposome is cleaned with ultra-pure water；Then, Avidin is added
After being incubated some minutes, excessive Avidin is washed away with ultra-pure water or solution, you can obtaining surface modification has the glass of Avidin
Interface.
2. a kind of method to interface Rapid Modification Avidin based on liposome according to claim 1, it is characterised in that
Phosphatide used is 1,2- dioleyl phosphatidyl cholines（DOPC）, DPPC (DPPC), two myristoyls
Phosphatid ylcholine（DMPC）, dilinoleoylphosphatidylcholine（DLPC）, two peanut phosphatidyl cholines（DAPC）Phosphatidyl courage
One kind or its combination in alkali.
3. a kind of method to interface Rapid Modification Avidin based on liposome according to claim 1, it is characterised in that
The liposomal particle size size of synthesis is 50 ~ 200 nm.
4. a kind of method to interface Rapid Modification Avidin based on liposome according to claim 1, it is characterised in that
Avidin used is Streptavidin.
5. a kind of method to interface Rapid Modification Avidin based on liposome according to claim 1, it is characterised in that
The ratio of phosphatide and biotinylated phosphatidyl ethanol is 80:1-120:1.
6. a kind of method to interface Rapid Modification Avidin based on liposome according to claim 1, it is characterised in that
According to the glass size modified, the volume of the general liposome that synthesis is added dropwise is at 50-300 microlitres.
7. a kind of method to interface Rapid Modification Avidin based on liposome according to claim 1, it is characterised in that
Liposome is incubated 5-10 min higher than phase transition temperature with slide, and incubation period liposome can not be dried.
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| CN201611029323.9A CN106754860A (en) | 2016-11-22 | 2016-11-22 | A kind of method to interface Rapid Modification Avidin based on liposome |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110108624A (en) * | 2019-05-08 | 2019-08-09 | 中国科学院化学研究所 | A kind of method of functionalization phosphatide preparation nanometer individual particle and the detection of this nanometer of individual particle |
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| CN1335505A (en) * | 2001-07-11 | 2002-02-13 | 上海晶泰生物技术有限公司 | Protein chip based on labeling streptavidin-biotin technology |
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| CN103555568A (en) * | 2013-10-18 | 2014-02-05 | 南京农业大学 | Sample capsule for detecting nucleic acid single molecule and preparation method thereof |
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2016
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| CN1335505A (en) * | 2001-07-11 | 2002-02-13 | 上海晶泰生物技术有限公司 | Protein chip based on labeling streptavidin-biotin technology |
| US8815577B2 (en) * | 2001-07-30 | 2014-08-26 | Meso Scale Technologies, Llc | Assay electrode having immobilized lipid/protein layers, methods of making the same and methods of using the same for luminescence test measurements |
| CN1597987A (en) * | 2004-08-03 | 2005-03-23 | 湖南大学 | Process for testing SARS virus genome segment by silicon shell nano particle |
| CN103411818A (en) * | 2013-08-27 | 2013-11-27 | 苏州大猫单分子仪器研发有限公司 | Modifying glass slide for fixing biomacromolecules and method thereof |
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| HUA, DY ET AL: "Biotinylated lipid membrane patterns supported by proteins for the recognition of streptavidined polystyrene microspheres", 《JOURNAL OF NANOSCIENCE AND NANOTECHNOLOGY》 * |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110108624A (en) * | 2019-05-08 | 2019-08-09 | 中国科学院化学研究所 | A kind of method of functionalization phosphatide preparation nanometer individual particle and the detection of this nanometer of individual particle |
| CN110108624B (en) * | 2019-05-08 | 2020-06-16 | 中国科学院化学研究所 | Method for preparing nano single particle by functionalized phospholipid and detection of nano single particle |
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Application publication date: 20170531 |