CN105862391B - The preparation method of BC@metal oxide composite nano fibers - Google Patents
The preparation method of BC@metal oxide composite nano fibers Download PDFInfo
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Abstract
The present invention provides a kind of preparation methods of BC@metal oxide composite nano fibers, include the following steps：(a) BC nanofibers are prepared；(b) in the surface clad oxide granular layer of BC nanofibers, and natural packing forms meso-hole structure, obtains being coated with the BC@metal oxide composite nano fibers of metal oxide particle；(c) with the BC metal oxide composite nano fibers obtained in base catalysis step (b), the BC metal oxide composite nano fibers that can be used for enriched phosphorus acidified protein or polypeptide are obtained.The present invention also provides the methods of BC metal oxides composite nano fiber enriched phosphorus acidified protein or polypeptide.Composite nano fiber prepared by the present invention can sensitive, rapidly enriched phosphorus acidified protein or polypeptide, highly practical, good separating effect is environmental-friendly, has a good application prospect.
Description
Technical field
The present invention relates to the specific enrichment technical fields of phosphorylated protein and polypeptide, and more particularly to one kind is with bacterial fibers
Plain (Bacterial Cellulose, BC) is the preparation method of the BC@metal oxide composite nano fibers of substrate.
Background technology
For protein phosphorylation as a kind of important protein post-translational modification, play in cellular activity is adjusted can not
Substituted role, such as：Signal transduction, cytoskeleton adjusting, gene expression, cell cycle, Apoptosis etc..Protein phosphoric acid
The imbalance of cell processes caused by the imbalance of change is the root place of numerous diseases.Therefore, in order to reach early diagnosis disease
Purpose, Many researchers focus be placed on research Intracellular phosphorylation protein content variation.However, phosphorylated protein exists
Intracellular existence form is dynamic change, and content is extremely low, in addition the influence of a large amount of non-phosphorylating albumen, is made directly
The the researching and analysing phosphorylated protein of the task is extremely arduous.Therefore, the enrichment of phosphorylated protein becomes particularly important before detection and analysis
With it is urgent.Recently, many pertinent literatures report various enrichment materials and corresponding concentration effect.Wherein, very much
All it is metal oxide, for example, TiO2, ZrO2, Fe2O3And Al2O3, wherein, the metal oxide TiO of column IV element2, ZrO2Most
Common and best results.Meanwhile also many methods can be used to improve the sensitivity of material, be increased by the way that meso-hole structure is made
It is one of them to add specific surface area so as to improve sensitivity.Big specific surface area and appropriate aperture can be proteins and peptides
Sufficient active site and disengaging space are provided in enrichment process.
In addition, including Matrix Assisted Laser Desorption time-of-flight mass spectrometry (MALDI-TOF-MS) and nanoliter liquid chromatogram
Mass-spectrometric technique including ionization tandem mass spectrum (Nano-LC-ESI-MS) become analyze phosphorylated protein and polypeptide can not or
Scarce tool.
Nowadays, by acetic acid bacteria, such as：Acetobacter xylinum, the bacteria cellulose (BC) of biosynthesis cause the pole of researcher
Big interest.Not only because this bacterial fibers are environmentally friendly materials, there is degradability, also as it is with future
Wide attribute, including：Good biocompatibility, higher mechanically and chemically stability, good retentiveness and hydrophily,
Big surface area and porosity, the characteristics such as building-up process is controllable, this causes bacteria cellulose or its modified cellulose to be formed
Fiber possess multiple use.BC fibers are normally used for biological medicine, food and advanced diaphragm etc., and for modified
BC fibers, BC fibers are passed through loads individual part frequently as fabulous substrate, best so as to expand the performance of BC fibers
Example is exactly the BC@TiO for possessing very high photo-catalysis capability2。
Recently, the TiO of different structure2Composite material is widely used in the enrichment of phosphorylated protein or phosphorylated polypeptide
Cheng Zhong.Moreover, Many researchers are tried one's best to increase the specific surface area of material to promote the accumulation ability of material and sensitivity,
And certain effect is also harvested.In addition, nano level TiO2Particle possesses particle bigger more even greater than micron order
Specific surface area, this feature is very attractive, but excessively tiny grain size allows their problem that is separated into, therefore, only solid
Determine or be wrapped in outside other compositions to form shell, the specific surface area that just its can be made big in this way possesses meaning in bio-separation.
Invention content
In order to solve the above technical problems, the object of the present invention is to provide a kind of BC@metal oxide composite nano fibers
Preparation method, this method can prepare it is repeated preferably, stable homogeneous is higher and the preferable composite Nano of biocompatibility
Fiber.The high sensitivity of composite nano fiber detection phosphorylated protein or polypeptide prepared by this method, selects enriching and again
Renaturation is preferable.
Present invention employs following technical schemes：
On the one hand, the present invention provides a kind of preparation method of BC@metal oxide composite nano fibers, including following
Step：
(a) BC nanofibers are prepared, which has stable chemical machinery performance；
(b) in the surface clad oxide granular layer of BC nanofibers, and natural packing forms meso-hole structure, obtains
It is coated with the BC@metal oxide composite nano fibers of metal oxide particle；
(c) it with the BC@metal oxide composite nano fibers obtained in base catalysis step (b), obtains can be used for enriched phosphorus
The BC@metal oxide composite nano fibers of acidified protein or phosphorylated polypeptide, the composite nano fiber have orderly mesoporous
Structure.The meso-hole structure of composite nano fiber can be improved with base catalysis, and then is formed useful to phosphorylated protein or polypeptide enrichment
Modified form BC@metal oxide composite nano fibers.
Further, step (d) is further included after step (c)：
BC@metal oxide composite nano fibers are filled in liquid-transfering gun pipette tips, are formed in liquid-transfering gun pipette tips shape
BC@metal oxide composite nano fibers.That is, Tip is made in the composite nano fiber after improvement.
Preferably, the composite nano fiber after improvement is filled in the liquid-transfering gun pipette tips of 200 μ L, forms specific enrichment
Use Tip.
Preferably, metal oxide is selected from titanium dioxide, zirconium dioxide, alundum (Al2O3), di-iron trioxide, stannic oxide
One or more of.
Preferably, in step (a), BC nanofibers are prepared through biological synthesis process using bacteria cellulose producing strains.
Specifically, acetobacter xylinum prepares BC nanofibers under fermentation conditions.
In one embodiment, it is highly preferred that in step (a), BC nanofibers are by bacteria cellulose producing strains
The Nano bacteria of acetobacter xylinum (Acetobacter xylinum) NUST5.2 biosynthesis under fermentation conditions.Bacterium is fine
Dimension element has good biocompatibility, physicochemical properties stabilization, large specific surface area, mechanical strength good and is conducive to operate etc. excellent
Point.In addition, bacteria cellulose surface is rich in hydroxyl, this provides suitable Targeting groups for surface sol-gel process.
Preferably, in step (a), a diameter of 15-80nm of BC nanofibers.
Preferably, in step (b), by situ synthesis in BC nanofiber surface coated metal oxide particles
Layer.
It is highly preferred that in step (b), the grain size of metal oxide particle is 5-15nm.
Further, in step (b), the mesoporous pore size of meso-hole structure is 6-10nm.
Preferably, in step (c), alkali in ammonium hydroxide, n,N-Dimethylformamide and tetramethylammonium hydroxide one
Kind is several.
On the other hand, the present invention also provides a kind of BC metal oxides composite nano fiber enriched phosphorus acidified proteins
Or the method for phosphorylated polypeptide, include the following steps：
(e) BC@metal oxide composite nano fibers are washed with the buffer solution of different pH value, is at being enriched with best shape
State；
(f) phosphorylated protein or phosphorylated polypeptide are added in BC@metal oxide composite nano fibers and are enriched with；
(g) phosphorylated protein or phosphorylated polypeptide are enriched with what is obtained in the buffer solution washing step (f) of different pH value
BC@metal oxide composite nano fibers, to remove the albumen of non-specific adsorption or polypeptide；And
(h) the pecific phosphorylation albumen or polypeptide being enriched on BC@metal oxide composite nano fibers are taken off with alkali cleaning.
Further, in step (e), buffer solution includes buffer solution A and buffer solution B, and wherein buffer solution A is volume fraction
Aqueous solution of the meter containing 0.4% trifluoroacetic acid (TFA) and 80% acetonitrile (ACN), buffer solution B is with volume fraction, and 25%
The mixed liquor of lactic acid and 75% buffer solution A.
Preferably, in step (e), after buffer solution A washes twice, buffer solution B washes twice again.
Further, in step (f), phosphorylated protein or polypeptide and buffer solution B are with 1:2 volume ratio is mixed to get mixed
Liquid is closed, which is added in liquid-transfering gun pipette tips (Tip) and is enriched with.
Preferably, in step (f), by means of centrifuge so that mixed liquor carries out richness in composite nano-fiber material
Collection.
Further, in step (g), washed successively with buffer solution B and buffer solution A respectively be enriched with phosphorylated protein or
The BC@metal oxide composite nano fibers of polypeptide.
Preferably, in step (g), after buffer solution B washes twice, buffer solution A washes twice.
The phosphorylated protein or polypeptide that the BC metal oxide composite nano fibers of the present invention are enriched with can be taken off with alkali cleaning
Come, and carry out Matrix Assisted Laser Desorption time-of-flight mass spectrometry (MALDI-TOF-MS) detection.
The mass spectrum auxiliary type BC metal oxides composite nano fiber of the present invention includes bacterial fibers (BC) and metal aoxidizes
Object (titanium dioxide, zirconium dioxide etc.) nano-particle layer, and composite nano fiber specific surface area and mesoporous is improved on this basis
Aperture.Bacterial fibers surface is rich in hydroxyl, and metal oxide nanoparticles are through hydro-thermal method growth in situ in bacterial fibers (BC) table
Face.In the present invention, using base catalysis modified surface metal oxide crystal property, specific enrichment phosphorylated protein and more
Peptide, the present invention is based on metal oxides (titanium dioxide) can form double coordinations with the phosphate radical of phosphorylated protein or phosphorylated polypeptide
The principle of key.
According to the above aspect of the present invention, compared with prior art, the present invention has at least the following advantages：
The present invention prepares bacteria cellulose by biological synthesis method, is then prepared by situ synthesis regular
, have uniform particle sizes metal oxide (titanium dioxide etc.) nano particle, and pass through appropriate bases (ammonium hydroxide etc.) catalysis, promote
The nano particles such as the metal oxide (titanium dioxide) of growth re-assembly with crystal further growth, crystallization, so as to crystallinity
Enhancing so as to reduce random titanium dioxide granule, makes the surface mesoporous structure optimization of composite nano fiber to enrichment acid
Change the optimum aperture of albumen and polypeptide, so as to improve the sensitivity of enrichment and shorten the time of enrichment process.It is provided by the invention
Mass spectrum auxiliary type BC@metal oxides composite nano fiber has highly practical, preferable conducive to separation, specific surface area and aperture
Feature, so as to provide a kind of material of novel sensitive auxiliary mass spectral analysis.
Above description is only the general introduction of technical solution of the present invention, in order to better understand the technological means of the present invention,
And can be implemented in accordance with the contents of the specification, below with presently preferred embodiments of the present invention and after attached drawing is coordinated to be described in detail such as.
Description of the drawings
Fig. 1 is the BC@mTiO of the embodiment of the present invention 12The preparation of composite nano fiber and its showing for enriched phosphorus acidified protein
It is intended to；
Fig. 2 is the transmission electron microscope shape appearance figure of the bacteria cellulose (BC) of the embodiment of the present invention 1；
Fig. 3 is the BC@mTiO for being coated with titania nanoparticles of the embodiment of the present invention 12Composite nano fiber it is saturating
Penetrate electron microscope shape appearance figure；
Fig. 4 is the scanning electron microscope shape appearance figure of the bacteria cellulose (BC) of the embodiment of the present invention 1；
Fig. 5 is the BC@mTiO for being coated with titania nanoparticles of the embodiment of the present invention 12Composite nano fiber is swept
Retouch electron microscope shape appearance figure；
Fig. 6 is the mixed solution (molar ratio 1 of beta-casein enzymolysis liquid and BSA enzymolysis liquids in the embodiment of the present invention 1:10) it passes through
BC@mTiO2Mass spectrogram before composite nano fiber enrichment；
Fig. 7 is the mixed solution (molar ratio 1 of beta-casein enzymolysis liquid and BSA enzymolysis liquids in the embodiment of the present invention 1:10) it passes through
BC@mTiO2Mass spectrogram after composite nano fiber enrichment；
Fig. 8 is the mixed solution (molar ratio 1 of beta-casein enzymolysis liquid and phosphorylated polypeptide sample in the embodiment of the present invention 2:
10) through BC@mZrO2Mass spectrogram before enrichment.
Fig. 9 is the mixed solution (molar ratio 1 of beta-casein enzymolysis liquid and phosphorylated polypeptide sample in the embodiment of the present invention 2:
10) through BC@mZrO2Mass spectrogram after enrichment.
Specific embodiment
With reference to the accompanying drawings and examples, the specific embodiment of the present invention is described in further detail.It should be understood that
It is that following embodiment is merely to illustrate the present invention, but is not limited to the scope of the present invention.
Embodiment 1
BC@mTiO2The preparation of the bacteria cellulose of package (mesoporous TiO 2) composite nano fiber and its in biomass
Application in spectrum.
Embodiment 1 provides a kind of preparation method of BC@metal oxide composite nano fibers, and wherein metal oxide is two
Titanium oxide, as shown in Figure 1, this method includes the following steps successively：
(a) BC nanofibers are prepared by the biological synthesis process of bacteria cellulose producing strains
BC nanofibers are by bacteria cellulose producing strains acetobacter xylinum (Acetobacter xylinum) NUST5.2 29
Cultivation and fermentation generates for 7 days under conditions of DEG C.Entire incubation is dynamic cultivation method, and this method can be carried effectively
High production rate.Therebetween, 21g sucrose, 10g yeast extracts, 20g D-Glucoses, 4g ammonium sulfate (NH4)2SO4, 2g biphosphates
Potassium (KH2PO4) and 0.4g magnesium sulfate (MgSO4) be dissolved in 1 liter of water, PH to 6.1 or so is adjusted to match with 2.5M sodium hydroxides (NaOH)
Culture medium is made, nutrition needed for bacterial growth is provided.The BC nanofibers of acquisition immerse washing, purifying in deionized water, Ran Houyong
3/1000ths sodium hydroxide (NaOH) and 3/1000ths hydrogen peroxide (H2O2) heating water bath 6 hours, remove bacterial residues.
Finally with deionized water rinsing several times, the base reagent on fiber is removed, is close to neutrality, and preserve in 4 DEG C of refrigerators.With before
Fiber is lyophilized using freeze dryer and is used, the diameter of prepared fiber is about 15-40nm.
(b) uniform TiO is formed by hydro-thermal method2Layer.
Hydro-thermal method refers to that in the pressure vessel of sealing water is solvent, and the chemistry carried out under conditions of high temperature and pressure is anti-
It should.Hydro-thermal method is to make those insoluble or indissoluble substance dissolving or reactions in atmospheric conditions using the aqueous solution of high temperature and pressure
Generate the lysate of the substance, generation convection current made by the temperature difference for controlling solution in autoclave, to form hypersaturated state and
The method of growth crystal is precipitated.
Specifically preparation method is：By dry BC nanofiber dispersions in second alcohol and water (1:1, v:V) in mixed solution,
Then, serial dehydration is carried out to it with the ethyl alcohol of various concentration, until concentration reaches 99%.Then, centrifugation removal supernatant solvent,
And add in 200 milliliters of Ti (OBu)4Ethanol solution, as titanium source.Meanwhile 2 milliliters of acetylacetone,2,4-pentanediones of addition prevent from hydrolyzing.3 hours
After mechanical agitation, reaction solution is transferred in polytetrafluoroethylene (PTFE) stainless steel high temperature reaction kettle (maximum volume is 200 milliliters), is heated to
180 DEG C, keep the temperature 8 hours in confined conditions.Treat that reaction finishes, temperature is cooled to room temperature, and is washed respectively with deionized water and ethyl alcohol
It washs 3 times.Finally, in vacuum drying chamber, 60 DEG C are dried overnight.Wherein, obtained BC@metal oxide composite nano fibers
The TiO on surface2Grain size be 5nm.
(c)BC@mTiO2The modification of composite nano fiber.
The nanofiber of the above-mentioned acquisitions of 50mg is scattered in (ethyl alcohol in the mixed solution of 60mL second alcohol and waters again：Water, 2:
1, v:V), and appropriate ammonium hydroxide (3mL) is added in, is transferred in polytetrafluoroethylene (PTFE) stainless steel high temperature reaction kettle that (capacity is after mixing
100mL), 160 DEG C are heated to, keeps the temperature 20 hours.After completion of the reaction, when the temperature to room temperature, the nanofiber water and second of gained
Alcohol washs several times respectively, and fiber is collected by centrifugation, and utilizes freeze dryer drying for standby.Obtained BC@metal oxide composite Nanos
The aperture of the meso-hole structure of fiber is 6nm.
(d)BC@mTiO2The preparation of composite nano fiber Tip (liquid-transfering gun pipette tips shape).
200 μ L liquid-transfering gun pipette tips are elected to be container to support BC@mTiO2Composite nano fiber forms liquid-transfering gun pipette tips shape
BC@mTiO2Composite nano fiber Tip.
It is as follows：First, appropriate absorbent cotton is put in Tip bottom ends to extend liquid by the time, so as to make sample
Product and BC@mTiO2Composite nano fiber comes into full contact with.Then, by the BC@mTiO of 1mg2Composite nano fiber is filled in absorbent cotton
It is upper to form complete BC@mTiO2Composite nano fiber Tip.Room temperature is positioned over to treat subsequently to be enriched with use.In order to facilitate centrifugation, use
Adapter auxiliary centrifugation is made (with reference to figure 1) in microcentrifugal tube.
BC@mTiO2Enrichment of the composite nano fiber to phosphorylated protein.
The preparation of standard protein solution
Beta-casein and bovine serum albumin are dissolved in respectively in 25mM ammonium bicarbonate aqueous solutions, form final concentration of 2mg/mL's
Protein standard liquid, and with 1M Tris solution tune pH to 8.0.Then it is denaturalized 10 minutes for 100 DEG C.It treats that temperature is cooled to room temperature, adds in
Trypsase is with 1：2.5 (enzymes：Substrate) ratio digested, 37 DEG C overnight, while add in 5% acetonitrile improve trypsase
Activity.Standard protein solution storage after enzymolysis is spare in -20 DEG C of refrigerators.
BC@mTiO2Enriched phosphorus acidified protein specifically includes following steps：
(e) loading
Before enrichment, above-mentioned manufactured composite nano fiber Tip is optimal richness with the buffer solution processing of two kinds of difference pH
Collection state, both buffer solutions are respectively：Buffer solution A is to contain 0.4% trifluoroacetic acid (TFA) and 80% second with volume fraction
The aqueous solution of nitrile (ACN), buffer solution B are with the mixed liquor of volume fraction, 25% lactic acid and 75% buffer solution A.Wherein,
Buffer solution B also serves as next sample-loading buffer.Pretreatment process and following washing process exist by centrifuge
It is centrifuged under 5000rpm rotating speeds 2 minutes, wherein each buffer solution washs 2 times.
(f) after pre-treatment, by buffer solution B and protein solution with 2：1 volume ratio mixing adds in liquid-transfering gun pipette tips
(Tip) it is enriched in.The process makes mixed liquor by enrichment material, and centrifuge 8 points with the rotating speed of 3500rpm by centrifuge
The sample that centrifugation obtains is moved on in Tip, is repeated 3 times by clock again.
(g) then, respectively with buffer solution B and buffer solution A successively washing composite nano fiber Tip, it is therefore an objective to which removing is sticked to
The non-phosphorylated polypeptide and reagent of fiber surface, each buffer solution wash 2 times.
(h) after washing, with the phosphorylated protein of the ammonium hydroxide of 50 μ L 5% elution specific adsorption, centrifugal speed, when
Between and number it is consistent with enrichment process.Finally eluent is lyophilized, and is put in -20 DEG C and saves backup.
Matrix Assisted Laser Desorption time-of-flight mass spectrometry analyzes (MALDI-TOF-MS)
The principle of MALDI is with laser irradiation sample and substrate formed cocrystallization film, and matrix is from swashing during laser irradiation
Energy is absorbed in light, passing to sample molecule makes its instant vaporization, and proton translocation to sample molecule is made its ionization, then
Into time-of-flight mass analyzer, it is detected further according to their own mass-to-charge ratio (m/z).
Detailed process is：By the sample of freeze-drying with 3 μ L buffer solutions C (acetonitrile/trifluoroacetic acid/water, 50%/0.1%/
49.9%, v/v/v) redissolve, then with isometric saturation CHCA matrix mixings.CHCA matrix is by alpha-cyano -4- hydroxyl meat
Cinnamic acid is dissolved in the saturated solution formed in TA30 solution, wherein, TA30 be containing volume fraction be 30% acetonitrile and 0.1% trifluoro
The aqueous solution of acetic acid.Then, mixing liquid is dripped on target plate with micropipette rifle, drips one layer of CHCA base again after natural air drying
Matter carries out MALDI-TOF mass spectral analyses after drying and crystallizing.
Wherein, the auxiliary mass spectrum type BC@mTiO of the present embodiment 12Composite nano fiber, including bacterial fibers (BC) and cladding
Titania nanoparticles on its surface layer, the compound fiber of receiving are supported by liquid-transfering gun pipette tips, auxiliary mass spectrum type BC@mTiO2It is multiple
Nanofiber Tip is closed to be made of above-mentioned steps a-d.Before the process of auxiliary mass spectrum specific enrichment phosphorylated protein or polypeptide includes
Processing, loading enrichment and elution analysis process, assist mass spectrum specific enrichment phosphorylated protein or polypeptide process by above-mentioned steps
E-h is completed.
BC@mTiO in the embodiment of the present invention 12Composite nano fiber, to the beta-casein enzymolysis liquid (10 of various concentration- 5M、10-6M、10-7M) Mass Spectrometer Method is done as a result, sensitivity reaches 1.7pmol.
Fig. 1 illustrates the BC@mTiO of embodiment 12The preparation of composite nano fiber and the schematic diagram of enriched phosphorus acidified protein.
Fig. 2, Fig. 3 are respectively bacteria cellulose (BC) and BC@mTiO in embodiment 12The transmission electron microscope of composite nano fiber
Shape appearance figure.Fig. 4, Fig. 5 are respectively bacteria cellulose (BC) and BC@mTiO in embodiment 12The scanning electron of composite nano fiber is shown
The shape appearance figure of micro mirror.Fig. 6, Fig. 7 are respectively beta-casein enzymolysis liquid and the molar ratio of BSA enzymolysis liquids is 1:10 mixed solution warp
BC@mTiO2Mass spectrogram before composite nano fiber enrichment, after enrichment.
Embodiment 2
BC@ZrO2Application in the preparation of composite nano fiber and its biological mass spectrometry.
Embodiment 2 provides a kind of preparation method of BC@metal oxide composite nano fibers, and wherein metal oxide is two
Zirconium oxide, this method include the following steps successively：
(a) preparation method of BC nanofibers is same as Example 1 in embodiment 2, the BC fibers prepared with reference to embodiment 1
A diameter of 50-80nm.
(b) uniform ZrO is formed by hydro-thermal method2Layer, wherein with zirconium-n-butylate as zirconium source.
Specifically preparation method is：By dry BC nanofiber dispersions in second alcohol and water (1:1, v:V) in mixed solution,
Then, serial dehydration is carried out to it with the ethyl alcohol of various concentration, until concentration reaches 99%.Then, centrifugation removal supernatant solvent,
And the ethanol solution of 250 milliliters of zirconium-n-butylates is added in, as zirconium source.Meanwhile 2.5 milliliters of acetylacetone,2,4-pentanediones of addition prevent from hydrolyzing.3 is small
When mechanical agitation after, reaction solution is transferred in polytetrafluoroethylene (PTFE) stainless steel high temperature reaction kettle to (maximum volume is 200 milliliters), heating
To 180 DEG C, 8 hours are kept the temperature in confined conditions.Treat that reaction finishes, temperature is cooled to room temperature, and is distinguished with deionized water and ethyl alcohol
Washing 3 times.Finally, in vacuum drying chamber, 60 DEG C are dried overnight.Wherein, obtained BC@metal oxides composite Nano is fine
Dimension table face ZrO2Grain size be 15nm.
(c)BC@mZrO2The modification of composite nano fiber.
The nanofiber of the above-mentioned acquisitions of 50mg is scattered in (ethyl alcohol in the mixed solution of 60mL second alcohol and waters again：Water, 2:
1, v:V), and appropriate tetramethylammonium hydroxide (3.5mL) is added in, is transferred to after mixing in polytetrafluoroethylene (PTFE) stainless steel high temperature reaction kettle
(capacity 100mL) is heated to 160 DEG C, keeps the temperature 20 hours.After completion of the reaction, when the temperature to room temperature, the nanofiber of gained is used
Water and ethyl alcohol wash several times respectively, and fiber is collected by centrifugation, and utilize freeze dryer drying for standby.Modified gained BC@mZrO2It is compound to receive
The meso-hole structure aperture of rice fiber is 10nm.
(d)BC@mZrO2The preparation of composite nano fiber Tip (liquid-transfering gun pipette tips shape).
200 μ L liquid-transfering gun pipette tips are elected to be container to support BC@mZrO2Composite nano fiber forms liquid-transfering gun pipette tips shape
BC@mZrO2Composite nano fiber Tip.
It is as follows：First, appropriate absorbent cotton is put in Tip bottom ends to extend liquid by the time, so as to make sample
Product and BC@mZrO2Composite nano fiber comes into full contact with.Then, by the BC@mZrO of 1mg2Composite nano fiber is filled in absorbent cotton
It is upper to form complete BC@mZrO2Composite nano fiber Tip.Room temperature is positioned over to treat subsequently to be enriched with use.In order to facilitate centrifugation, use
Adapter auxiliary centrifugation is made in microcentrifugal tube.
BC@mZrO2Enrichment of the composite nano fiber to phosphorylated protein.
The preparation of phosphorylated polypeptide sample
From A549 cell extraction phosphorylated polypeptide samples, extraction step includes：
(1) it after cell is handled using trypsase, is washed 3 times with 4 DEG C of PBS.
(2) cell is scattered in prepared lysate (being prepared with reference to product manual) in advance, ice bath 20min.
(3) it using the thorough smudge cells of cell crushing instrument, is carried out under ice bath.
(4) 10min is centrifuged in 4 DEG C of 14800rmp, takes supernatant.
(5) supernatant 50mM ammonium bicarbonate solns are diluted, 37 DEG C of 12h is incubated with pancreatin.
BC@mZrO2Enriched phosphorus acidified protein specifically includes following steps：
(e) loading
Before enrichment, above-mentioned manufactured composite nano fiber Tip is optimal richness with the buffer solution processing of two kinds of difference pH
Collection state, both buffer solutions are respectively：Buffer solution A is to contain 0.4% trifluoroacetic acid (TFA) and 80% second with volume fraction
The aqueous solution of nitrile (ACN), buffer solution B are with the mixed liquor of volume fraction, 25% lactic acid and 75% buffer solution A.Wherein,
Buffer solution B also serves as next sample-loading buffer.Pretreatment process and following washing process exist by centrifuge
It is centrifuged under 5000rpm rotating speeds 2 minutes, wherein each buffer solution washs 2 times.
(f) after pre-treatment, by buffer solution B and protein solution with 2：1 volume ratio mixing adds in liquid-transfering gun pipette tips
(Tip) it is enriched in.The process makes mixed liquor by enrichment material, and centrifuge 8 points with the rotating speed of 3500rpm by centrifuge
The sample that centrifugation obtains is moved on in Tip, is repeated 3 times by clock again.
(g) then, respectively with buffer solution B and buffer solution A successively washing composite nano fiber Tip, it is therefore an objective to which removing is sticked to
The non-phosphorylated polypeptide and reagent of fiber surface, each buffer solution wash 2 times.
(h) after washing, with the phosphorylated protein of the 5% ammonium hydroxide elution specific adsorption of 50 μ L, centrifugal speed, time
And number is consistent with enrichment process.Finally eluent is lyophilized, and is put in -20 DEG C and saves backup.
Matrix Assisted Laser Desorption time-of-flight mass spectrometry analyzes (MALDI-TOF-MS)
The principle of MALDI is with laser irradiation sample and substrate formed cocrystallization film, and matrix is from swashing during laser irradiation
Energy is absorbed in light, passing to sample molecule makes its instant vaporization, and proton translocation to sample molecule is made its ionization, then
Into time-of-flight mass analyzer, it is detected further according to their own mass-to-charge ratio (m/z).
Detailed process is：By the sample of freeze-drying with 3 μ L buffer solutions C (acetonitrile/trifluoroacetic acid/water, 50%/0.1%/
49.9%, v/v/v) redissolve, then with isometric saturation CHCA matrix mixings.CHCA matrix is by alpha-cyano -4- hydroxyl meat
Cinnamic acid is dissolved in the saturated solution formed in TA30 solution, wherein, TA30 be containing volume fraction be 30% acetonitrile and 0.1% trifluoro
The aqueous solution of acetic acid.Then, mixing liquid is dripped on target plate with micropipette rifle, drips one layer of CHCA base again after natural air drying
Matter carries out MALDI-TOF mass spectral analyses after drying and crystallizing.
Wherein, the auxiliary mass spectrum type BC@mZrO of the present embodiment 22Composite nano fiber, including bacterial fibers (BC) and cladding
Zirconium dioxide nanoparticles on its surface layer, the compound fiber of receiving are supported by liquid-transfering gun pipette tips, auxiliary mass spectrum type BC@mZrO2It is multiple
Nanofiber Tip is closed to be made of above-mentioned steps a-d.Before the process of auxiliary mass spectrum specific enrichment phosphorylated protein or polypeptide includes
Processing, loading enrichment and elution analysis process, assist mass spectrum specific enrichment phosphorylated protein or polypeptide process by above-mentioned steps
E-h is completed.
BC@ZrO in the embodiment of the present invention 22Composite nano fiber is to beta-casein enzymolysis liquid (from institute in embodiment 1
Prepare) and phosphorylated polypeptide sample (1:1、1:10、1:100) mixed solution of different mol ratio does Mass Spectrometer Method, specifically
Property reaches 1:100.
The BC@mZrO of embodiment 22The preparation of composite nano fiber and the process and schematic diagram and reality of enriched phosphorus acidified protein
It is similar to apply example 1.
Fig. 8, Fig. 9 are respectively beta-casein enzymolysis liquid and the molar ratio of phosphorylated polypeptide sample is 1:10 mixed solution warp
BC@mZrO2Mass spectrogram before composite nano fiber enrichment and after enrichment.
As can be seen from the above embodiments：Enrichment and context of detection in phosphorylated protein or polypeptide, BC@of the invention gold
Belonging to oxide composite nano fiber not only has higher specificity and sensitivity, and Mass Spectrometer Method collection of illustrative plates also has higher noise
Than the present invention provides feasible new method for field of biological detection.In terms of material, BC@metal oxides of the invention are answered
Closing nanofiber not only has many advantages, such as big specific surface area, orderly meso-hole structure, also has and is simple to operate and friendly to environment.
The above is only the preferred embodiment of the present invention, is not intended to restrict the invention, it is noted that for this skill
For the those of ordinary skill in art field, without departing from the technical principles of the invention, can also make it is several improvement and
Modification, these improvements and modifications also should be regarded as protection scope of the present invention.
Claims (8)
1. a kind of preparation method of BC@metal oxide composite nano fibers, which is characterized in that include the following steps：
（a）Prepare BC nanofibers；
（b）In the surface clad oxide granular layer of BC nanofibers, and natural packing forms meso-hole structure, is coated
There are the BC@metal oxide composite nano fibers of metal oxide particle；The grain size of metal oxide particle is 5-15 nm, is situated between
The mesoporous pore size of pore structure is 6-10 nm；
（c）With base catalysis step（b）In obtained BC metal oxide composite nano fibers, obtain that enriched phosphorus can be used for be acidified
The BC@metal oxide composite nano fibers of albumen or phosphorylated polypeptide.
2. the preparation method of BC@metal oxide composite nano fibers according to claim 1, which is characterized in that in step
Suddenly（c）Step is further included later（d）：BC@metal oxide composite nano fibers are filled in liquid-transfering gun pipette tips, are formed in shifting
The BC@metal oxide composite nano fibers of liquid rifle pipette tips shape.
3. the preparation method of BC@metal oxide composite nano fibers according to claim 1, it is characterised in that：It is described
Metal oxide is selected from one or more of titanium dioxide, zirconium dioxide, alundum (Al2O3), di-iron trioxide, stannic oxide.
4. the preparation method of BC@metal oxide composite nano fibers according to claim 1, it is characterised in that：In step
Suddenly（a）In, using bacteria cellulose producing strains BC nanofibers are prepared through biological synthesis process.
5. the preparation method of BC@metal oxide composite nano fibers according to claim 4, it is characterised in that：In step
Suddenly（a）In, acetobacter xylinum prepares BC nanofibers under fermentation conditions.
6. the preparation method of BC@metal oxide composite nano fibers according to claim 1, it is characterised in that：In step
Suddenly（b）In, by situ synthesis in BC nanofiber surface coated metal oxide stratum granulosums.
7. the preparation method of BC@metal oxide composite nano fibers according to claim 1, it is characterised in that：In step
Suddenly（c）In, alkali is selected from one or more of ammonium hydroxide, n,N-Dimethylformamide and tetramethylammonium hydroxide.
8. the BC@metal oxide composite nano fiber enrichment acids prepared by the preparation method described in a kind of claims 1 or 2
Change the method for albumen or phosphorylated polypeptide, which is characterized in that include the following steps：
（e）BC@metal oxide composite nano fibers are washed with buffer solution, are at enrichment optimum state；
（f）Phosphorylated protein or phosphorylated polypeptide are added in BC@metal oxide composite nano fibers and are enriched with；
（g）With buffer solution washing step（f）In obtain be enriched with phosphorylated protein or the BC@metal oxides of phosphorylated polypeptide
Composite nano fiber, to remove the albumen of non-specific adsorption or polypeptide；
（h）The pecific phosphorylation albumen or polypeptide being enriched on BC@metal oxide composite nano fibers are taken off with alkali cleaning.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102008902A (en) * | 2010-10-13 | 2011-04-13 | 天津大学 | Bacterial cellulose-inorganic nanoparticle hybrid membrane and preparation method thereof |
| CN103908979A (en) * | 2014-04-10 | 2014-07-09 | 南京理工大学 | Supported nano TiO2 catalyst and preparation method thereof |
-
2016
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102008902A (en) * | 2010-10-13 | 2011-04-13 | 天津大学 | Bacterial cellulose-inorganic nanoparticle hybrid membrane and preparation method thereof |
| CN103908979A (en) * | 2014-04-10 | 2014-07-09 | 南京理工大学 | Supported nano TiO2 catalyst and preparation method thereof |
Non-Patent Citations (2)
| Title |
|---|
| Rapid enrichment of phosphopeptides by SiO2–TiO2 composite fibers;He Xiaomei et al.;《Analyst》;20130729(第138期);第5495-5502页 * |
| 细菌纤维素杂化纳米材料的制备及性能研究;杨加志;《中国博士学位论文全文数据库 工程科技I辑》;20111115(第11期);第B020-41页 * |
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