CN105079806B - A kind of carbon nanomaterial and its preparation method and application that polypeptide is directly modified - Google Patents
A kind of carbon nanomaterial and its preparation method and application that polypeptide is directly modified Download PDFInfo
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- CN105079806B CN105079806B CN201410201129.9A CN201410201129A CN105079806B CN 105079806 B CN105079806 B CN 105079806B CN 201410201129 A CN201410201129 A CN 201410201129A CN 105079806 B CN105079806 B CN 105079806B
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Abstract
The present invention provides a kind of carbon nanomaterials that polypeptide is directly modified, the carbon nanomaterial that the polypeptide is directly modified includes peptide molecule and carbon nanomaterial, wherein, the peptide molecule is connected with carbon nanomaterial by covalent bond, the carbon nanomaterial that the polypeptide is directly modified is product obtained by the carbon nanomaterial using peptide molecule reduction-oxidation state, wherein, the peptide molecule contains reduction acidic amino acid, the carbon nanomaterial of the oxidation state contains surface active groups, the surface active groups of the carbon nanomaterial of the oxidation state are carboxyl, epoxy group or hydroxyl.The carbon nanomaterial that polypeptide provided by the invention is directly modified has preferable stability, and the activity of polypeptide and the dispersibility of carbon nanomaterial are not substantially change.The present invention also provides a kind of preparation method and applications of carbon nanomaterial that polypeptide is directly modified.
Description
Technical field
The present invention relates to production of polysilicon equipment technical fields, and in particular to a kind of carbon nanomaterial that polypeptide is directly modified
And its preparation method and application.
Background technique
Carbon nanomaterial has special physicochemical property, carbon nanomaterial (such as carbon nanotube, graphene, carbon nano-particle
Deng) there are very strong absorbing properties, surface can be used in the local thermal imaging of tumour after biological functional is modified, light
Heat cure, photoacoustic imaging and fluorescence sense etc..Mainly there is physical absorption currently based on the bio-modification of carbon nanomaterial
With two kinds of technologies of covalent coupling.
Physical absorption, which passes through, utilizes the molecule (such as organic dyestuff probe) and pi-electron carbon nanometer abundant with phenyl ring conjugation
Between material π-π interact self assembly and be adsorbed onto together；This method is simple and convenient, still, since such assembling is not
Covalent coupling, stability are easy the pH by solution, ionic strength, the influence of the factors such as salinity.
Covalent coupling is at present to change carbon nanomaterial first to the common technology of carbon nanomaterial bio-modification
Processing (such as: oxidation), allows the active group in its surface, then in the coupling technology by some classics (such as: EDC is activated)
By large biological molecule (albumen, nucleic acid and polypeptide etc.) covalent coupling to the surface of carbon nanomaterial；The method of modifying stability is good,
But Bioconjugation process will affect the dispersibility of carbon nanomaterial and the activity of biomolecule.
Therefore, it is necessary to which the stability for providing material after a kind of bio-modification is preferable, and to the dispersibility of carbon nanomaterial
Influence the method for bio-modification carbon nanomaterial small, small to the activity influence of biomolecule.
Summary of the invention
For the defect for overcoming the above-mentioned prior art, first aspect present invention provides a kind of carbon nanometer that polypeptide is directly modified
Material, the carbon nanomaterial that the polypeptide is directly modified have preferable stability, the activity of polypeptide and point of carbon nanomaterial
Scattered property is not substantially change.Second aspect of the present invention provides a kind of preparation side of carbon nanomaterial that polypeptide is directly modified
Method, the preparation method are passed through the method that a step restores, surface are in the carbon of oxidation state using the polypeptide with reducing property
Nano material is restored, and polypeptide is directly coupled to the surface of carbon nanomaterial.It is more that third aspect present invention provides one kind
The application for the carbon nanomaterial that peptide is directly modified.
In a first aspect, the present invention provides a kind of carbon nanomaterial that polypeptide is directly modified, what the polypeptide was directly modified
Carbon nanomaterial includes peptide molecule and carbon nanomaterial, wherein the peptide molecule and carbon nanomaterial pass through covalent bond phase
Even, the carbon nanomaterial that the polypeptide is directly modified is production obtained by the carbon nanomaterial using peptide molecule reduction-oxidation state
Object, wherein the peptide molecule contains reduction acidic amino acid, and the carbon nanomaterial of the oxidation state contains surface active groups,
The surface active groups of the carbon nanomaterial of the oxidation state are carboxyl, epoxy group or hydroxyl.
Preferably, the carbon nanomaterial of the oxidation state is the oxidation carbon nanomaterial of zero dimension.
Under this optimum condition, the oxidation carbon nanomaterial of the zero dimension is since it is without spatial orientation, polypeptide point
It is not influenced by space structure when son functions.So peptide molecule is on the surface of the oxidation carbon nanomaterial of the zero dimension
Distribution be random uniform, to make the carbon nanomaterial after modification that there is more stable property.
In addition, the oxidation carbon nanomaterial size of zero dimension is more uniform, and partial size only has tens to receive compared with multidimensional carbon material
Rice with peptide molecule there is better size compatibility will have better cell-penetrating energy since size is relatively smaller
Power will have the advantage become apparent in terms of biologic applications.
It is further preferred that the oxidation carbon nanomaterial of the zero dimension is fullerene.
Preferably, the carbon nanomaterial of the oxidation state is oxidation carbon nano-particle.
It is further preferred that oxidation carbon nano-particle of the present invention is prepared by following step:
1) candle ash is collected；
2) the candle ash for taking out certain mass, is added isometric DMF solvent and concentrated nitric acid, at a certain temperature back flow reaction
For a period of time；
3) reaction solution obtained by step (2) is separated by gradient centrifugation, collects dark brown precipitating, and use certain volume
Water dissolution precipitating after, ultrasonic disperse processing to get arrive the solution containing the oxidized form carbon nano-particle.
It is further preferred that the candle ash is that black smog not sufficiently combusted above the flame of candle is attached to porcelain
Atrament on brick.
It is further preferred that the step of gradient centrifugation separates are as follows:
A) 6000 revs/min of the slow-speed of revolution are centrifuged 5 minutes, and large-sized particle is precipitated and is removed, and collect upper layer black liquor simultaneously
Ultrasonic disperse is handled 2 hours；
B) pH value of black liquor obtained by step (a) is adjusted to neutrality with diluted sodium hydroxide solution, then passes through liquid
9000 revs/min are crossed, is centrifuged 5 minutes, further by relatively large sized particle precipitation and separation, collects upper layer dark brown transparent liquid
Body；
C) it by dark brown transparency liquid obtained by step (b) with 13000 revs/min, is centrifuged 10 minutes, collects dark brown precipitating,
And precipitating and constant volume are dissolved with the water of certain volume, ultrasonic disperse handles 30 minutes to get to containing the oxidized form carbon nanometer
The solution of grain.
It is further preferred that the candle ash is that black smog not sufficiently combusted above the flame of candle is attached to porcelain
Atrament on brick.
Under this optimum condition, the oxidation carbon nano-particle can be made, the oxidation carbon nano-particle includes zero dimension
Aoxidize carbon nanomaterial.
Preferably, the carbon nanomaterial of the oxidation state is graphene oxide.
Preferably, the carbon nanomaterial of the oxidation state is oxide/carbon nanometer tube.
Preferably, the reduction acidic amino acid is tyrosine, cysteine, histidine or tryptophan.
Preferably, the reduction acidic amino acid that the present invention uses includes but is not limited to aromatic amino acid.
Preferably, the peptide molecule containing reduction acidic amino acid is the polypeptide containing 5~40 amino acid.
Preferably, the peptide molecule containing reduction acidic amino acid is the polypeptide with site targeting.
Preferably, the peptide molecule containing reduction acidic amino acid is that neurotoxic peptide, RGD cyclic peptide or antibacterial are more
Peptide.
It is further preferred that the amino acid sequence of the neurotoxic peptide includes the amino as shown in SEQ ID NO:1
Acid sequence.
It is further preferred that the amino acid sequence of the neurotoxic peptide is as shown in SEQ ID NO:1.
Specifically, the RGD cyclic peptide is the circular polypeptides containing arginyl-glycyl-aspartic acid tripeptides, wherein institute
State arginyl-glycyl-aspartic acid i.e. Arg-Gly-Asp (RGD).
It is further preferred that the RGD cyclic peptide is c (RGDfK).
Specifically, the Pub Chem Commpound of the c (RGDfK) CID is 10196873.
It is further preferred that the antibacterial polypeptide is nisin.
It is further preferred that the Pub Chem Commpound of the nisin CID is 16219761.
Preferably, the size for the carbon nanomaterial that polypeptide is directly modified is 30~60nm.
The present invention by the carbon nanomaterial directly modified of polypeptide go back original surface using the polypeptide with reducing property
Peptide molecule is coupled to carbon nanomaterial surface by the carbon nanomaterial in oxidation state, a direct step.In the coupling process, institute
It states and is formed between the active group on the carbon nanomaterial surface of reduction acidic amino acid and the oxidation state by redox reaction
Covalent bond, compares physical absorption, and the stability of resulting peptide modified carbon nanomaterial is more preferable.
In addition, comparing traditional covalent coupling technology, the present invention requires no the EDC activation of biomolecule, without right
Carbon nanomaterial carries out the modification such as PEG, and polypeptid covalence can be coupled to the surface of carbon nanomaterial, resulting peptide modified
The bioactivity of the relatively good holding peptide molecule of the energy of carbon nanomaterial, and bio-modification process is substantially reduced to carbon material
Size, surface nature, water solubility etc. influence, to reduce influence of the bio-modification process to carbon nanomaterial dispersibility；Therefore,
The dispersibility and water solubility of peptide modified carbon nanomaterial provided by the invention are preferable.
Second aspect, the present invention provides a kind of preparation method of carbon nanomaterial that polypeptide is directly modified, including it is as follows
Step:
1) mass ratio is taken to be in the carbon nanomaterial of oxidation state for the peptide molecule of 1~4:1~20 and surface, wherein institute
It states peptide molecule and contains reduction acidic amino acid, the surface is in the surface active groups that contain of oxidation state, the oxidation state
The surface active groups of carbon nanomaterial are carboxyl, epoxy group or hydroxyl；
2) carbon nanomaterial that the surface is in oxidation state is added in buffer, after ultrasonic treatment, is added described more
Peptide molecule is simultaneously uniformly mixed so as to obtain mixture；
3) gained mixture be stirred to react at normal temperature 8~24 hours, reaction products therefrom with ultra-filtration centrifuge tube carry out from
After the heart, washing, buffer is added and is ultrasonically treated, obtains the carbon nanomaterial that the polypeptide is directly modified.
Preferably, in the step (1), the carbon nanomaterial of the oxidation state is the oxidation carbon nanomaterial of zero dimension.
It is further preferred that the oxidation carbon nanomaterial of the zero dimension is fullerene.
Preferably, in the step (1), the carbon nanomaterial of the oxidation state is oxidation carbon nano-particle.
It is further preferred that oxidation carbon nano-particle of the present invention is prepared by following step:
1) candle ash is collected；
2) the candle ash for taking out certain mass, is added isometric DMF solvent and concentrated nitric acid, at a certain temperature back flow reaction
For a period of time；
3) reaction solution obtained by step (2) is separated by gradient centrifugation, collects dark brown precipitating, and use certain volume
Water dissolution precipitating after, ultrasonic disperse processing to get arrive the solution containing the oxidized form carbon nano-particle.
It is further preferred that the candle ash is that black smog not sufficiently combusted above the flame of candle is attached to porcelain
Atrament on brick.
It is further preferred that the step of gradient centrifugation separates are as follows:
A) 6000 revs/min of the slow-speed of revolution are centrifuged 5 minutes, and large-sized particle is precipitated and is removed, and collect upper layer black liquor simultaneously
Ultrasonic disperse is handled 2 hours；
B) pH value of black liquor obtained by step (a) is adjusted to neutrality with diluted sodium hydroxide solution, then passes through liquid
9000 revs/min are crossed, is centrifuged 5 minutes, further by relatively large sized particle precipitation and separation, collects upper layer dark brown transparent liquid
Body；
C) it by dark brown transparency liquid obtained by step (b) with 13000 revs/min, is centrifuged 10 minutes, collects dark brown precipitating,
And precipitating and constant volume are dissolved with the water of certain volume, ultrasonic disperse handles 30 minutes to get to containing the oxidized form carbon nanometer
The solution of grain.
It is further preferred that the candle ash is that black smog not sufficiently combusted above the flame of candle is attached to porcelain
Atrament on brick.
Under this optimum condition, the oxidation carbon nano-particle can be made, the oxidation carbon nano-particle includes zero dimension
Aoxidize carbon nanomaterial.
Preferably, in the step (1), the carbon nanomaterial of the oxidation state is graphene oxide.
Preferably, in the step (1), the carbon nanomaterial of the oxidation state is oxide/carbon nanometer tube.
Preferably, in the step (1), the reduction acidic amino acid is tyrosine, cysteine, histidine or color ammonia
Acid.
Preferably, in the step (1), the reduction acidic amino acid that the present invention uses includes but is not limited to aromatic series amino
Acid.
Preferably, in the step (1), the peptide molecule containing reduction acidic amino acid is to contain 5~40 amino
The polypeptide of acid.
Preferably, in the step (1), the peptide molecule containing reduction acidic amino acid is with site targeting
Polypeptide.
Preferably, the peptide molecule containing reduction acidic amino acid is that neurotoxic peptide, RGD cyclic peptide or antibacterial are more
Peptide.
It is further preferred that the amino acid sequence of the neurotoxic peptide includes the amino as shown in SEQ ID NO:1
Acid sequence.
It is further preferred that the amino acid sequence of the neurotoxic peptide is as shown in SEQ ID NO:1.
Preferably, in the step (2) or (3), the buffer is HEPES buffer solution, the HEPES buffer solution
For the conventional use of 4- hydroxyethyl piperazineethanesulfonic acid buffer solution in laboratory.
It is further preferred that the concentration of the HEPES buffer solution is 0.02mol/L, pH in the step (2) or (3)
Value is 7.4.
Preferably, in the step (2), the concrete operations of the ultrasonic treatment are as follows: surface is in oxidation state first
Carbon nanomaterial is dissolved in HEPES buffer solution, and certain time is ultrasonically treated under Ultrasound Instrument, after material is evenly dispersed, then
Suitable HEPES buffer solution is added, continues ultrasonic treatment a period of time, peptide molecule is then added.
It is further preferred that the dosage of suitable HEPES buffer solution is that every 1mg peptide molecule is dissolved in final volume
For in 2~3ml HEPES buffer solution.
It is further preferred that the condition of the ultrasonic treatment are as follows: power 100W, ultrasound 15 minutes.
Preferably, in the step (3), the mode of the stirring is magnetic agitation.
Preferably, in the step (3), the molecular cut off 100kD of the ultra-filtration centrifuge tube.
Preferably, in the step (3), the step that reaction products therefrom is centrifuged with ultra-filtration centrifuge tube, is washed
Suddenly include:
Ultra-filtration centrifuge tube is added in reaction products therefrom, is then centrifuged for 2~4 times, is washed every time with HEPES buffer solution；
Wherein, the condition of the centrifugation is to be centrifuged 10 minutes under 5000r/m revolving speed.
By being centrifuged repeatedly washing, free, not to be modified carbon nanomaterial surface peptide molecule can be separated dry
Only.
Preferably, in the step (3), the carbon nanomaterial certain volume that will finally modify a lot of peptides
The dissolution of HEPES buffer solution is subsequently placed in 4 degree of refrigerators and saves backup after being ultrasonically treated a period of time.
It is further preferred that in the step (3), the condition of the ultrasonic treatment are as follows: power 100W, ultrasound 10 minutes.
Preferably, the size for the carbon nanomaterial that the polypeptide is directly modified is 30~60nm.
The present invention also provides the preparation method of carbon nanomaterial directly modified of polypeptide use the peptide molecule of reproducibility
As reducing agent, while the biological function of peptide molecule is remained, eliminating in traditional chemical modification first to receive carbon
The step of rice material and biomolecule are activated, simplifies operating procedure, saves production cost.
In addition, during traditional protein macromolecule modifies carbon nanomaterial, due to the molecular weight of albumen
Greatly, space structure is complicated, influences greatly, to be easy to cause nano material aggregate and precipitate to the surface nature of carbon nanomaterial.This hair
The peptide molecule of bright use has smaller size, and structure is more flexible, low in cost；And peptide molecule is to carbon nanometer material after modification
The influences such as the surface nature (such as charge, size) of material are smaller, therefore, peptide molecule modification after carbon nanomaterial stability more
It is good.
The third aspect, the present invention also provides a kind of carbon nanomaterials that polypeptide is directly modified to prepare tumour medicine, light
Application in acoustic imaging contrast agent or photo-thermal therapy preparation.
The carbon nanomaterial that polypeptide provided by the invention is directly modified is with multi-functional: not only having peptide molecule tumour
The selectively targeted function of tissue, simultaneously because therefore the optical property that carbon nanomaterial is excellent can be applied to preparation tumour medicine
Object, photoacoustic imaging contrast agent or photo-thermal therapy preparation.
The present invention provides a kind of polypeptide directly modify carbon nanomaterial, and its preparation method and application, have with
It is lower the utility model has the advantages that
(1) peptide modified carbon nanomaterial provided by the invention not only has preferable bioactivity, but also has
Preferable stability and dispersibility；
(2) the present invention also provides the preparation method of carbon nanomaterial directly modified of polypeptide using reproducibility polypeptide point
Son is used as reducing agent, eliminates the step that be first activated to carbon nanomaterial and biomolecule in traditional chemical modification
Suddenly, operating procedure is simplified, production cost is saved；
(3) the present invention also provides a kind of carbon nanomaterials that polypeptide is directly modified to prepare tumour medicine, photoacoustic imaging
Application in contrast agent or photo-thermal therapy preparation.
Detailed description of the invention
Fig. 1 is the UV-visible absorption spectrum for the peptide modified carbon nano-particle that the embodiment of the present invention 2 provides；
Fig. 2~Fig. 4 is the transmission electron microscope picture for the peptide modified carbon nano-particle that the embodiment of the present invention 2 provides；
Fig. 5 is the carbon nano-particle that the embodiment of the present invention 1 provides and the peptide modified carbon nano-particle that embodiment 2 provides
Water-soluble pictorial diagram.
Specific embodiment
Following will be combined with the drawings in the embodiments of the present invention, and technical solution in the embodiment of the present invention carries out clear, complete
Site preparation description, it is clear that described embodiments are only a part of the embodiments of the present invention, instead of all the embodiments.It is based on
Embodiment in the present invention, it is obtained by those of ordinary skill in the art without making creative efforts every other
Embodiment shall fall within the protection scope of the present invention.
Preparation oxidation carbon nano-particle
Embodiment 1
A kind of preparation method for aoxidizing carbon nano-particle is present embodiments provided, is included the following steps:
1) not sufficiently combusted black smog is collected above the flame of candle with the smooth ceramic tile of clean surface, will be adhered to
Atrament (candle ash, main component is carbon) medication spoon on ceramic tile scrapes and is stored in vial；
2) the candle ash for taking out certain mass, is added isometric DMF solvent and concentrated nitric acid, at a certain temperature back flow reaction
For a period of time；
3) reaction solution obtained by step (2) is separated by gradient centrifugation, the step of gradient centrifugation separation are as follows:
A) 6000 revs/min of the slow-speed of revolution are centrifuged 5 minutes, and large-sized particle is precipitated and is removed, and collect upper layer black liquor simultaneously
Ultrasonic disperse is handled 2 hours；
B) pH value of black liquor obtained by step (a) is adjusted to neutrality with diluted sodium hydroxide solution, then passes through liquid
9000 revs/min are crossed, is centrifuged 5 minutes, further by relatively large sized particle precipitation and separation, collects upper layer dark brown transparent liquid
Body；
C) it by dark brown transparency liquid obtained by step (b) with 13000 revs/min, is centrifuged 10 minutes, collects dark brown precipitating,
And with the water of certain volume dissolution precipitating and constant volume, ultrasonic disperse handle 30 minutes to get arrive oxidized form carbon nano-particle solution,
4 degree of refrigerators save backup.
Reduction-oxidation carbon nano-particle
Embodiment 2
Present embodiments provide a kind of neurotoxic peptide (chlorotoxin, CTX) carbonoxide nanometer using reproducibility
The preparation method of particle, includes the following steps:
1) it is molten that the carbon nanomaterial that the surface for taking 0.5mg embodiment 1 to prepare is in oxidation state is dissolved in 1mLHEPES buffering
It in liquid, is ultrasonically treated 15 minutes, makes material fully dispersed, the HEPES buffer solution of 1.5mL volume is then added, continue ultrasound
Then processing 5 minutes is added the CTX of 4mg, obtains mixture, wherein the amino acid sequence of the CTX such as SEQ ID NO:1 institute
Show；
2) by mixture obtained by step (1) under 25 degree of room temperature, magnetic agitation is reacted 24 hours, obtains reaction product, institute
State the carbon nanomaterial for having peptide molecule in reaction product containing surface modification；
3) it by reaction product obtained by step (2) under 5000r/m revolving speed, is centrifugated 10 minutes, is centrifuged repeatedly 3 times every time
It is rinsed with HEPES buffer solution, so that the free polypeptide removal for not being modified material surface is clean, is finally repaired surface
It is decorated with the HEPES buffer solution dissolution of the carbon nanomaterial 1mL volume of peptide molecule and constant volume, is then ultrasonically treated 10 points
Clock obtains the oxidation carbon nano-particle that the CTX is directly modified, and size is 30~60nm, is placed in 4 degree of refrigerators and saves backup.
To absolutely prove beneficial effects of the present invention, the present invention also provides polypeptide (CTX) modified carbons that embodiment 2 provides
The UV-visible absorption spectrum of nano particle, as shown in Figure 1.Curve 1 is punished in 230nm or so and 278nm in Fig. 1
There is not 1 characteristic absorption peak, the characteristic absorption peak of 230nm or so is the characteristic peak of carbon nano-particle (CNPs) after CTX modification,
Characteristic absorption peak at 278nm is the characteristic peak of CTX polypeptide, illustrates that the embodiment of the present invention 2 has been prepared what CTX was directly modified
Aoxidize carbon nano-particle.
To further illustrate beneficial effects of the present invention, the peptide modified carbon provided the present invention also provides embodiment 2 is received
The transmission electron microscope picture of rice grain, as shown in figs. 2 to 4, by Fig. 2~4 it is found that illustrating that CTX prepared by the embodiment of the present invention 2 is directly repaired
The oxidation carbon nano-particle of decorations has preferable dispersibility；
To further explain beneficial effects of the present invention, the present invention also provides the carbon nano-particles that embodiment 1 provides
And the peptide modified carbon nano-particle water solubility pictorial diagram that embodiment 2 provides, as shown in figure 5, as shown in Figure 5: peptide modified
The dispersibility and water solubility of front and back nano material are kept well, and there is no significant changes；Secondly, carbon nanometer material after modification
The color of material is changed into carbon black color (test tube 2) (carbon black color be the unoxidized color of carbon material) from dark brown (test tube 1), this also from
Side further demonstrates CTX polypeptide can be carbon nano materials modified, can go back the carbon nanomaterial that original surface is in oxidation state.
Embodiment 3~6
The step of the present embodiment 3~7 is with embodiment 2 are essentially identical, and difference is only that the carbon nanometer material of step (1) oxidation state
Material is different from the mass ratio of CTX dosage, and difference is as shown in the table:
Embodiment 8
Present embodiments provide a kind of preparation side of Nisin modified oxidation carbon nano-particle using reproducibility
Method includes the following steps:
1) it is molten that the carbon nanomaterial that the surface for taking 0.5mg embodiment 1 to prepare is in oxidation state is dissolved in 1mLHEPES buffering
It in liquid, is ultrasonically treated 15 minutes, makes material fully dispersed, the HEPES buffer solution of 1.5mL volume is then added, continue ultrasound
Then processing 5 minutes is added the nisin of 4mg, obtains mixture, wherein the Pub of the nisin
Chem Commpound CID is 16219761；
2) by mixture obtained by step (1) under 25 degree of room temperature, magnetic agitation is reacted 24 hours, obtains reaction product；
3) it by reaction product obtained by step (2) under 5000r/m revolving speed, is centrifugated 10 minutes, is centrifuged repeatedly 3 times every time
It is rinsed with HEPES buffer solution, so that the free polypeptide removal for not being modified material surface is clean, is finally repaired surface
It is decorated with the HEPES buffer solution dissolution of the carbon nanomaterial 1mL volume of peptide molecule and constant volume, is then ultrasonically treated 10 points
Clock obtains the oxidation carbon nano-particle that the nisin is directly modified, and size is 30~60nm, is placed in 4 degree of refrigerators and protects
It deposits spare.
Embodiment 9
Present embodiments provide a kind of preparation side of c (RGDfK) cyclic peptide modification oxidation carbon nano-particle using reproducibility
Method includes the following steps:
1) it is molten that the carbon nanomaterial that the surface for taking 0.5mg embodiment 1 to prepare is in oxidation state is dissolved in 1mLHEPES buffering
It in liquid, is ultrasonically treated 15 minutes, makes material fully dispersed, the HEPES buffer solution of 1.5mL volume is then added, continue ultrasound
Then processing 5 minutes is added c (RGDfK) cyclic peptide of 4mg, obtains mixture, wherein the Pub of c (RGDfK) cyclic peptide
Chem Commpound CID is 10196873；
2) by mixture obtained by step (1) under 25 degree of room temperature, magnetic agitation is reacted 24 hours, obtains reaction product；
3) it by reaction product obtained by step (2) under 5000r/m revolving speed, is centrifugated 10 minutes, is centrifuged repeatedly 3 times every time
It is rinsed with HEPES buffer solution, so that the free polypeptide removal for not being modified material surface is clean, is finally repaired surface
It is decorated with the HEPES buffer solution dissolution of the carbon nanomaterial 1mL volume of peptide molecule and constant volume, is then ultrasonically treated 10 points
Clock obtains the oxidation carbon nano-particle that the c (RGDfK) cyclic peptide is directly modified, and size is 30~60nm, is placed in 4 degree of refrigerators and protects
It deposits spare.
The above is a preferred embodiment of the present invention, it is noted that for those skilled in the art
For, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications are also considered as
Protection scope of the present invention.
Claims (9)
1. a kind of carbon nanomaterial that polypeptide is directly modified, the carbon nanomaterial that the polypeptide is directly modified include peptide molecule and
Carbon nanomaterial, wherein the peptide molecule is connected with carbon nanomaterial by covalent bond, which is characterized in that the polypeptide is straight
The carbon nanomaterial of modification is connect as product obtained by the carbon nanomaterial using peptide molecule reduction-oxidation state, wherein described
Peptide molecule contains reduction acidic amino acid, and the peptide molecule containing reduction acidic amino acid is neurotoxic peptide, RGD cyclic peptide
Or antibacterial polypeptide, the amino acid sequence of the neurotoxic peptide are SEQ ID NO:1；The RGD cyclic peptide is c (RGDfK), and
Pub Chem Commpound CID of the c (RGDfK) is 10196873；The antibacterial polypeptide is nisin,
And the Pub Chem Commpound of the nisin CID is 16219761, the carbon nanomaterial of the oxidation state
Containing surface active groups, the surface active groups of the carbon nanomaterial of the oxidation state are carboxyl, epoxy group or hydroxyl.
2. the carbon nanomaterial that polypeptide as described in claim 1 is directly modified, which is characterized in that the carbon nanometer of the oxidation state
Material is oxidation carbon nano-particle, graphene oxide or oxide/carbon nanometer tube.
3. the carbon nanomaterial that polypeptide as described in claim 1 is directly modified, which is characterized in that the reduction acidic amino acid is
Tyrosine, cysteine, histidine or tryptophan.
4. the carbon nanomaterial that polypeptide as described in claim 1 is directly modified, which is characterized in that the polypeptide was directly modified
The size of carbon nanomaterial is 30~60nm.
5. a kind of preparation method for the carbon nanomaterial that polypeptide is directly modified, which comprises the steps of:
1) mass ratio is taken to be in the carbon nanomaterial of oxidation state for the peptide molecule of 1~4:1~20 and surface, wherein described more
Peptide molecule contains reduction acidic amino acid, the peptide molecule containing reduction acidic amino acid be neurotoxic peptide, RGD cyclic peptide or
Antibacterial polypeptide, the amino acid sequence of the neurotoxic peptide are SEQ ID NO:1；The RGD cyclic peptide is c (RGDfK), and institute
Stating Pub Chem Commpound CID of c (RGDfK) is 10196873；The antibacterial polypeptide is nisin, and
Pub Chem Commpound CID of the nisin is 16219761, and the surface is in containing for oxidation state
Surface active groups, the surface active groups of the carbon nanomaterial of the oxidation state are carboxyl, epoxy group or hydroxyl；
2) carbon nanomaterial that the surface is in oxidation state is added in buffer, after ultrasonic treatment, the polypeptide point is added
Son is simultaneously uniformly mixed so as to obtain mixture；
3) gained mixture is stirred to react 8~24 hours at normal temperature, and reaction products therefrom is centrifuged with ultra-filtration centrifuge tube, is washed
After washing, buffer is added and is ultrasonically treated, obtains the carbon nanomaterial that the polypeptide is directly modified.
6. the preparation method for the carbon nanomaterial that polypeptide as claimed in claim 5 is directly modified, which is characterized in that the step
(1) in, the carbon nanomaterial of the oxidation state is oxidation carbon nano-particle, graphene oxide or oxide/carbon nanometer tube.
7. the preparation method for the carbon nanomaterial that polypeptide as claimed in claim 5 is directly modified, which is characterized in that the step
(1) in, the reduction acidic amino acid is tyrosine, cysteine, histidine or tryptophan.
8. the preparation method for the carbon nanomaterial that polypeptide as claimed in claim 5 is directly modified, which is characterized in that the step
(3) in, the size for the carbon nanomaterial that the polypeptide is directly modified is 30~60nm.
9. a kind of carbon nanomaterial that polypeptide according to any one of claims 1-4 is directly modified is preparing tumour medicine, light
Application in acoustic imaging contrast agent or photo-thermal therapy agent.
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