CN108483426A - A method of the luminous carbon dots of hypotoxicity multi-mode are prepared based on functional polypeptide - Google Patents
A method of the luminous carbon dots of hypotoxicity multi-mode are prepared based on functional polypeptide Download PDFInfo
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- CN108483426A CN108483426A CN201810172324.1A CN201810172324A CN108483426A CN 108483426 A CN108483426 A CN 108483426A CN 201810172324 A CN201810172324 A CN 201810172324A CN 108483426 A CN108483426 A CN 108483426A
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- VNWKTOKETHGBQD-UHFFFAOYSA-N methane Chemical compound C VNWKTOKETHGBQD-UHFFFAOYSA-N 0.000 title claims abstract description 92
- 238000000034 method Methods 0.000 title claims abstract description 47
- 229920001184 polypeptide Polymers 0.000 title claims abstract description 18
- 108090000765 processed proteins & peptides Proteins 0.000 title claims abstract description 18
- 102000004196 processed proteins & peptides Human genes 0.000 title claims abstract description 18
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims abstract description 25
- 229910052799 carbon Inorganic materials 0.000 claims abstract description 25
- RWSXRVCMGQZWBV-WDSKDSINSA-N glutathione Chemical compound OC(=O)[C@@H](N)CCC(=O)N[C@@H](CS)C(=O)NCC(O)=O RWSXRVCMGQZWBV-WDSKDSINSA-N 0.000 claims abstract description 25
- 239000000843 powder Substances 0.000 claims abstract description 18
- 108010024636 Glutathione Proteins 0.000 claims abstract description 17
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 17
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims abstract description 14
- 229910052757 nitrogen Inorganic materials 0.000 claims abstract description 14
- 239000012298 atmosphere Substances 0.000 claims abstract description 12
- NINIDFKCEFEMDL-UHFFFAOYSA-N Sulfur Chemical compound [S] NINIDFKCEFEMDL-UHFFFAOYSA-N 0.000 claims abstract description 10
- 239000005864 Sulphur Substances 0.000 claims abstract description 7
- 238000005979 thermal decomposition reaction Methods 0.000 claims abstract description 7
- 238000000197 pyrolysis Methods 0.000 claims description 17
- 239000000047 product Substances 0.000 claims description 11
- 230000008569 process Effects 0.000 claims description 10
- 239000012299 nitrogen atmosphere Substances 0.000 claims description 8
- 239000008367 deionised water Substances 0.000 claims description 6
- 229910021641 deionized water Inorganic materials 0.000 claims description 6
- 238000000502 dialysis Methods 0.000 claims description 6
- 238000001035 drying Methods 0.000 claims description 5
- 239000012528 membrane Substances 0.000 claims description 5
- 239000000706 filtrate Substances 0.000 claims description 4
- 229960003180 glutathione Drugs 0.000 claims description 4
- 238000001914 filtration Methods 0.000 claims description 2
- 238000000227 grinding Methods 0.000 claims description 2
- 238000000746 purification Methods 0.000 claims description 2
- 239000002904 solvent Substances 0.000 claims description 2
- 229910052717 sulfur Inorganic materials 0.000 abstract description 10
- 230000005284 excitation Effects 0.000 abstract description 8
- 238000000967 suction filtration Methods 0.000 abstract description 5
- 238000001291 vacuum drying Methods 0.000 abstract description 5
- 239000012535 impurity Substances 0.000 abstract description 2
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 abstract 1
- 125000002887 hydroxy group Chemical group [H]O* 0.000 abstract 1
- 238000001727 in vivo Methods 0.000 abstract 1
- 239000000243 solution Substances 0.000 description 24
- 238000012360 testing method Methods 0.000 description 14
- 238000002360 preparation method Methods 0.000 description 11
- 230000000052 comparative effect Effects 0.000 description 9
- 229910052760 oxygen Inorganic materials 0.000 description 8
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 7
- 235000003969 glutathione Nutrition 0.000 description 7
- 239000001301 oxygen Substances 0.000 description 7
- 238000004833 X-ray photoelectron spectroscopy Methods 0.000 description 6
- 238000010792 warming Methods 0.000 description 6
- 230000000694 effects Effects 0.000 description 5
- 238000010438 heat treatment Methods 0.000 description 5
- 239000000463 material Substances 0.000 description 5
- 239000000203 mixture Substances 0.000 description 5
- 229910052573 porcelain Inorganic materials 0.000 description 5
- 239000007787 solid Substances 0.000 description 5
- 239000007864 aqueous solution Substances 0.000 description 4
- 238000004630 atomic force microscopy Methods 0.000 description 4
- 210000004027 cell Anatomy 0.000 description 4
- 239000007788 liquid Substances 0.000 description 4
- 239000004570 mortar (masonry) Substances 0.000 description 4
- 238000002390 rotary evaporation Methods 0.000 description 4
- 238000002604 ultrasonography Methods 0.000 description 4
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 description 3
- 230000001419 dependent effect Effects 0.000 description 3
- 238000001514 detection method Methods 0.000 description 3
- 125000000524 functional group Chemical group 0.000 description 3
- 238000003384 imaging method Methods 0.000 description 3
- 230000001590 oxidative effect Effects 0.000 description 3
- 239000002245 particle Substances 0.000 description 3
- 229910052698 phosphorus Inorganic materials 0.000 description 3
- 238000004321 preservation Methods 0.000 description 3
- 230000001105 regulatory effect Effects 0.000 description 3
- 239000011593 sulfur Substances 0.000 description 3
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 2
- LFVGISIMTYGQHF-UHFFFAOYSA-N ammonium dihydrogen phosphate Chemical compound [NH4+].OP(O)([O-])=O LFVGISIMTYGQHF-UHFFFAOYSA-N 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 230000001276 controlling effect Effects 0.000 description 2
- 231100000135 cytotoxicity Toxicity 0.000 description 2
- 230000003013 cytotoxicity Effects 0.000 description 2
- 238000010348 incorporation Methods 0.000 description 2
- 239000002096 quantum dot Substances 0.000 description 2
- 239000002994 raw material Substances 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- PWKSKIMOESPYIA-UHFFFAOYSA-N 2-acetamido-3-sulfanylpropanoic acid Chemical compound CC(=O)NC(CS)C(O)=O PWKSKIMOESPYIA-UHFFFAOYSA-N 0.000 description 1
- 229910014033 C-OH Inorganic materials 0.000 description 1
- 229920000049 Carbon (fiber) Polymers 0.000 description 1
- 229910014570 C—OH Inorganic materials 0.000 description 1
- WHUUTDBJXJRKMK-UHFFFAOYSA-N Glutamic acid Natural products OC(=O)C(N)CCC(O)=O WHUUTDBJXJRKMK-UHFFFAOYSA-N 0.000 description 1
- 239000004471 Glycine Substances 0.000 description 1
- XUJNEKJLAYXESH-REOHCLBHSA-N L-Cysteine Chemical compound SC[C@H](N)C(O)=O XUJNEKJLAYXESH-REOHCLBHSA-N 0.000 description 1
- WHUUTDBJXJRKMK-VKHMYHEASA-N L-glutamic acid Chemical compound OC(=O)[C@@H](N)CCC(O)=O WHUUTDBJXJRKMK-VKHMYHEASA-N 0.000 description 1
- AFCARXCZXQIEQB-UHFFFAOYSA-N N-[3-oxo-3-(2,4,6,7-tetrahydrotriazolo[4,5-c]pyridin-5-yl)propyl]-2-[[3-(trifluoromethoxy)phenyl]methylamino]pyrimidine-5-carboxamide Chemical compound O=C(CCNC(=O)C=1C=NC(=NC=1)NCC1=CC(=CC=C1)OC(F)(F)F)N1CC2=C(CC1)NN=N2 AFCARXCZXQIEQB-UHFFFAOYSA-N 0.000 description 1
- CPDVMAVJZXIERD-GEMLJDPKSA-N N[C@H](C(=O)O)CCC(=O)N[C@@H](CS)C(=O)NCC(=O)O.[N] Chemical compound N[C@H](C(=O)O)CCC(=O)N[C@@H](CS)C(=O)NCC(=O)O.[N] CPDVMAVJZXIERD-GEMLJDPKSA-N 0.000 description 1
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 description 1
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 1
- 108010087230 Sincalide Proteins 0.000 description 1
- 238000002441 X-ray diffraction Methods 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 238000000862 absorption spectrum Methods 0.000 description 1
- 150000001336 alkenes Chemical class 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 239000003963 antioxidant agent Substances 0.000 description 1
- 230000003078 antioxidant effect Effects 0.000 description 1
- 235000006708 antioxidants Nutrition 0.000 description 1
- 238000000089 atomic force micrograph Methods 0.000 description 1
- DLGYNVMUCSTYDQ-UHFFFAOYSA-N azane;pyridine Chemical compound N.C1=CC=NC=C1 DLGYNVMUCSTYDQ-UHFFFAOYSA-N 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 230000005540 biological transmission Effects 0.000 description 1
- 239000000090 biomarker Substances 0.000 description 1
- 210000002798 bone marrow cell Anatomy 0.000 description 1
- 229910052793 cadmium Inorganic materials 0.000 description 1
- BDOSMKKIYDKNTQ-UHFFFAOYSA-N cadmium atom Chemical compound [Cd] BDOSMKKIYDKNTQ-UHFFFAOYSA-N 0.000 description 1
- 239000004917 carbon fiber Substances 0.000 description 1
- 238000010609 cell counting kit-8 assay Methods 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 230000010261 cell growth Effects 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- 239000007795 chemical reaction product Substances 0.000 description 1
- 238000005345 coagulation Methods 0.000 description 1
- 230000015271 coagulation Effects 0.000 description 1
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 description 1
- 235000018417 cysteine Nutrition 0.000 description 1
- 231100000263 cytotoxicity test Toxicity 0.000 description 1
- 238000005034 decoration Methods 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 210000004443 dendritic cell Anatomy 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 238000000295 emission spectrum Methods 0.000 description 1
- 238000003912 environmental pollution Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 230000002349 favourable effect Effects 0.000 description 1
- 238000000799 fluorescence microscopy Methods 0.000 description 1
- 235000013922 glutamic acid Nutrition 0.000 description 1
- 239000004220 glutamic acid Substances 0.000 description 1
- 229910021389 graphene Inorganic materials 0.000 description 1
- 230000003760 hair shine Effects 0.000 description 1
- -1 has two big methods Chemical compound 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 229910001385 heavy metal Inorganic materials 0.000 description 1
- 238000002173 high-resolution transmission electron microscopy Methods 0.000 description 1
- 238000001027 hydrothermal synthesis Methods 0.000 description 1
- 230000010354 integration Effects 0.000 description 1
- 231100000053 low toxicity Toxicity 0.000 description 1
- 238000002844 melting Methods 0.000 description 1
- 230000008018 melting Effects 0.000 description 1
- 229910052751 metal Inorganic materials 0.000 description 1
- 239000002184 metal Substances 0.000 description 1
- 150000002739 metals Chemical class 0.000 description 1
- 239000002086 nanomaterial Substances 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 239000005416 organic matter Substances 0.000 description 1
- 239000011574 phosphorus Substances 0.000 description 1
- 238000001420 photoelectron spectroscopy Methods 0.000 description 1
- 238000004375 physisorption Methods 0.000 description 1
- 239000002574 poison Substances 0.000 description 1
- 231100000614 poison Toxicity 0.000 description 1
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 1
- 150000003233 pyrroles Chemical class 0.000 description 1
- 239000000700 radioactive tracer Substances 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 230000000630 rising effect Effects 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 239000000741 silica gel Substances 0.000 description 1
- 229910002027 silica gel Inorganic materials 0.000 description 1
- IZTQOLKUZKXIRV-YRVFCXMDSA-N sincalide Chemical compound C([C@@H](C(=O)N[C@@H](CCSC)C(=O)NCC(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC=1C=CC=CC=1)C(N)=O)NC(=O)[C@@H](N)CC(O)=O)C1=CC=C(OS(O)(=O)=O)C=C1 IZTQOLKUZKXIRV-YRVFCXMDSA-N 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 230000006641 stabilisation Effects 0.000 description 1
- 238000013112 stability test Methods 0.000 description 1
- 238000011105 stabilization Methods 0.000 description 1
- 239000004575 stone Substances 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 230000007704 transition Effects 0.000 description 1
- 229910052724 xenon Inorganic materials 0.000 description 1
- FHNFHKCVQCLJFQ-UHFFFAOYSA-N xenon atom Chemical compound [Xe] FHNFHKCVQCLJFQ-UHFFFAOYSA-N 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C01—INORGANIC CHEMISTRY
- C01B—NON-METALLIC ELEMENTS; COMPOUNDS THEREOF; METALLOIDS OR COMPOUNDS THEREOF NOT COVERED BY SUBCLASS C01C
- C01B32/00—Carbon; Compounds thereof
- C01B32/15—Nano-sized carbon materials
- C01B32/182—Graphene
- C01B32/184—Preparation
-
- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
- C09K11/00—Luminescent, e.g. electroluminescent, chemiluminescent materials
- C09K11/08—Luminescent, e.g. electroluminescent, chemiluminescent materials containing inorganic luminescent materials
- C09K11/65—Luminescent, e.g. electroluminescent, chemiluminescent materials containing inorganic luminescent materials containing carbon
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- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Inorganic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Materials Engineering (AREA)
- Nanotechnology (AREA)
- Carbon And Carbon Compounds (AREA)
- Luminescent Compositions (AREA)
Abstract
The present invention relates to a kind of methods preparing the luminous carbon dots of hypotoxicity multi-mode based on functional polypeptide, using reduced glutathione as carbon source, nitrogen source, sulphur source, using N2It is pyrolyzed and prepares under atmosphere, thermal decomposition product is ground, ultrasonic disperse, suction filtration, vacuum drying can obtain carbon dots powder in water.With in the made luminous carbon dots of the method, impurities atom N, S has played important function in carbon dots luminescent behavior, this makes in service band, both exists and excites independent wave band there is also excitation dependence wave band, this will widen its application range significantly.In addition, being widely present in vivo due to reduced glutathione, contains great amount of hydroxy group, carboxyl etc. and the stronger group of organism compatibility by carbon dots surface obtained by carbon source of reduced glutathione so that it is water-soluble preferably, convenient for expanding biologic applications.
Description
Technical field
The invention belongs to micro-nano biotechnologies, and in particular to a kind of preparation of the fluorescent carbon point of hypotoxicity multimodes emitting
Method
Background technology
Fluorescent carbon point due to its excellent bio-compatibility, water solubility, can be repaiied as a kind of novel function nano material
The features such as decorations property, causes the extensive concern in the fields such as biomedicine, material science.Compared with conventional metals chalkogenide quantum dot,
It does not contain cadmium or other heavy metals, and serious health damage will not be caused to biosystem.But due to common carbon dots itself
Chemical attribute and the surface functional groups institute having is electrically charged and distribution, make its during biomarker to biological microenvironment and
Cell behavior can still cause potential threat and influence, such as the generation interference cell growth cycle of active oxygen.
Currently, the preparation of carbon dots mainly has two big methods, i.e. method and from bottom to top method from top to bottom, the former is generally basede on stone
The larger carbon structures such as black alkene, carbon pipe, carbon fiber, are cut or are peeled off into nanometer little particle, otherwise such method needs are special
The experiment conditions such as testing equipment or a large amount of strong acid-bases of needs, Strong oxdiative object, and low yield.The latter is directly original with organic matter
Material, carbon dots are prepared using modes such as hydro-thermal reaction, high temperature pyrolysis, and for this method since abundant raw material, type are various, equipment tests item
Part requires relatively low, has been used as main way to prepare carbon dots, but the made carbon dots of such method be generally separated it is more difficult and past
It is further passivated toward needs to improve fluorescent stabilization performance.In addition, the fluorescent carbon point prepared by this kind of bottom-to-top method,
The luminescent behavior that one mode is often only presented in entire service band, that is, excite dependent behavior.
Recent lot of documents report points out, element doping become it is a kind of be used for regulating and controlling carbon dots luminescent behavior have efficacious prescriptions
Formula.Such as：A kind of nitrogen phosphate and sulfur doping of Chinese patent document CN104987863A or codope carbon dots and its batch controllable preparation
Carbon source, nitrogen source, phosphorus source and sulphur source are uniformly mixed by methods and applications, obtain mixture, and the molar ratio of C, N, P, S are in mixture
1:(0~0.8):(0~0.4):(0~0.4), and be 0 when the content difference of N, P, S, mixture is heated in air atmosphere
To melting, reaction product silica gel chromatograph post separation is removed unreacted original by cooled to room temperature after reacting 3~60min
Material to get to nitrogen phosphate and sulfur doping or codope carbon dots.Although this method has effectively achieved element doping, but its carbon dots system
Carbon source composition involved in standby process is excessive, this is but also the separating technology of next step product becomes complicated.In addition its air atmosphere
Under preparation condition the oxygen-containing groups of a large amount of different shapes will be introduced on its carbon dots surface, such surface features are in biologic applications
In will generate larger oxidative pressure (active oxygen in biosystem to biosystem especially in being related to molecular biology field
Generate), this will be unfavorable for the expansion of biologic applications.
Chinese patent document CN106929014A discloses a kind of preparation of solid state fluorescence carbon dots and its in latent fingerprint detection
The application of aspect belongs to materials synthesis and fingerprinting detection technique field.The present invention solid state fluorescence carbon dots, be with glutathione
Nitrogen/sulphur source, citric acid are that carbon source is synthesized using microwave method, and materials safety is easy to get, and preparation method is easy quickly, particle safety nothing
Poison.Although the method is accelerated using conventional aqueous microwave method aggregate velocity, member is adulterated in its reaction process GSH-PX activity
The incorporation degree of element is not high, is distributed in surface group more so that effect of the doped chemical in luminescent behavior regulation and control is significantly
Weaken so that its luminous characteristics of the method products therefrom have single weak excitation dependent behavior.In addition its reaction atmosphere is still
For air atmosphere, still there are problems that Surface oxygen-containing groups transition introducing here, the hidden danger of oxidative pressure will limit its
Application in many biosystems.
Invention content
In view of the deficiencies of the prior art, bio-safety hidden danger and hair existing for the common carbon dots that especially prepared by existing method
The problems such as light behavior is single, the present invention, as carbon source, while also being made using a large amount of existing reduced glutathiones in organism
For nitrogen source and sulphur source, in N2Under atmosphere, by high temperature pyrolysis self-assembling method, the introducing of the stringent excessive oxygen-containing group of limitation is real
The luminous carbon dots of multi-mode are prepared in effective incorporation of existing impurity element.This method is easy to operate, without any in whole preparation process
The fluorescent carbon point of environmental pollution, preparation has higher yield and good bio-compatibility.
Technical scheme is as follows：
A method of the luminous carbon dots of hypotoxicity multimode being prepared based on functional polypeptide, using reduced glutathione as carbon
Source, nitrogen source and sulphur source, deionized water are solvent, are obtained in a nitrogen atmosphere to reduced glutathione pyrolysis, purification.
, according to the invention it is preferred to, based on the method that functional polypeptide prepares the luminous carbon dots of hypotoxicity multimode, including step
It is as follows：
(1) by reduced glutathione powder in N2Atmosphere high temperature is pyrolyzed；
(2) it is add to deionized water supersound process after grinding the thermal decomposition product that step (1) obtains, obtains turbid solution；
(3) turbid solution is filtered, filtrate obtains fluorescent carbon point solution through dialysis；
(4) by the concentration of fluorescent carbon point solution, drying to get the luminous carbon dots of hypotoxicity multimode.
, according to the invention it is preferred to, N in step (1)2Atmosphere high temperature pyrolysis process is：Use N2Progress displaced air, and
It is pyrolyzed under normal pressure.As pressure rising can take extraction N in enclosure space in being pyrolyzed temperature-rise period2Mode keep normal
Pressure.
, according to the invention it is preferred to, the temperature being pyrolyzed in step (1) is 240-260 DEG C, pyrolysis time 1-3h；Pyrolysis
It is down to room temperature naturally afterwards, temperature-fall period does not intervene enclosure space pressure change.
, according to the invention it is preferred to, the relative molecular mass of the reduced glutathione described in step (1) is
307.32。
, according to the invention it is preferred to, a concentration of 0.8-1.2mg/mL of turbid solution described in step (2)；
Preferably, the ultrasonic power of the supersound process is 200W, frequency 80KHz.
, according to the invention it is preferred to, the aperture of filtering filter membrane used is 0.22 μm in step (3)；
Preferably, the molecular cut off of dialysis is 500Da, dialysis time 20-25h.
, according to the invention it is preferred to, dry temperature described in step (4) is 45-50 DEG C.
According to the present invention, a kind of preferred embodiment, including steps are as follows：
(1) it takes reduced glutathione to be transferred to porcelain boat and shares uniformly；It is then charged into after being sealed in atmosphere tube type stove
Use N2It rinses 10 times, last N2Atmosphere keeps normal pressure, is warming up to 240-260 DEG C with 5 DEG C/min rates, is down to naturally after being pyrolyzed 2h
Room temperature；
(2) thermal decomposition product of step (1) is transferred to grind into powder state in mortar from taking-up in porcelain boat；Then disperse
In deionized water, it is ultrasonically treated 30min, ultrasonic power 200W, frequency 80KHz obtain turbid solution；
(3) the turbid solution suction filtration after supersound process is detached into obtain filter residue and filtrate, 0.22 μm of filter sizes；Filtrate is placed in
In bag filter, bag filter is put into deionized water by molecular cut off 500Da, and dialysis Inner liquid is taken after the 24-48h that dialyses, is obtained glimmering
Light carbon dots solution；
(4) it will be transferred in vacuum drying chamber and be dried after fluorescent carbon point solution concentrated by rotary evaporation, drying temperature：40-50
DEG C, drying time 12-24h obtains solid carbon dots powder.
The present invention is raw material using the reduced glutathione being widely present in organism, with anti-oxidant and integration solution
Toxic action.Glutathione is made of glutamic acid, cysteine and glycine, it means that itself has contained N, S element and rich
Rich bio-compatibility functional group.Therefore, the carbon dots prepared by this method have the characteristics that low toxicity and bio-compatibility are strong, and
Its luminescent behavior is regulated and controled by foreign atom again so that optics behavior is different from conventional fluorescent carbon dots, this characteristic contributes in life
Object technical applications play the effect of bigger.
Solid carbon dots powder produced by the present invention is dispersed among in water, three months or more is can save under the conditions of 4 DEG C without coagulation,
Longer time can save at 4 DEG C for obtained solid carbon dots powder after drying.
Beneficial effects of the present invention：
The present invention, to potential problems such as the issuable oxidative pressures of biosystem, chooses reduction for the conventional carbon dots that prepare
Property glutathione as carbon source, under oxygen-free environment pyrolysis be assembled into the laminar carbon dots with graphite-phase, even size distribution,
Structure is no different with graphene quantum dot, and surface is low with water-soluble and the preferable functional group of bio-compatibility, toxicity.In carbon dots
In operating wavelength range, both existed and excited independent wave band there is also exciting to rely on wave band, to sum up feature can widen carbon dots in cell
The application of multiple biological fields such as imaging, Nano medication load tracer.
Description of the drawings
Fig. 1 is the TEM photos of 2 gained carbon dots of the embodiment of the present invention.
Fig. 2 is the high-resolution TEM photos of 2 gained carbon dots of the embodiment of the present invention.
Fig. 3 is the grain size distribution of prepared carbon dots in the embodiment of the present invention 2.
Fig. 4 is the TEM photos of 1 gained carbon dots of comparative example of the present invention.
Fig. 5 is the TEM photos of 2 gained carbon dots of comparative example of the present invention.
Fig. 6 is the atomic force microscopy (AFM) of carbon dots made from the embodiment of the present invention 2.
Fig. 7 is the height distribution map of the atomic force microscopy selected location of carbon dots made from the embodiment of the present invention 2.
Fig. 8 is the Absorption and emission spectra of prepared carbon dots in the embodiment of the present invention 2；Wherein：A, abosrption spectrogram, b, c,
D is launching light spectrogram.
Fig. 9 is photoelectron spectroscopy (XPS) figure of prepared carbon dots in the embodiment of the present invention 2.Wherein：A, the full spectrograms of XPS, b,
The swarming figure of C1s, the swarming figure of c, N1s, the swarming figure of d, O1s, the swarming figure of e, S2p.
Figure 10 is the light stability test figure of prepared carbon dots in the embodiment of the present invention 2.
Figure 11 is the cytotoxicity test figure of prepared carbon dots in the embodiment of the present invention 2.
Figure 12 is the cell imaging picture of prepared carbon dots in the embodiment of the present invention 2.
Specific implementation mode
Technical solution of the present invention is further described with reference to specific embodiment, but not limited to this.
Characterization in embodiment：X-ray photoelectron spectroscopy (XPS), X-ray diffraction are carried out to the carbon dots powder of acquisition
(XRD) phenetic analysis, specific sample morphology, which is subject to, meets test equipment.Carbon dots morphology observation choice of powder sample be dissolved in from
Aqueous solution after sub- water ultrasound 5-10min carries out transmission electron microscope (TEM) respectively, atomic force microscope (AFM) characterizes carbon
The horizontal and vertical Size Distribution of point, Fluorescence Spectrometer and ultraviolet-uisible spectrophotometer characterize its optical property respectively.Knot
By：The carbon dots prepared with the method are flaky, and water dispersible is preferable, even size distribution, have multi-mode luminous characteristics.
The pyrolysis of 1 functional polypeptide of embodiment prepares the preparation of water-soluble carbon dots
500mg reduced glutathiones are packed into porcelain boat and are shared uniformly, moves into atmosphere tube type stove and closes, vacuumize and fill
N2It is repeated 10 times and finally keeps normal pressure N2Atmosphere.240 DEG C are warming up to 5 DEG C/min of heating rate, heat preservation is dropped naturally after 2 hours
To room temperature, carbonarius thermal decomposition product is transferred to grind into powder state in mortar.Then this powder is distributed to 40-50mL
Ultrasound 30min, ultrasonic power 200W, frequency 80KHz obtain turbid solution in ionized water；By gained turbid solution aperture
0.22 μm of filter membrane suction filtration detaches to obtain light yellow transparent solution；This solution is fitted into 500Da bag filters and is dialysed for 24 hours, will be dialysed
Dry 12h under the conditions of 50 DEG C of vacuum drying chamber of immigration after Inner liquid concentrated by rotary evaporations；Finally obtain water soluble fluorescence carbon dots powder.
The pyrolysis of 2 functional polypeptide of embodiment prepares the preparation of water-soluble carbon dots
500mg reduced glutathiones are packed into porcelain boat and are shared uniformly, moves into atmosphere tube type stove and closes, vacuumize and fill
N2It is repeated 10 times and finally keeps normal pressure N2Atmosphere.250 DEG C are warming up to 5 DEG C/min of heating rate, heat preservation is dropped naturally after 2 hours
To room temperature, carbonarius thermal decomposition product is transferred to grind into powder state in mortar.Then this powder is distributed to 40-50mL
Ultrasound 30min, ultrasonic power 200W, frequency 80KHz obtain turbid solution in ionized water；By gained turbid solution aperture
0.22 μm of filter membrane suction filtration detaches to obtain light yellow transparent solution；This solution is fitted into 500Da bag filters and is dialysed for 24 hours, will be dialysed
Dry 12h under the conditions of 50 DEG C of vacuum drying chamber of immigration after Inner liquid concentrated by rotary evaporations；Finally obtain water soluble fluorescence carbon dots powder.
The pyrolysis of 3 functional polypeptide of embodiment prepares the preparation of water-soluble carbon dots
500mg reduced glutathiones are packed into porcelain boat and are shared uniformly, moves into atmosphere tube type stove and closes, vacuumize and fill
N2It is repeated 10 times and finally keeps normal pressure N2Atmosphere.260 DEG C are warming up to 5 DEG C/min of heating rate, heat preservation is dropped naturally after 2 hours
To room temperature, carbonarius thermal decomposition product is transferred to grind into powder state in mortar.Then this powder is distributed to 40-50mL
Ultrasound 30min, ultrasonic power 200W, frequency 80KHz obtain turbid solution in ionized water；By gained turbid solution aperture
0.22 μm of filter membrane suction filtration detaches to obtain light yellow transparent solution；This solution is fitted into 500Da bag filters and is dialysed for 24 hours, will be dialysed
Dry 12h under the conditions of 50 DEG C of vacuum drying chamber of immigration after Inner liquid concentrated by rotary evaporations；Finally obtain water soluble fluorescence carbon dots powder.
Comparative example 1
As described in Example 2, unlike：
Pyrolytic process is to be warming up to 200 DEG C with 5 DEG C/min of heating rate.
Comparative example 2
As described in Example 2, unlike：
Pyrolytic process is to be warming up to 300 DEG C with 5 DEG C/min of heating rate.
Comparative example 3
As described in Example 2, unlike：
Pyrolysis feed is L-cysteine, it is one of main composition of glutathione.
3 obtained product of comparative example is almost without fluorescence.Infer that reason may be single amino according to its unstressed configuration phenomenon
Sour structure its smaller unit will bear higher steric hindrance in self assembling process under pyrolytic conditions can not be effectively from group
Dress up fluorescent carbon point.Thus it can also show that this method for pyrolysis synthesis fluorescent carbon point has raw molecule size and its surface state
It is selective.
Test example 1
The product of embodiment 2, comparative example 1-2 is made into the aqueous solution and ultrasonic disperse of carbon dots concentration 0.05mg/mL, to upper
It states three samples and carries out TEM morphology observations, as shown in Fig. 1,2,3,4,5.
By Fig. 1,2,3 it is found that 2 gained carbon dots of the embodiment of the present invention are well dispersed, even size distribution, grain size again smaller than
5nm and there is graphite-phase lattice fringe.
As shown in Figure 4, the larger (≤20nm of carbon dots particle size that comparative example 1 obtains) and there is reunion.
As shown in Figure 5,2 gained carbon dots Size Distribution of comparative example is uneven.
Therefore, pyrolysis temperature has vital influence for carbon dots quality.Pyrolysis temperature is too low, excessively high is all unfavorable for
To the carbon dots of high quality.
Test example 2
The atomic force microscope images of carbon dots are made in testing example 2, as shown in Figure 6.The height distribution of carbon dots is observed, such as
Shown in Fig. 7.In conjunction with the lateral dimension distribution in Fig. 3, it is known that this carbon dots is existed with platelet-type morphology, shows temperature model of the present invention
Carbon dots obtained by lower pyrolysis are enclosed since its specific surface state makes its favorable dispersibility, appearance and size all be conducive in biological skill
Carry out application in art field.
Test example 3
Carbon dots made from embodiment 2 are made into the aqueous solution of carbon dots concentration 0.05mg/mL, carry out ultravioletvisible absorption and glimmering
Light spectrum property is tested.As shown in Figure 8.Wherein：A, abosrption spectrogram, b, c, d are launching light spectrogram.
As shown in Figure 8, for excitation wavelength in 300-340nm wave bands, carbon dots luminescent behavior shows as excitation independently, transmitted wave
It is long to be located at 382nm；After excitation wavelength is more than 340nm, in 360-420nm wave bands, carbon dots luminescent behavior shows as excitation and relies on,
Moving range is smaller, and launch wavelength is near 450nm；And when Ji hair Bo Chang≤430nm, excitation dependent behavior is still presented, and move
Dynamic wider range.
Test example 4
The x-ray photoelectron spectroscopy of carbon dots made from testing example 2, as shown in Figure 9；Wherein：A is the full spectrograms of XPS, from
It can be seen from the figure that, prepared carbon dots contain carbon, nitrogen, oxygen, element sulphur；B is the swarming figure of C1s, and wherein 284.5ev indicates C-
C/C=C exists, and 285.4ev indicates that C-N/C-O/C-S exists, and 287.7ev indicates that C=O/C=N exists；C is the swarming of N1s
Figure, wherein 399ev indicate that the presence of pyridine nitrogen, 399.7ev indicate that the presence of pyrroles's nitrogen, 402.3ev indicate the presence of N-H；D is
The swarming figure of O1s, wherein 530.98ev indicate that C-O-C/C-OH exists, and 531.8ev indicates that the presence of C=O, 533.92ev indicate
The presence of oxygen physisorption；E is the swarming figure of S2p, and wherein 163.4ev and 164.5ev indicates thiophenic sulfur (- C-S-) respectively
2P3/2With 2P1/2Presence, 168ev indicate O-S presence.
As shown in Figure 9, gained carbon dots are mainly made of tetra- kinds of elements of C, N, O, S under 2 pyrolytical condition of embodiment, adulterate N, S
The presence of element shows that N, S element in reduced glutathione have been enter into inside carbon dots, and has been produced to the fluorescence property of carbon dots
Raw important regulating and controlling effect (multi-mode shines).
Test example 5
The photostability of carbon dots solution is made in testing example 2, and the product of embodiment 2 is made into carbon dots concentration 0.05mg/
The aqueous solution of mL is subsequently placed in and its fluorescence property is irradiated and measured under 450W xenon lamps, as shown in Figure 10, excitation wavelength 350nm,
Detection wavelength is 450nm, it is known that the fluorescence intensity of carbon dots is held essentially constant, and photobleaching phenomenon does not occur.
Test example 6
The cytotoxicity of carbon dots made from testing example 2, is tested, as a result as shown in figure 11 by CCK-8 kits.By
Figure 11 it is found that under at concentrations up to 600 μ g/mL to Hela cell culture 48h after cell activity still 80% or more, illustrate made
It is relatively low to obtain carbon dots cytotoxicity.
Test example 7
Carbon dots made from embodiment 2 are used for mouse bone marrow cells source Dendritic Cells fluorescence imaging with the concentration of 200 μ g/mL
(culture 1h), as shown in figure 12.As shown in Figure 12, imaging effect is good.
Claims (10)
1. a kind of method preparing the luminous carbon dots of hypotoxicity multimode based on functional polypeptide, which is characterized in that this method is to restore
Type glutathione is carbon source, nitrogen source and sulphur source, and deionized water is solvent, reduced glutathione is pyrolyzed in a nitrogen atmosphere,
Purification obtains.
2. the method according to claim 1 for preparing the luminous carbon dots of hypotoxicity multimode based on functional polypeptide, including step
It is as follows：
(1) by reduced glutathione powder in N2Atmosphere high temperature is pyrolyzed；
(2) it is add to deionized water supersound process after grinding the thermal decomposition product that step (1) obtains, obtains turbid solution；
(3) turbid solution is filtered, filtrate obtains fluorescent carbon point solution through dialysis；
(4) by the concentration of fluorescent carbon point solution, drying to get the luminous carbon dots of hypotoxicity multimode.
3. the method according to claim 2 for preparing the luminous carbon dots of hypotoxicity multimode based on functional polypeptide, feature exist
In N in step (1)2Atmosphere high temperature pyrolysis process is：Use N2Displaced air is carried out, and is pyrolyzed under normal pressure.
4. the method according to claim 2 for preparing the luminous carbon dots of hypotoxicity multimode based on functional polypeptide, feature exist
In the temperature being pyrolyzed in step (1) is 240-260 DEG C, pyrolysis time 1-3h.
5. the method according to claim 2 for preparing the luminous carbon dots of hypotoxicity multimode based on functional polypeptide, feature exist
In the relative molecular mass of the reduced glutathione described in step (1) is 307.32.
6. the method according to claim 2 for preparing the luminous carbon dots of hypotoxicity multimode based on functional polypeptide, feature exist
In a concentration of 0.8-1.2mg/mL of turbid solution described in step (2).
7. the method according to claim 2 for preparing the luminous carbon dots of hypotoxicity multimode based on functional polypeptide, feature exist
In the ultrasonic power being ultrasonically treated described in step (2) is 200W, frequency 80KHz.
8. the method according to claim 2 for preparing the luminous carbon dots of hypotoxicity multimode based on functional polypeptide, feature exist
In the aperture of filtering filter membrane used is 0.22 μm in step (3).
9. the method according to claim 2 for preparing the luminous carbon dots of hypotoxicity multimode based on functional polypeptide, feature exist
In the molecular cut off dialysed in step (3) is 500Da, dialysis time 20-25h.
10. the method according to claim 2 for preparing the luminous carbon dots of hypotoxicity multimode based on functional polypeptide, feature exist
In dry temperature described in step (4) is 45-50 DEG C.
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