CN107857248A - Preparation method, carbon-based nano particle and its application of carbon-based nano particle - Google Patents
Preparation method, carbon-based nano particle and its application of carbon-based nano particle Download PDFInfo
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- CN107857248A CN107857248A CN201711057803.0A CN201711057803A CN107857248A CN 107857248 A CN107857248 A CN 107857248A CN 201711057803 A CN201711057803 A CN 201711057803A CN 107857248 A CN107857248 A CN 107857248A
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Abstract
The invention provides a kind of preparation method of carbon-based nano particle, including：(1) blade of deciduous tree is carbonized to obtain carbon source；(2) oxidant is added in carbon source and carries out oxidation reaction, be then centrifugally separating to obtain supernatant liquor；(3) supernatant liquor is filtered, collects filtrate；(4) filtrate is dried, obtains carbon-based nano particle.Present invention also offers the carbon-based nano particle prepared using preceding method, and the application of the carbon-based nano particle in bioanalysis.
Description
Technical field
The invention belongs to field of nanometer material technology, in particular it relates to a kind of preparation method of carbon-based nano particle, by
The application of carbon-based nano particle and the carbon-based nano particle made from this method.
Background technology
The synthesizing new biomedicine that is found to be of c-based nanomaterial with photoluminescent property is brought newly with fluorescent material
Hope.Because c-based nanomaterial has very high chemical inertness, good biocompatibility, and very low cell toxicant
Property, thus it is very likely turned into the alternative materials of the fluorescence quantum based on metallic element in biomedical sector.
At present, the method for synthesizing carbon-based nano particle mainly has laser ablation method, pyrolysismethod, electrolytic oxidation etc., still
These method generally existings need the carbon-based nano particle that the equipment of specialty, operating method are complicated, prepare exist fluorescence efficiency it is low,
The problems such as stability difference.
The content of the invention
The goal of the invention of the present invention is the defects of being directed to prior art, there is provided a kind of preparation side of carbon-based nano particle
The application of method, carbon-based nano particle obtained by this method and the carbon-based nano particle.
On the one hand, the invention provides a kind of preparation method of carbon-based nano particle, comprise the following steps：
(1) blade of deciduous tree is carbonized to obtain carbon source；
(2) oxidant is added in carbon source and carries out oxidation reaction, be then centrifugally separating to obtain supernatant liquor；
(3) supernatant liquor is filtered, collects filtrate；
(4) filtrate is dried, obtains carbon-based nano particle.
On the other hand, the invention provides a kind of carbon-based nano particle, it uses above-mentioned preparation method to be made.
On the other hand, the application the invention provides carbon-based nano particle in bioanalysis.
Technical scheme has the advantages that：
The method of the present invention is mitigating treatment of urban garbage burden using the fallen leaves blade of common deciduous tree as raw material
Develop new application material simultaneously.The method of the present invention can obtain carbon-based nano by low-temperature carbonization and acid activation oxidation
Particle, technique is simple, mild condition.
The carbon-based nano particle of the present invention is the carbon-based nano-particle of water soluble fluorescence, has the maximum excitation close to visible ray
Wavelength, at least two independent fluorescent emission sites be present, and there is good ph stability, photostability and ionic strength
Stability, so that it can be widely applied in bioanalysis.
Brief description of the drawings
Fig. 1 is the XRD diffraction spectrograms of the blade of white birch and weeping willow, wherein, (a) is the fallen leaves blade of white birch, and (b) is weeping willow
Fallen leaves blade.
Fig. 2 is the C of the blade of white birch and weeping willow1sXPS spectrum figure, wherein, the C of the fallen leaves blade of white birch and weeping willow1sXPS spectrum
Scheme essentially identical, the two coincidence, therefore only show a curve.
Fig. 3 is transmission electron microscope photo, wherein, (a) is Def-CNPs-1 transmission electron microscope photo, and (b) is Def-CNPs-2
Transmission electron microscope photo.
Fig. 4 is fluorescence excitation and launching light spectrogram, wherein, ex1 and em1 represent exciting for the Def-CNPs-1 aqueous solution respectively
And emission spectrum, ex2 and em2 represent the excitation and emission spectra of the Def-CNPs-2 aqueous solution respectively.
Fig. 5 shown with multiple results for exciting peak value to excite sample to obtain in PLE, wherein, (a) is Def-
CNPs-1 result, (b) are Def-CNPs-2 results.
Fig. 6 shows that increase 10nm gradually excites the knot of Def-CNPs-1 samples every time since 320nm by excitation wavelength
Fruit.
Fig. 7 shows that increase 10nm gradually excites the knot of Def-CNPs-2 samples every time since 320nm by excitation wavelength
Fruit.
Fig. 8 shows the fluorescence of sample with the situation of change of solution ph, wherein, (a) is Def-CNPs-1 change feelings
Condition, (b) are Def-CNPs-2 situations of change.
Fig. 9 shows the result that real-time fluorescence test is carried out to sample.
Figure 10 shows the change of sample fluorescence intensity in the sodium-chloride water solution of various concentrations, wherein, (a) is Def-
CNPs-1 result, (b) are Def-CNPs-2 results.
Figure 11 shows the bioactivity testing result of sample.
Figure 12 is Laser Scanning Confocal Microscope photo of the sample with blank control to human embryonic kidney cell, wherein, (a) is Def-
For CNPs-1 to the Laser Scanning Confocal Microscope photo of human embryonic kidney cell, (b) is that copolymerization of the Def-CNPs-2 to human embryonic kidney cell is burnt
Microphotograph, (d) are Laser Scanning Confocal Microscope photo of the blank control to human embryonic kidney cell.
Embodiment
In order to be fully understood by the purpose of the present invention, feature and effect, by following embodiments, the present invention is made detailed
Describe in detail bright.For the process of the present invention in addition to the description below, remaining uses the conventional method or device of this area.Following nouns
Term is unless otherwise stated, be respectively provided with the implication that those skilled in the art are generally understood that.
In a first aspect, the invention provides a kind of preparation method of carbon-based nano particle, using the blade of deciduous tree as carbon
Source, by low-temperature carbonization and the process of oxidation processes, it is prepared for the carbon-based nano-particle of fluorescence with good aqueous solubility.In carbon
During change, cellulose and protein molecule in leaf are pyrolyzed, and self structure is destroyed and forms carbon distribution.Carbon distribution sample
It is oxidized in oxidant, the particle reunited together is decomposed to form independent carbon-based nano particle, and is scattered in the aqueous solution
In.Specifically, the preparation method of carbon-based nano particle of the invention comprises the following steps：
(1) blade of deciduous tree is carbonized to obtain carbon source
Deciduous tree is the arbor all to be come off in annual autumn and winter or arid season leaf, and the fallen leaves for referring generally to temperate zone are tall
Wood.The fallen leaves of deciduous tree are mainly made up of cellulose and a small amount of protein, wherein containing substantial amounts of C, H, O element and on a small quantity
The content of the essential elements such as the N, metallic element of Si elements and denier, C, H, O accounts for more than the 90% of fallen leaves gross mass.In this hair
Mainly the method for the present invention is illustrated using the blade of white birch and weeping willow as raw material in bright it should be appreciated that this
It is exemplary.
Inventor has found that carbon mainly exists with graphite-structure in the blade of deciduous tree by studying, and is specifically
Mainly with sp2The graphite-like structure that hydridization is formed is present.As shown in figure 1, in the XRD diffraction spectrograms of the blade of white birch and weeping willow all
An only broad graphite peaks are along with an acromion, and stone mill peak is located at 26.5 °, and acromion is located at 43.5 °, respectively close to stone
(002) and (010) crystal face of ink, illustrate that carbon mainly exists with the structure of graphite in both fallen leaves.In addition, such as Fig. 2 institutes
Show, the C of the fallen leaves of white birch and weeping willow1sXPS spectrum figure is essentially identical, and an only main peak is located at 284.6eV, and is combined with to height
The slight tailing peak in energy region, illustrates the carbon in two kinds of blades mainly with sp2The graphite-like structure that hydridization is formed is present,
This from XRD with characterizing it is concluded that consistent.There is the characteristics of cost is cheap, simple and easy to get using this raw material, and fall
The graphite-like structure of carbon advantageously forms the structure carbon-based nano particle stable with photoluminescent property in leaf.
Preferably, before being carbonized, first fallen leaves are cleaned up with deionized water, and removes petiole and only retains leaf
Piece.
Preferably, carbonization refers to be carbonized in 300 DEG C of -600 DEG C of constant temperature 1-4 hours.It is highly preferred that the carbonization includes
Following steps：The blade of deciduous tree is placed in tube furnace, in a nitrogen atmosphere, by temperature with 1-10 DEG C/min heating speed
Degree rises to 300 DEG C -600 DEG C, and constant temperature 1-4 hours by room temperature, is then cooled to room temperature, that is, obtains carbon source.
In a kind of particularly preferred embodiment, blade is placed in tube furnace, in the nitrogen gas slowly flowed
Under atmosphere, 400 DEG C are risen to from room temperature with 1 DEG C/min programming rate, room temperature is naturally cooled to after 3 hours in 400 DEG C of constant temperature.
(2) oxidation reaction
Oxidant is added in the carbon source prepared in above-mentioned steps (1) and carries out oxidation reaction.Oxidant can be nitric acid,
The Binary Mixtures of 98% concentrated sulfuric acid or 98% concentrated sulfuric acid with 8mol/L nitric acid according to volume ratio for 1: 1-1: 5 formation.Its
In, nitric acid is preferably the nitric acid that concentration is 4-10mol/L, and more preferably concentration is 5mol/L nitric acid.According to carbon source and oxygen
The w/v of agent is 1-10g: 100-500mL oxidant to be added in carbon source, stirring reaction 6-48 hours.With laggard
Row is centrifuged, and obtained supernatant liquor enters to be operated in next step.
Preferably, in order to further improve the yield of carbon-based nano particle, oxidation reaction can be carried out several times, such as be divided
Carry out twice, i.e., oxidant is added to progress first time oxidation reaction in carbon source according to the above ratio, reaction is carried out after completing
Centrifuge, obtain the first supernatant liquor and solid precipitation, then, add oxidant in being precipitated in proportion to solid again and carry out
Second of oxidation reaction, reaction are centrifuged after completing, and the second supernatant liquor are obtained, by the first supernatant liquor and second
Supernatant liquor merges, and then enters back into and operates in next step.
In a kind of particularly preferred embodiment, the fallen leaves (i.e. carbon source) for taking 1g to be carbonized are placed in round-bottomed flask,
Add 100mL 5mol/L nitric acid to be stirred at room temperature 6 hours, resulting solution centrifuges, and the black precipitate of collection adds
Reaction 6 hours is stirred at room temperature in 5mol/L nitric acid, and resulting solution centrifuges again, and the upper strata being centrifugally separating to obtain twice is clear
Liquid merges.When containing 98% sulfuric acid in oxidant, added and the equimolar barium chloride solution of sulfuric acid into filtrate.
(3) filter
The supernatant liquor being centrifugally separating to obtain in step (2) is filtered and collects filtrate.Preferably, use is fixed at a slow speed
Amount filter paper is filtered.Wherein, quantitative filter paper refers to the filter paper that aperture is 1-3 microns at a slow speed.
(4) dry
Filtrate is dried, obtains carbon-based nano particle.The present invention does not have particular/special requirement to dry method, every normal
Drying means, such as freeze-drying, rotary evaporation etc., are used equally in the present invention.Those skilled in the art are according to reality
It is required to the suitable drying means of reasonable selection.
Generally, it is considered that, it is necessary to the pH value of filtrate is adjusted to neutral range (such as pH value be 7) after filtration step,
Then dialysed, can ensure that obtained carbon-based nano particle has photoluminescent property steady in a long-term, including ph stability
Property, ion stability, photostability, and the property by excited by visible light, so that it can be used in such as bioluminescence mark
In the biological detection of note.But inventor omits regulation pH value and thoroughly by studying discovery beyond expectation, after filtration step
Analysis operation, directly filtrate is dried and prepares carbon-based nano particle, due to the oxidizing acid in filtrate in the drying process by
Thermally decompose and steam with moisture, be not only able to rapidly remove unnecessary reactant from carbon-based nano particle, and also retains
The particle-stabilised photoluminescent property of carbon-based nano, and reduce the loss that dialysis operation causes carbon-based nano particle, so that carbon
The preparation method of base nano-particle is easier, and improves yield.
Second aspect, the invention provides a kind of carbon-based nano particle, it is made using the preparation method of first aspect.
The carbon-based nano particle of the present invention is spherical in shape, and particle diameter is 2-8nm, and spacing of lattice is 0.32-0.34nm, close to graphite
(002) crystal face, illustrate a certain degree of graphite-like structure in carbon-based nano particle be present, i.e., in carbonisation to a certain degree
On save the graphite-like structure of fallen leaves carbon itself.
The maximum excitation wavelength of the carbon-based nano particle of the present invention is 320-420nm, is sufficiently close to visible-range.This hair
Bright carbon-based nano particle has at least two fluorescent emission sites, for example, two kinds, three kinds, four kinds.
In addition, the carbon-based nano particle of the present invention has good ph stability, photostability and ionic strength stable
Property.
It is spherical in shape the invention provides a kind of carbon-based nano particle in a kind of particularly preferred embodiment, directly
Footpath is 5nm, and spacing of lattice 0.33nm, maximum excitation wavelength is 391nm-394nm, and close to visible ray, it is only to have at least two
Vertical fluorescent emission site, and there is good ph stability, photostability and ionic strength stability.
Basic sign, photoluminescent property and the stability of carbon-based nano particle based on the present invention, the present invention is in the third aspect
Application of the carbon-based nano particle in bioanalysis is provided, wherein, bioanalysis is bioactivity detection and/or cell fluorescence
Mark.
Embodiment
The present invention is further illustrated below by the mode of embodiment, but does not therefore limit the present invention to described reality
Apply among a scope.The experimental method of unreceipted actual conditions in the following example, conventionally and condition, or according to business
Product specification selects.
Embodiment 1
White birch is fallen leaves and cleaned with deionized water, petiole is removed and only retains blade.Blade is placed in tube furnace, slow
Under the nitrogen atmosphere of flowing, 400 DEG C are risen to from room temperature with 1 DEG C/min programming rate, it is naturally cold after 400 DEG C of constant temperature 3 hours
But to room temperature.The fallen leaves for taking 1g to be carbonized are placed in round-bottomed flask, are added 100mL 5mol/L nitric acid and are stirred at room temperature 6 hours.Institute
Solution centrifugal separation is obtained, the black precipitate of collection adds 5mol/L nitric acid and reaction 6 hours is stirred at room temperature, and resulting solution is again
Centrifuge, the supernatant liquor being centrifugally separating to obtain twice is merged.Subsequent supernatant liquor is filtered with quantitative filter paper at a slow speed,
Gained filtrate rotary evaporation is dried, and obtained sample is labeled as Def-CNPs-1.
Embodiment 2
The method that embodiment 2 uses is same as Example 1, differs only in using weeping willow fallen leaves as raw material, finally obtains
Sample is labeled as Def-CNPs-2.
Embodiment 3
White birch is fallen leaves and cleaned with deionized water, petiole is removed and only retains blade.Blade is placed in tube furnace, slow
Under the nitrogen atmosphere of flowing, 600 DEG C are risen to from room temperature with 2 DEG C/min programming rate, it is naturally cold after 600 DEG C of constant temperature 2 hours
But to room temperature.The fallen leaves for taking 5g to be carbonized are placed in round-bottomed flask, are added 100mL 98% (weight) concentrated sulfuric acid and are stirred at room temperature 24
Hour.Resulting solution centrifuges, and collects supernatant liquor, adds 400mL 4.6moL/L barium chloride solution.Subsequent upper strata is clear
Liquid is filtered with quantitative filter paper at a slow speed, and gained filtrate rotary evaporation is dried, and obtained sample is labeled as Def-CNPs-3.
Embodiment 4
Weeping willow is fallen leaves and cleaned with deionized water, petiole is removed and only retains blade.Blade is placed in tube furnace, slow
Under the nitrogen atmosphere of flowing, 300 DEG C are risen to from room temperature with 1 DEG C/min programming rate, it is naturally cold after 300 DEG C of constant temperature 4 hours
But to room temperature.The fallen leaves for taking 8g to be carbonized are placed in round-bottomed flask, are added 98% concentrated sulfuric acid and are according to volume ratio with 8mol/L nitric acid
1:The 1 Binary Mixtures 300mL formed, is stirred at room temperature 24 hours.Resulting solution centrifuges, and collects supernatant liquor, adds
Enter 500mL 5.6mol/L barium chloride solution.Subsequent supernatant liquor is filtered with quantitative filter paper at a slow speed, gained filtrate rotary evaporation
Dry, obtained sample is labeled as Def-CNPs-4.
The structure of the carbon-based nano particle of experimental example one
Def-CNPs-1 and Def-CNPs-2 carbon-based nano particles are proceeded as follows respectively：Take the carbon-based nano of preparation
Particle about 10mg, 50 milliliters of chromatogram methanol are added, ultrasonic 5-10 minutes, disperse carbon-based nano uniform particle.Dispersion liquid is added dropwise
To the copper mesh of 400 mesh, next drop is added dropwise again after methanol drying, about 5-10 drops are added dropwise altogether.Then copper mesh is placed in vacuum
Sample is made in dry overnight at room temperature in drying box.
Obtained sample is delivered into Changchun applied chemistry study and carries out the inspection of JEOL JEM-3010 transmission electron microscopes
Survey, as a result as shown in Figure 3.
(a) and (b) is Def-CNPs-1 and Def-CNPs-2 transmission electron microscope photo respectively in Fig. 3.Can be with from photo
Find out, all samples it is spherical in shape, diameter is about 5nm.This it appears that Def-CNPs-1 and Def-CNPs-2 in photo
The spacing of lattice of sample, it is 0.33nm through measurement, close to (002) crystal face of graphite, illustrates to exist in sample a certain degree of
Graphite-like structure.Two kinds of samples save the graphite-like structure of fallen leaves carbon itself to a certain extent in carbonisation.
Above-mentioned detection has also been carried out to Def-CNPs-3 and Def-CNPs-4 carbon-based nanos particle, has finally determined Def-
CNPs-3 and Def-CNPs-4 diameter is 6nm and 8nm respectively, and spacing of lattice is 0.33nm and 0.32nm respectively.
The photoluminescent property of experimental example dicarbonyl nano-particle
Def-CNPs-1 and Def-CNPs-2 carbon-based nano particles are proceeded as follows respectively：Take 10mg carbon-based nano grains
Son dissolves and constant volume is in 2mL deionized waters.Sample drop is added in cuvette, sample volume is the 3/4 of cuvette volume.Will
Cuvette is put in XRF, and (XRF model Perkin-Elmer LS 55 used in this experiment, other have glimmering
The instrument of light spectral detection is used equally for this test) darkroom in, setting excite with launch wavelength scanning range, excite and launch
Slit width, speed is swept, click on test, record excitation and emission spectra.Wherein, excitation wavelength range 320-420nm；Launch wavelength
Scope 360-800nm；Excite with transmite slit width 2.5-15nm, sweep fast 100-500nm/min.
Fig. 4 is the fluorescence excitation and launching light spectrogram of Def-CNPs-1 the and Def-CNPs-2 aqueous solution.Can from figure
Go out, exciting for two kinds of samples is basically identical with emission spectrum peak shape, and emission spectra peak width is wealthy and symmetry is good, excites spectral peak pair
Title property is very poor, has obvious more excitation peak features.The maximum excitation wavelength of Def-CNPs-1 samples is 394nm, corresponding hair
It is 497nm to penetrate spectrum peak, and Def-CNPs-2 maximum excitation wavelength is 391nm, and corresponding transmitting spectrum peak is 476nm.By
It is easy to kill cell in ultraviolet light, so maximum excitation wavelength is close to visible ray, this feature makes carbon-based nano particle of the invention
More suitable for the application in terms of cell marking or pharmaceutical indications.
From figure 5 it can be seen that by taking Def-CNPs-1 as an example, the emission spectrum spectral peak for exciting to obtain with 354nm is equally wide
It is wealthy and there is good symmetry.But found after examining, with the spectral peak for exciting to obtain with maximum excitation wavelength (394nm)
Compare, 354nm excites the symmetry decrease to some degree of obtained peak shape, is equally existed so in Def-CNPs-2 samples
Phenomenon.After this explanation changes excitation wavelength, the response site in Def-CNPs-1 is changed, i.e., in carbon-based nano particle
It there may be a variety of fluorescent emission sites.
From fig. 6 it can be seen that when being excited with 320nm, transmitting peak intensity is very low, peak value 435nm.Work as excitation wavelength
Most strong emission peak 495nm is obtained when increasing to 390nm, and continue increase excitation wavelength when, transmitting peak-to-peak value continue red shift and
Intensity gradually reduces, during fluorometric investigation, when excitation wavelength increases or decreases certain numerical value, if the wavelength of emission peak
Also red shift or the blue shift of respective value occurs, then this emission peak is generally identified as sample to being dissipated caused by exciting light
Penetrate peak.In Def-CNPs-1 samples, excitation wavelength increase 110nm, launch wavelength red shift 95nm, it can exclude to be by sample
Caused by scattering, i.e. Def-CNPs-1 is implicitly present in the emission peak phenomenon dependent on excitation wavelength.This also illustrates Def-
A variety of luminescence sites in CNPs-1 samples be present.
For Def-CNPs-2 samples, such as Fig. 7, can lead to the same conclusion.It is worth noting that, when excitation wavelength by
Although when 330nm increases to 350nm sample fluorescence intensity gradually increase, excitation wavelength continue to increase to 360nm and
During 370nm, the fluorescence intensity of sample has but occurred substantially to reduce.Continuing increase excitation wavelength, then fluorescence intensity is increased up again
Reach most strong emission peak (476nm).It is this while excitation wavelength increase, fluorescence intensity occur two minor peaks phenomenon it is obvious
Different from Def-CNPs-1 samples, it is thought that because there are at least two independent fluorescence in inventor in Def-CNPs-2 samples
Caused by emission sites.And produce two peak values excitation wavelength just with two peak values in Def-CNPs-2 PLE
It is corresponding, illustrate that the two excite peak value to correspond to two kinds of maximum excitation energy for exciting site.
Above-mentioned detection has also been carried out to Def-CNPs-3 and Def-CNPs-4 carbon-based nanos particle, and the knot finally given
Fruit is consistent with above-mentioned Def-CNPs-1 and Def-CNPs-2 result.
The stability of the carbon-based nano particle of experimental example three
3.1 ph stability
Def-CNPs-1 and Def-CNPs-2 carbon-based nano particles are proceeded as follows respectively：The carbon for taking 100mg to prepare
Base nanoparticle dissolution is simultaneously settled in 10mL deionized waters, and 0.01mol/L sodium hydroxides (nitric acid) are added dropwise dropwise while stirring
Solution, the pH value of regulation system, the pH value of solution are determined with pH meter.As a result it is as shown in Figure 8.
As can be seen from Figure 8, change of the fluorescence of Def-CNPs-1 and Def-CNPs-2 samples for system pH becomes
Gesture is identical.When Def-CNPs-1 and Def-CNPs-2 samples are present in acid condition, its photoluminescence peak occurs a certain degree of
Blue shift, and intensity decreases with the enhancing of acidity.When being present in alkalescence condition, red shift then occurs for photoluminescence peak, by force
Degree substantially reduces with the enhancing of alkalescence.But no matter the acid-base value of system continues to increase or reduced, the emission wavelength of sample
No longer change, i.e., sample has fixed emission wavelength for Acid-Base System.Therefore the carbon-based nano particle of the present invention can be with
For qualitatively judging the acid-base property of residing system.
3.2 photostability
Use power to irradiate Def-CNPs-1 and Def-CNPs-2 samples for a long time for 8w uviol lamp, and use fluorescence light
Spectrometer (model Perkin-Elmer LS 55) carries out real-time fluorescence test to it, as a result as shown in Figure 9.
It can be seen in figure 9 that after Continuous irradiation 4 hours, the fluorescence of Def-CNPs-1 and Def-CNPs-2 samples is strong
For degree all without obvious quenching phenomenon occurs, illustrating the carbon-based nano particle of the present invention all has good photostability.
3.3 ionic strength stability
Detection architecture is used as using the sodium-chloride water solution of the various concentrations closer to human body environment.Respectively to Def-CNPs-1
Proceeded as follows with Def-CNPs-2 carbon-based nano particles：Take 100mg prepare carbon-based nano particle dissolving and constant volume in
In 10mL deionized waters, the sodium chloride for weighing different quality is dissolved in the carbon-based nano particle solution of preparation, is prepared different dense
The sodium-chloride water solution of degree.Then detected with XRF (model Perkin-Elmer LS 55).As a result such as Figure 10 institutes
Show.
It can be seen from fig. 10 that in the sodium-chloride water solution of various concentrations, Def-CNPs-1 and Def-CNPs-2 samples
Product fluorescence intensity is without occurring significant change, even if when the concentration of sodium chloride reaches high 5mol/L, fluorescence intensity is still
So keep stable.The aqueous solution of the carbon-based nano particle of this explanation present invention all has good ionic strength stability.
Above-mentioned detection has also been carried out to Def-CNPs-3 and Def-CNPs-4 carbon-based nanos particle, and the knot finally given
Fruit is consistent with above-mentioned Def-CNPs-1 and Def-CNPs-2 result.
The biological applications of the carbon-based nano particle of experimental example four
The bioactivity detection of 4.1 carbon-based nano particles
Def-CNPs-1 the and Def-CNPs-2 samples of various concentrations are added in the culture medium of human cervical carcinoma cell, sample
The concentration of product is up to 1000 μ g/mL, and culture carries out the detection of cell survival rate using MTT colorimetric methods after 24 hours.Wherein,
MTT colorimetric method operating methods are as follows：
Human cervical carcinoma cell is incubated at the DMEM nutrient solutions containing 10% calf serum, 1% penicillin and 1% streptomysin
In, it is seeded to the density of 5 × 103 cells/wells in 96 well culture plates.Culture plate is moved into incubator, at 37.0 DEG C, 5%
CO2And cultivated under the conditions of saturated humidity 24 hours and be paved with bottom hole to cell monolayer.Orifice plate is then taken out, 10 μ L are added per hole and are contained
After the DMEM nutrient solutions of various concentrations carbon-based nano particle, orifice plate is put back in incubator and continues culture 24 hours.Will with PBS
MTT is configured to 5mg/mL solution.Orifice plate is taken out again, and careful inhale discards nutrient solution in hole, adds 10 μ L 5mg/mL's in every hole
Incubator is put back to after MTT solution and continues culture 4 hours.Then orifice plate is taken out, carefully removes culture supernatants.Added in per hole
150 μ L dimethyl sulphoxide solutions, vibration 10min make the purple crystal of generation fully dissolve.It is 570nm to select wavelength, enzyme-linked
The absorbance in each hole is determined in immune detector.To be added without the cell experiment of carbon-based nano particle under same culture conditions
As blank control.Cell survival rate is the suction of cell in absorbance and blank assay with the cell of carbon-based nano particle processing
The ratio of luminosity.As a result it is as shown in figure 11.
It can be seen from fig. 11 that no matter in low concentration (50 μ g/mL) or the carbon-based nano of high concentration (1000 μ g/mL)
In the presence of particle, cell all shows good survival rate.Especially when the concentration of carbon-based nano particle reaches 1000 μ g/mL
When, cell still has more than 80% survival rate.And usual quanta point material, such as the ZnO material of polymer overmold, it is higher than in concentration
Cell survival rate just significantly reduces during 300 μ g/mL.Therefore carbon-based nano particle has relative to the higher life of quanta point material
Thing activity, it may be said that be a kind of biological nontoxic material.The bioactivity for comparing Def-CNPs-1 and Def-CNPs-2 samples can be with
It was found that both do not have significant difference.For the sign of both fluorescent properties with reference to more than, led to two kinds of fallen leaves for raw material
Crossing the carbon-based nano particle that the method for carbonization/oxidation obtains has similar chemical property, illustrates that this synthetic method has one
Fixed universality.
The cell fluorescence mark application of 4.2 carbon-based nano particles
The Secondary Culture human embryonic kidney cell in 6 orifice plates.When the saturation degree of cell reaches 60%, added not in every hole
With the carbon-based nano particle of concentration, continue culture 6 hours.It is then centrifuged for separating unlabelled carbon-based nano particle, it is heavy to retain
The cell in shallow lake.Cell is redispersed in PBS afterwards three times with PBS, centrifugation, 4 DEG C of black outs preserve.Before imaging experiment, take
The scattered suspended drop of one drop cell cover glass mounting, is carried out carefully on slide with Laser Scanning Confocal Microscope with 405nm exciting lights
Born of the same parents' imaging experiment.As a result it is as shown in figure 12.
In figure 12 it can be seen that, i.e., it is added without carbon-based receive under equal cell culture condition in blank assay (i.e. (d))
Rice corpuscles, cell do not have a fluorescence signal, and Def-CNPs-1 and Def-CNPs-2 samples by with it is all saturating after cell co-culture
Cell membrane has entered cell interior, and shows obvious fluorescence signal.Due to Def-CNPs-1 and Def-CNPs-
2 samples are in post synthesis all without the further functionalization of progress, therefore inventor thinks that both carbon-based nano particles are mainly
Cell is entered by concentration diffusion.Def-CNPs-1 and Def-CNPs-2 samples are existed entirely in cytoplasm.
Above-mentioned detection has also been carried out to Def-CNPs-3 and Def-CNPs-4 carbon-based nanos particle, and the knot finally given
Fruit is consistent with above-mentioned Def-CNPs-1 and Def-CNPs-2 result.
Characterized by having carried out detailed photoluminescent property to Def-CNPs-1 and Def-CNPs-2 samples, characterization result card
Bright, carbon-based nano particle of the invention has the typical emission peak property for depending on excitation wavelength, and this is mainly due to carbon-based
Exist in nano-particle caused by a variety of luminescence sites.Also shown in the excitation wavelength dependence of Def-CNPs-2 samples
The phenomenon of multiple emission peak extreme values, further demonstrate a variety of luminescence sites in carbon-based nano particle be present.Def-CNPs-1 and
Def-CNPs-2 samples show different fluorescence responses to different pH environments, under acid or alkalescence condition respectively
With the emission wavelength not changed with pH value, it can be used for the acid-base value of qualitative detection system.Def-CNPs-1 and Def-CNPs-
2 samples also show good photostability and the stability for high concentration inorganic ion.Thin to human cervical carcinoma
In the biological activity test of born of the same parents, Def-CNPs-1 and Def-CNPs-2 samples all show good bioactivity, especially when
The bioactivity for being substantially better than traditional quanta point material has been embodied when sample concentration is higher.Def-CNPs-1 and Def-CNPs-2
Sample also has good performance in cell labeling experiment, without in the case of further surface-functionalized, Def-
CNPs-1 and Def-CNPs-2 samples can be entered in cytoplasm through cell membrane, and all show obvious fluorescence
Signal.Fluorescent probe molecule can be used as by illustrating the carbon-based nano particle of the present invention, applied to cell imaging and biological detection
Deng in research.No matter Def-CNPs-1 and Def-CNPs-2 samples mark in photoluminescent property or bioactivity and cell fluorescence
Aspect all shows identical property, illustrates there is good fluorescence property with cheap biological material fallen leaves for Material synthesis
The method of carbon-based nano be feasible, and a greater variety of fallen leaves raw materials can be extended to.
The present invention is hereinbefore disclosed with preferred embodiment, but it should be understood by those skilled in the art that, these
Embodiment is only used for describing the present invention, and should not be construed as limiting the scope of the present invention.It should be noted that every implement with these
Example equivalent change and displacement, all should be set to be covered by scope of the presently claimed invention.Therefore, protection scope of the present invention
It should be defined by the scope defined in claims.
Claims (11)
1. a kind of preparation method of carbon-based nano particle, it is characterised in that comprise the following steps：
(1) blade of deciduous tree is carbonized to obtain carbon source；
(2) oxidant is added in carbon source and carries out oxidation reaction, be then centrifugally separating to obtain supernatant liquor；
(3) supernatant liquor is filtered, collects filtrate；
(4) filtrate is dried, obtains carbon-based nano particle.
2. preparation method according to claim 1, it is characterised in that in step (1), carbonization is in 300 DEG C of -600 DEG C of perseverances
Warm 1-4 hours are carbonized.
3. preparation method according to claim 1, it is characterised in that in step (1), deciduous tree is white birch and/or hung down
Willow.
4. preparation method according to claim 1, it is characterised in that in step (2), oxidant is appointing in following substances
It is a kind of：Nitric acid (preferred concentration is 4-10mol/L nitric acid), 98% concentrated sulfuric acid, or 98% concentrated sulfuric acid are pressed with 8mol/L nitric acid
It is 1 according to volume ratio:1-1:5 Binary Mixtures formed.
5. preparation method according to claim 1, it is characterised in that in step (2), according to carbon source and the weight of oxidant
Volume ratio is 1-10g:Oxidant is added in carbon source by 100-500mL, stirring reaction 6-48 hours.
6. preparation method according to claim 1, it is characterised in that step (2) further comprises：Oxidant is added to
First time oxidation reaction is carried out in carbon source, the first supernatant liquor and solid precipitation are centrifugally separating to obtain, in being precipitated then to solid
Oxidant is added again and carries out second of oxidation reaction, is centrifugally separating to obtain the second supernatant liquor, by the first supernatant liquor and the
Two supernatant liquors merge into the supernatant liquor.
7. a kind of carbon-based nano particle, it is characterised in that be made using the preparation method described in claim any one of 1-6.
8. carbon-based nano particle according to claim 7, it is characterised in that the particle diameter of the carbon-based nano particle is 2nm-
8nm, spacing of lattice are 0.32nm-0.34nm.
9. carbon-based nano particle according to claim 7, it is characterised in that the maximum excitation ripple of the carbon-based nano particle
Length is 320nm-420nm.
10. application of the carbon-based nano particle described in claim any one of 7-9 in bioanalysis.
11. application according to claim 10, it is characterised in that the bioanalysis is bioactivity detection and/or thin
Born of the same parents' fluorescence labeling.
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