CN105802621A - N-CQDs (nitrogen-doped carbon quantum dots) with high fluorescence quantum yield as well as preparation method and application of N-CQDs - Google Patents

Abstract

The invention discloses N-CQDs (nitrogen-doped carbon quantum dots) with high fluorescence quantum yield and a preparation method of the N-CQDs. Alanine is taken as a carbon source, ethanediamine is taken as a surface passivator, and the N-doped carbon quantum dots with high fluorescence quantum yield is successfully prepared with a simple one-step hydrothermal method. Compared with carbon quantum dots synthesized with other protein or amino acid as a carbon source, the nitrogen content of the prepared N-CQDs can reach 61.1%, the average fluorescence lifetime is 4.43 ns, the highest fluorescence quantum yield can reach 46.2% and approaches the fluorescence quantum yield of the carbon quantum dots prepared with a laser ablation method and an electric arc method, and the N-doped carbon quantum dots have low cytotoxicity and excellent biocompatibility and has wide application value in biosensing and bioimaging. The invention further discloses a high quenching effect of NADH (nicotinamide adenine dinucleotide) on fluorescence of the N-CQDs. On the basis that NADH has high quenching effect on fluorescence of the N-CQDs, high-sensitivity fluorescence biosensing for detecting NADH is established, the linear detection range is as low as 80 mu M, and the limit of detection is 25.1 nM.

Description

The nitrogen-doped carbon quantum dot of a kind of high-fluorescence quantum yield, preparation method and applications

Technical field

The invention belongs to the preparing technical field of fluorescent carbon nano material, relate to the nitrogen-doped carbon of a kind of high-fluorescence quantum yield Quantum dot, preparation method and applications, be specifically related to a kind of with alanine as carbon source, ethylenediamine as passivator, use one One-step hydrothermal prepares the method for nitrogen-doped carbon quantum dot, and concrete application includes the hypersensitive to nicotinamide adenine dinucleotide Detection and the fluorescence imaging to living cells.

Background technology

Carbon quantum dot can be improved its fluorescence property by Organic substance surface passivation.Nitrogen-atoms can improve the table of carbon quantum dot Planar defect also strengthens its fluorescence intensity and fluorescence quantum yield, thus being prepared as of nitrogen-doped carbon quantum dot (N-CQDs) The study hotspot prepared for carbon quantum dot.At present the synthetic method of carbon quantum dot include laser ablation, concentrated acid oxidizing process, Electrochemical preparation method, organic carbon method, template etc..But above-mentioned synthetic method needs precursor costly, and Complicated process of preparation, the quantum yield of product is relatively low.

Hydro-thermal method is a kind of method of novel preparation carbon quantum dot, carbon source is placed in airtight hydrothermal reaction kettle, under high temperature Produce environment under high pressure in hydrothermal reaction kettle, make carbon source generation carbonization form carbon quantum dot, have low cost, productivity high, after Processing the advantages such as simple, oneself is widely used in carbon quantum dot preparation field at present.Zhou Ming seminar is with glucose and glycine For mixed carbon source, glycine is simultaneously as nitrogen source, in 180 DEG C of hydro-thermal method one-step synthesis N-CQDs, when glucose and sweet When the mass ratio of propylhomoserin is 2:1, quantum yield is the highest, but only 6.57% (Li Xiaofeng etc. nitrogen-doped carbon quantum dot Synthesis, sign and the application [J] in cell imaging thereof. Materials Science and Engineering journal, 2015,1 (33): 41-45.).Wei Great seminar first with ammonium citrate simultaneously as carbon source and nitrogen source, in 160 DEG C of hydro-thermal method one-step synthesis N-CQDs, its Relative quantum yields is 13.5%, and fluorescence lifetime is 10.6ns, and quantum yield is not the highest, and reason is that its nitrogen element contains Amount is only 5.35%, and carbon content is up to 68.75%, the lowest (Yang Z, the et al. of carbon quantum dot surface nitrating degree Nitrogen-doped,carbon-rich,highly photoluminescent carbon dots from ammonium citrate[J]. Nanoscale,2014,6(3):1890-1895.).Existing hydro-thermal method prepares N-CQDs, uses Polyethylene Glycol (PEG) more Or polymine (PEI) is as passivator, technique wastes time and energy and fluorescence quantum yield is the highest.In sum, compel The little Organic substance of the molecular weight to be found that is essential, to substitute traditional passivation agents, studies green economy, the simple method of technique Prepare the nitrogen-doped carbon quantum dot being applicable to bio-sensing and bio-imaging.

Nicotinamide adenine dinucleotide (NADH) is NAD⁺/ NADH rely on dehydrogenase reaction involved by auxiliary because of Son, plays a significant role in endocellular metabolism approach and energy shift.NADH with include cancer, parkinson combine Closing disease and have directly related property in interior multiple disease and cell failure with death etc., its activity directly characterizes metabolic pathway Operating and the physiological status of cell, thus to its concentration and the focus of dynamic observation always metabolism research, right NADH content carries out highly sensitive detection provides important evidence by the early diagnosis for related significant disease.In recent years, research Multiple high accuracy, high-sensitivity analysis technology are applied to the detection of the metabolite such as NADH, especially fluorescence analysis by person Technology and electrochemical analysis techniques.Zhu person of outstanding talent seminar is prepared for a kind of nitrogen-doped graphene (Gai P P, et al.NADH dehydrogenase-like behavior of nitrogen-doped graphene and its application in NAD⁺-dependent dehydrogenase biosensing[J].Biosensors and Bioelectronics,2014,62: 170-176.), having the characteristic being similar to nadh dehydrogenase, it is 0.1～4.00 to the linear detection range of NADH MM, detection limit is 300nM.Esmaeil Shams seminar is prepared for a kind of NiO nanoparticle (Sharifi E, et al.Electrocatalytic activity of nickel oxide nanoparticles as mediatorless system for NADH and ethanol sensing at physiological pH solution[J].Biosensors and Bioelectronics,2013,45: 260-266.), and constructing the electrochemical sensor detecting NADH, it is 0～1 to the linear detection range of NADH MM, detection is limited to 106nM.

In the method for existing detection NADH, electrochemical process is optics based on NADH or chemical property, it is adaptable to Vitro detection, needs to carry out sample pretreatment, operation complexity before detection.Comparing electrochemical process, fluorescence analysis is applied Relatively broad, have that sensitivity is strong, specificity high, in vitro on Research foundation, may be directly applied to live body thin Born of the same parents.But in the fluorescence detection method of existing NADH, sensitivity and the resolution of fluorescent probe all have much room for improvement, Detection range and detection limit thereof to NADH optimize further.

Summary of the invention

The deficiency the highest for existing nitrogen-doped carbon quantum dot fluorescence quantum yield, preparation technology is complicated, the present invention provides A kind of cytotoxicity is low, biocompatibility is good nitrogen-doped carbon quantum dot of high-fluorescence quantum yield and preparation method thereof And application.The preparation method of the nitrogen-doped carbon quantum dot of the present invention is simple, green economy, and prepared N-CQDs can be real The now highly sensitive detection to nicotinamide adenine dinucleotide (NADH), it is possible to realize the fluorescence imaging to living cells.

Technical scheme is as follows:

A kind of preparation method of the N-CQDs of high-fluorescence quantum yield, with ethylenediamine (EDA) as surface passivator, with Alanine (Ala) is carbon source, is synthesized by one step hydro thermal method, specifically comprises the following steps that

By alanine ultrasonic dissolution in ultra-pure water, it is slowly added to passivator ethylenediamine while stirring, obtains mixed solution, Afterwards mixed solution is proceeded in autoclave, hydro-thermal reaction 6～48h at 150 DEG C～250 DEG C, after reaction terminates, Naturally cool to room temperature, remove solvent through dialysis, rotary evaporation, i.e. obtain the N-CQDs of high-fluorescence quantum yield, its In, the mol ratio of alanine and ethylenediamine is 4:1～20.

Preferably, described alanine is 4:1～10 with the mol ratio of ethylenediamine.

Preferably, described hydrothermal temperature is 200 DEG C, and the response time is 6～24h.

Preferably, in described dialysis procedure, the molecular cut off of bag filter is 1000Da, and dialysis time is 24h.

Preferably, it is 45 DEG C that temperature is steamed in described rotation.

On the other hand, the present invention also provides for the N-CQDs of the high-fluorescence quantum yield that above-mentioned preparation method obtains at nicotiamide Application in adenine-dinucleotide detection.

Nitrogen-doped carbon quantum dot prepared by the present invention, its fluorescence can be by NADH quencher, the c/s-diol functional group of NADH With the carboxyl generation esterification on N-CQDs surface, hinder N-CQDs surface void and interelectric radiation recombination, and then Make the fluorescent quenching of N-CQDs.

On the other hand, the present invention also provides for the N-CQDs of the high-fluorescence quantum yield that above-mentioned preparation method obtains alive thin Application in born of the same parents' fluorescence imaging.

Preferably, described living cells is human breast cancer cell (MCF-7).

Compared with prior art, the present invention has a following remarkable result:

(1) with alanine as raw material, green economy, and there is good biocompatibility；

(2) with ethylenediamine as passivator, compared with conventional passivation agent PEG or PEI, its molecular weight is little, and alanine is high Temperature carbonization is formed while the surface of carbon quantum dot is modified with ethylenediamine by carbon quantum dot and carries out, and makes N-CQDs surface contain The functional group that O-H, N-H, C-OH, C=O, C=C, C-N and C-O etc. are abundant, thus a step obtains N doping Fluorescent carbon quantum dot, step is simple；

(3) with other protein or aminoacid be carbon source synthesis carbon quantum dot compared with, N-CQDs prepared by the present invention Nitrogen content up to 61.1%, the mean fluorecence life-span is 4.43ns, and fluorescence quantum yield reaches as high as 46.2%, close to With the fluorescence quantum yield of carbon quantum dot prepared by laser ablation and arc process, and there is low cytotoxicity and good life The thing compatibility, is with a wide range of applications in bio-sensing and bio-imaging；

(4) N-CQDs of the present invention is in the detection of NADH is applied, its linear detection range as little as 80 μMs, inspection Survey limit up to 25.1nM, it is possible to realize the overdelicate detection of NADH.

Accompanying drawing explanation

Fig. 1 is low resolved transmittance Electronic Speculum figure (a) and the high resolution TEM figure of the N-CQDs of embodiment 1 preparation (b)。

Fig. 2 is the particle size distribution figure of the N-CQDs of embodiment 1 preparation.

Fig. 3 is the x-ray photoelectron power spectrum of the N-CQDs of embodiment 1 preparation, and wherein (a) is full spectrogram, (b) Being the swarming figure of carbon, (c) is the swarming figure of nitrogen element, and (d) is the swarming figure of oxygen element.

Fig. 4 is the infrared spectrogram of the N-CQDs of embodiment 1 preparation.

Fig. 5 is the constitutional diagram of the ultra-violet absorption spectrum of N-CQDs, fluorescence excitation and the emission spectrum of embodiment 1 preparation.

Fig. 6 is the emission spectrum figure corresponding under different excitation wavelengths for N-CQDs of embodiment 1 preparation.

Fig. 7 is the fluorescence decay curve figure of the N-CQDs of embodiment 1 preparation.

Fig. 8 is the mechanism of action figure of NADH quencher N-CQDs fluorescence.

Fig. 9 is the N-CQDs for preparing of the present invention fluoroscopic examination result figure to NADH, and (a) is variable concentrations NADH Under N-CQDs fluorescence emission spectrum, (b) is the graph of a relation of fluorescence intensity and NADH concentration.

Figure 10 is the N-CQDs for preparing of the present invention cytotoxicity experiment result figure to A549 and MCF-7 cell.

Figure 11 is the fluorescent microscopic imaging figure after N-CQDs Yu the MCF-7 cell incubation that the present invention prepares, (a) be Light field microgram, (b) is the microgram under 365nm excitation source irradiates, and (c) figure is at 460nm excitation source Microgram under Zhao Sheing.

Detailed description of the invention

For the ease of understanding content of the present invention, below in conjunction with embodiment and accompanying drawing, the present invention is made the most in detail Explanation.

In the present invention, the fluorescence quantum yield of N-CQDs is determined by the following procedure:

Using relative method, reference is quinoline sulfate solution (0.1mol/L H₂SO₄Dissolve quinoline), its quantum yield is 0.54. Comprise the concrete steps that 5 Concentraton gradient being respectively provided with quinoline sulfate and N-CQDs, and the solution ultraviolet light absorption of each gradient Degree is all below 0.1；Meanwhile, under maximum excitation wavelength, measure the fluorescence emission spectrum of each solution, calculate emission peak Integration；Using the UV absorption of solution as abscissa, map using the integrated value of emission peak as vertical coordinate, fitting a straight line, Try to achieve slope.Pass through equation below:

${\mathrm{\φ}}_x={\mathrm{\φ}}_{st}(K_x/K_{st})({\mathrm{\η}}_x^2/{\mathrm{\η}}_{st}^2)$

Wherein, φ represents quantum yield, and K is the slope of above-mentioned fitting a straight line, η be solvent refractive index (water as solvent, η=1.33).St refers to standard reference quinoline sulfate, and x refers to N-CQDs solution.

Embodiment 1

A kind of method of nitrogen-doped carbon quantum dot preparing the present invention, comprises the steps:

Step 1, weigh 2.673g alanine (Ala) and be placed in the clean beaker of 50mL, add 30.0mL ultra-pure water, put Entering the ultrasonic 2min of Ultrasound Instrument, be slowly added to 500 μ L ethylenediamine (EDA) while stirring, the mol ratio of Ala with EDA is 4:1；

Step 2, above-mentioned mixed liquor transferred in 50mL politef inner liner of reaction kettle and install sealing steel bushing, being placed in Blowing-type adds in hot tank, is progressively heated at 200 DEG C and constant temperature 6h；

After step 3, reaction terminate, naturally cool to room temperature, obtain sepia clear solution；

Step 4, the bag filter of solution molecular cut off MWCO=1000Da dialyse in ultra-pure water 24h remove the most anti- The precursor answered；

Carbon quantum dot solution after step 5, dialysis rotary evaporation under the conditions of 45 DEG C removes aqueous solvent, obtains pressed powder, It is described N-CQDs.

Step 6, take a small amount of solid N-CQDs and be dissolved in ultra-pure water, obtain the N-CQDs of mass concentration.With quinoline sulfate Solution is reference, and the fluorescence quantum yield recording N-CQDs is 18.7%.

Embodiment 2

A kind of method preparing nitrogen-doped carbon quantum dot, embodiment 2 and embodiment 1 are not uniquely both EDA in step 1 Consumption be the mol ratio of 2.0mL, Ala and EDA be 4:4, the fluorescence quantum yield of the N-CQDs prepared is 33.3%.

Embodiment 3

A kind of method preparing nitrogen-doped carbon quantum dot, embodiment 3 and embodiment 2 are not uniquely both hydro-thermal temperature in step 2 Degree is 150 DEG C, and the fluorescence quantum yield of the N-CQDs prepared is 8.64%.

Embodiment 4

A kind of method preparing this nitrogen-doped carbon quantum dot, embodiment 4 and embodiment 2 are not uniquely both hydro-thermal in step 2 Temperature is 250 DEG C, and the fluorescence quantum yield of the N-CQDs prepared is 11.9%.

In conjunction with the embodiments 2～4, preferably hydrothermal temperature 200 DEG C.

Embodiment 5

A kind of method preparing nitrogen-doped carbon quantum dot, it is anti-that embodiment 5 and embodiment 2 are not uniquely both hydro-thermal in step 2 Being 12h between Ying Shi, the fluorescence quantum yield of the N-CQDs prepared is 37.5%.

Embodiment 6

A kind of method preparing nitrogen-doped carbon quantum dot, it is anti-that embodiment 6 and embodiment 2 are not uniquely both hydro-thermal in step 2 Being 24h between Ying Shi, the fluorescence quantum yield of the N-CQDs prepared is 27.6%.

Embodiment 7

A kind of method preparing nitrogen-doped carbon quantum dot, it is anti-that embodiment 7 and embodiment 2 are not uniquely both hydro-thermal in step 2 Being 48h between Ying Shi, the fluorescence quantum yield of the N-CQDs prepared is 10.2%.

In conjunction with the embodiments 2～7, the preferably hydro-thermal reaction time is 6h.

Embodiment 8

A kind of method preparing nitrogen-doped carbon quantum dot, embodiment 8 and embodiment 1 are not uniquely both EDA in step 1 Consumption be the mol ratio of 5.0mL, Ala and EDA be 4:10, the fluorescence quantum yield of the N-CQDs prepared is 46.2%.

Embodiment 9

A kind of method preparing nitrogen-doped carbon quantum dot, embodiment 9 and embodiment 1 are not uniquely both EDA in step 1 Consumption be the mol ratio of 10.0mL, Ala and EDA be 4:20, the fluorescence quantum yield of the N-CQDs prepared is 16.5%.

In conjunction with the embodiments 1～2 and embodiment 8～9, it is considered to prepared Nitrogen doped carbon quantum dot as the universality of fluorescent probe, And the green economy demand of preparation technology, the consumption selecting passivator EDA is 500 μ L, fed intake Ala's Yu EDA Mol ratio is 4:1.

Embodiment 10

The N-CQDs of embodiment 1 preparation is carried out phenetic analysis, and concrete steps include:

(1). transmission electron microscope observing

Pattern and the size of prepared N-CQDs are to be carried out by transmission electron microscope (TEM, Philips TECNAI-12) Test, voltage is 300kV.Before doing Electronic Speculum, first the nitrogen-doped carbon quantum dot of 10 μ L is added drop-wise to the 200 ultra-thin carbon of purpose Supporting, on film copper mesh, to put and evaporate aqueous solvent at room temperature, can carry out Electronic Speculum operation, result sees Fig. 1 and Fig. 2.Such as figure Shown in, N-CQDs is uniform-spherical, distribution of sizes 6～9nm, average-size about 8nm.

(2) .X X-ray photoelectron spectroscopy X

Crystal structure and the surface functional group of prepared N-CQDs are by x-ray photoelectron power spectrum (XPS, the U.S. Thermo Fisher Scientific company) to test, result is as shown in Figure 3.The N-CQDs of preparation is can be seen that from a figure Containing elements such as C, O, N, content is respectively 61.1%, 24.6% and 14.3%, and its nitrogen content reaches existing nitrogen-doped carbon The nitrogen content level of quantum dot.By b figure understand N-CQDs C1s main peak can be divided into 284.5eV, 286.0eV, 286.5 4 peaks such as eV, 288.0eV, respectively correspondence functional group C-C, C-N, C-O, C=N/C=O；The N of c figure_1s=Swarming The most corresponding C-N-C, N-H, show nitrogen doped in prepared carbon quantum dot；D schemes O_1sSwarming correspondence respectively C=O, C-OH/C-O-C.

(3). infrared spectrum

The surface group of prepared N-CQDs and structure composition are to be carried out by TENSOR27 type FT-IR spectrogrph Test, sample preparation uses KBr pressed disc method, and result is as shown in Figure 4.As seen from the figure, prepared N-CQDs surface is contained O-H, N-H, C-OH, C=O, C=C, C-N and C-O functional group, this is consistent with XPS test result.

(4). ultraviolet spectra

Prepared N-CQDs carries out ultraviolet spectra test, instrument be UV, visible light spectrophotometer (UV-3600, Shimadzu, Japan), result such as Fig. 5's is shown in solid.N-CQDs has strong absorption at 320nm, the most gradually weakens To visual field, this absworption peak is caused by the n-π * transition of the C=O generation on N-CQDs surface.

(5). fluorescence spectrum

The prepared fluorescence excitation of N-CQDs, emission spectrum be by spectrofluorophotometer (Edinburg analytical tool, FLS920) testing, its result is as shown in dotted line and Fig. 6 in Fig. 5；N-CQDs fluorescence lifetime is according to time phase Closing single photon technique, measured by combination fluorescence lifetime instrument (Edinburg analytical tool, FLS920), its result is such as Shown in Fig. 7, figure inserts the content analysis fluorescence lifetime of N-CQDs；Knowable to Fig. 5 illustration, the dilute water of N-CQDs Solution is yellow under natural light, at λ_ex=320nm exhibits blue fluorescence；The emission peak meeting of N-CQDs as can be seen from Figure 6 Changing along with the change of excitation wavelength, have excitation wavelength dependency, its maximum excitation wavelength is 320nm, corresponding Penetrating main peak is 390nm；The mean fluorecence life-span of N-CQDs is 4.43ns as can be seen from Figure 7.

(6). cytotoxicity characterizes

The N-CQDs of preparation is carried out cytotoxicity sign, is assessed by MTT experiment.First disappear respectively with 0.05% pancreatin Change Human Lung Cancer cell A549 and human breast cancer cell MCF-7, with containing 10% hyclone and 1% penicillin/strepto- The DMEM in high glucose culture medium of element is made into certain density cell suspension, and is inoculated in 96 porocyte culture plates, every hole 100μL；Culture plate is placed in cell culture incubator (37 DEG C, 5%CO₂) until the cell number in each hole increases to 4 × 10³Individual； Taking Tissue Culture Plate prepared by previous step, discard each hole supernatant, PBS rinse cell once, is subsequently adding above-mentioned Culture medium, the N-CQDs solution of the additional 10 μ L of wherein matched group every hole 100 μ L, experimental group every hole 90 μ L is (corresponding eventually Concentration is respectively 10,25,50,100,200,400 μ g/mL), at 37 DEG C, 5%CO₂Under the conditions of continue cultivation 24 h；Take Tissue Culture Plate prepared by previous step, add the MTT solution of 20 μ L 5mg/mL to every hole, at cell culture incubator In hatch 4h；Then wash away culture medium with MTT solution, add 150 μ L DMSO, by gained mixture shaken at room temperature About 10min；Each hole absorbance (OD) at wavelength 490nm is measured by microplate reader, record also result, according to Below equation calculating cell survival rate:

$CellViability\left(\%\right)=\left(\frac{{\mathrm{OD}}_{Treated}}{{\mathrm{OD}}_{Control}}\right)\×100\%$

Result as shown in Figure 10, when preparation N-CQDs at concentrations up to 400 more than μ g/mL time, A549 and MCF-7 The survival rate of cell have dropped 13% and 17% respectively, shows that N-CQDs has less cytotoxicity；And work as N-CQDs Concentration less than 200 μ g/mL, its cytotoxicity can be ignored, therefore controls the dosage of N-CQDs, can realize its application In cell and organism.

Embodiment 11

Nitrogen-doped carbon quantum dot prepared by the present invention, its fluorescence can by NADH quencher, quenching mechanism as shown in Figure 8, The c/s-diol functional group of NADH and the carboxyl generation esterification on N-CQDs surface, hinder N-CQDs surface holes Cave and interelectric radiation recombination, make the fluorescent quenching of N-CQDs.

The nitrogen-doped carbon quantum dot of a kind of present invention of utilization is applied to detect NADH, and concrete steps include:

Take the N-CQDs of 50 μ L purification in 5.0mL centrifuge tube, add variable concentrations (0 μM, 10 μMs, 20 μMs, 40μM、80μM、140μM、200μM、300μM、400μM、500μM、600μM、700μM、1000 μM) NADH solution (keep volume be all 3.0mL, solvent is the PBS at 0.1M pH 7.4)；Mixed After even 10min, the fluorescence exciting lower record to launch main peak each sample at 390nm at maximum excitation wavelength 320nm is sent out Penetrate spectrum；All experiments are the most at room temperature carried out.

Result sees Fig. 9, as shown in a schemes, along with the increase of NADH concentration, prepared N-CQDs fluorescence intensity with Reduction, show that NADH can the fluorescence of quencher N-CQDs.B figure show the concentration of NADH in the range of 0～80 μM with The fluorescence intensity change of N-CQDs is linear, and detection is limited to 25.1nM.

The method of N-CQDs detection NADH prepared by the present invention compared with the existing methods, divide by detection range and detection limit Do not reduce 5～150 times and 10～200 times.

Embodiment 12

A kind of nitrogen-doped carbon quantum dot utilizing the present invention includes for cell imaging, concrete steps:

First with 0.05% trypsinization MCF-7 cell, with the high sugar containing 10% hyclone and 1% penicillin/streptomycin DMEM culture medium is made into certain density cell suspension, and is inoculated in 96 porocyte culture plates, every hole 200 μ L； Culture plate is placed in cell culture incubator (37 DEG C, 5%CO₂) until the cell number in each hole increases to 1 × 10⁵Individual；Take one The Tissue Culture Plate of step preparation, discards each hole supernatant, and PBS rinse cell once, adds 200 μ L 200 μ g/mL N-CQDs (solvent is above-mentioned DMEM in high glucose culture medium), continues to cultivate 24h；With 4% paraformaldehyde, cell is fixed In fluorescent microscope slide, excess N-CQDs by with temperature PBS solution wash three times remove；Then distinguish At ultraviolet light (λ_ex=365nm) and blue light (λ_ex=460nm) under observe and take pictures.

As shown in figure 11, (a) is in bright field microgram to fluorescence imaging result, and (b) is to irradiate at 365nm excitation source Under microgram, (c) figure be 460nm excitation source irradiate under microgram.Prepared N-CQDs enters MCF-7 Cell, and under above-mentioned light source activation, show its fluorescence color, and the form and survival rate to cell has no significant effect, Therefore this N-CQDs can be used for labelling MCF-7 cell, and has potential in the biomarker and bio-imaging of living cells Using value.

Claims (9)

1. the preparation method of the nitrogen-doped carbon quantum dot of a high-fluorescence quantum yield, it is characterised in that specifically comprise the following steps that

By alanine ultrasonic dissolution in ultra-pure water, it is slowly added to passivator ethylenediamine while stirring, obtains mixed solution, Afterwards mixed solution is proceeded in autoclave, hydro-thermal reaction 6～48h at 150 DEG C～250 DEG C, after reaction terminates, Naturally cool to room temperature, remove solvent through dialysis, rotary evaporation, i.e. obtain the nitrogen-doped carbon quantum of high-fluorescence quantum yield Point, wherein, the mol ratio of alanine and ethylenediamine is 4:1～20.

The preparation method of the nitrogen-doped carbon quantum dot of a kind of high-fluorescence quantum yield the most according to claim 1, it is special Levying and be, described alanine is 4:1～10 with the mol ratio of ethylenediamine.

The preparation method of the nitrogen-doped carbon quantum dot of a kind of high-fluorescence quantum yield the most according to claim 1, it is special Levying and be, described hydrothermal temperature is 200 DEG C, and the response time is 6～24h.

The preparation method of the nitrogen-doped carbon quantum dot of a kind of high-fluorescence quantum yield the most according to claim 1, it is special Levying and be, in described dialysis procedure, the molecular cut off of bag filter is 1000Da, and dialysis time is 24h.

The preparation method of the nitrogen-doped carbon quantum dot of a kind of high-fluorescence quantum yield the most according to claim 1, it is special Levying and be, it is 45 DEG C that temperature is steamed in described rotation.

The preparation method system of the nitrogen-doped carbon quantum dot of a kind of high-fluorescence quantum yield the most according to claims 1 to 5 The nitrogen-doped carbon quantum dot obtained.

The preparation method of the nitrogen-doped carbon quantum dot of a kind of high-fluorescence quantum yield the most according to claim 6 prepares Nitrogen-doped carbon quantum dot nicotinamide adenine dinucleotide detect in application.

The preparation method of the nitrogen-doped carbon quantum dot of a kind of high-fluorescence quantum yield the most according to claim 6 prepares The application in live cell fluorescent imaging of the nitrogen-doped carbon quantum dot.

The preparation method of the nitrogen-doped carbon quantum dot of a kind of high-fluorescence quantum yield the most according to claim 8 prepares The application in live cell fluorescent imaging of the nitrogen-doped carbon quantum dot, it is characterised in that described living cells is human breast carcinoma Cell.