CN108007909A - Carbon quantum dot is in detection Fe after the modification of 4- naphthyl -3- thiosemicarbazides3+In application - Google Patents

Carbon quantum dot is in detection Fe after the modification of 4- naphthyl -3- thiosemicarbazides3+In application Download PDF

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CN108007909A
CN108007909A CN201711378764.4A CN201711378764A CN108007909A CN 108007909 A CN108007909 A CN 108007909A CN 201711378764 A CN201711378764 A CN 201711378764A CN 108007909 A CN108007909 A CN 108007909A
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quantum dot
carbon quantum
thiosemicarbazides
naphthyl
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傅政
贺建同
刘园
贾丰春
乔月纯
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Henan Institute of Technology
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    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
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    • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
    • G01N2021/6432—Quenching

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Abstract

Carbon quantum dot is in detection Fe after being modified the invention discloses 4 naphthyl, 3 thiosemicarbazides3+In application.The present invention has synthesized carbon quantum dot first by using hydro-thermal method.Then, by introducing big aromatic group naphthyl in thiosemicarbazides molecule, 4 naphthyl, 3 thiosemicarbazides (NTSC) has been synthesized.Carbon quantum dot surface is modified with 4 naphthyl, 3 thiosemicarbazides, to study the fluorescence property of carbon quantum dot and the recognition reaction to ion after 4 naphthyl, 3 thiosemicarbazides is modified.Found by studying, not only fluorescent emission is controllable for the carbon quantum dot after modification, but also good light stability, solution influenced small by pH value, ionic strength and buffering.Relative to other metal ions, the carbon quantum dot after modification is to Fe3+With special selectivity., the reason for causing this phenomenon, may be related with the naphthyl in 4 naphthyl, 3 thiosemicarbazides molecule and the carbon quantum dot after thiosemicarbazides modification often has copper ion level identification effect.Work as Fe3+When concentration is in the range of 0 to 0.20 μM, fluorescence intensity and Fe3+Concentration is in good correlation, and detection is limited to 1.68nM.It is worth noting that, the probe water solubility is fabulous, cytotoxicity is low, biocompatibility and permeability of cell membrane are good, can also be to intracellular Fe3+It is detected analysis.

Description

Carbon quantum dot is in detection Fe after the modification of 4- naphthyl -3- thiosemicarbazides3+In application
Technical field
The present invention relates to detection technique, and in particular to carbon quantum dot is in detection Fe after the modification of 4- naphthyl -3- thiosemicarbazides3+In Application.
Background technology
As the highest transition metal of content in cell, iron plays key player in human body, and many physiology courses are such as： The transport of oxygen and metabolism, transcriptional control and electric transmission etc. are required to the participation of iron.Iron deficiency and iron content is excessive can cause Various diseases, such as：Anaemia, parkinsonism, Alzheimer's disease and cancer etc..And serum levels of iron can objectively evaluator The nutrition condition of body and some relevant diseases of diagnosis.At present, measuring the method for iron has very much, such as colorimetric method, atomic absorption spectrophotometry Photometry, inductively coupled plasma mass spectrometry etc.-.However, there are analysis time length in these methods, detection needs essence The defects of close instrument, sample preparation procedure is complicated.In contrast, fluorescence analysis due to cost is low, high sensitivity, reaction The advantages such as speed is fast, a finer selection is provided for measure iron.Now, the iron ion probe based on different materials, such as：It is organic Dyestuff, semiconductor-quantum-point, fluorescence metal nano-cluster etc., have been developed that utilization.But the fluorescence probe of these reports is still deposited ：The drawbacks such as complex synthetic route, water-soluble low, light resistance be poor and toxic.Therefore, design is with low cytotoxicity, Gao Shui Dissolubility, light resistance and biocompatibility iron ion probe have become more and more important and urgent.
In recent years, fluorescent carbon quantum dot due to synthesis is simple, good water solubility, fast light Bleachability, strong chemical inertness, Hypotoxicity and biocompatibility and attracted the notice of people, the iron ion probe for also having come into being different based on this, such as： Qu etc. makees raw material with dopamine, and the carbon quantum dot of favorable optical performance has been synthesized using hydro-thermal method, and detects Fe3+And DOPA Amine；Zhao etc. goes out nitrogen, the high fluorescent carbon quantum dot of sulphur codope using ionic liquid as Material synthesis, and have detected Fe with it3+、 Cu2+And cell imaging；Wen etc. makees raw material using cotton for the first time, and having synthesized fluorescence quantum yield with pyrolysis microwave method is 14.8% carbon quantum dot.Fe is detected with it3+, detect and be limited to 27 μM；The oligosaccharide such as Hamed makees carbon source, and alkali makees additive, adopts Carbon quantum dot has been synthesized with thermal oxide approach, Fe in biological sample is detected with it3+, detection limit is up to 6.05 μM etc..But at this A little carbon quantum dots based on fluorescence probe in, some fluorescence quantum yields are low, and some poor specificities, some sensitivity is low, has Modification in nano material, in order to further widen its application field, is had the material of Selective recognition effect by detection limit height etc., It is the good method for realizing object specific detection.Zhao etc. modifies carbon quantum dot with polyethyleneimine, has obtained fluorescent quantum Yield is up to 42.5%, the iron ion fluorescence probe that fluorescence signal strengthens under alkaline environment, and detects tap water and river water with it In Fe3+, RSD<3.0%；Qu etc. is modified TPEA on carbon quantum dot surface by covalent bond, has obtained the intracellular Cu of detection2+ Probe, experiment shows that the probe has preferable selectivity and relatively low test limit；Zhu etc. has synthesized AE-TPEA, and by its As Cu2+Specific receptors, be bonded on the composite nano materials of carbon quantum dot, obtained fluorescence based on carbon quantum dot and visited Pin, is used for Cu in active somatic cell by it2+Measure, about 1.0 μM of test limit.
The content of the invention
In order to solve the deficiencies in the prior art, carbon quantum dot exists after being modified the present invention provides 4- naphthyl -3- thiosemicarbazides Detect Fe3+Application.
The technical scheme is that：Carbon quantum dot Fe in cell is detected after the modification of 4- naphthyl -3- thiosemicarbazides3+In Using.
Another object of the present invention is to carbon quantum dot after providing the modification of 4- naphthyl -3- thiosemicarbazides in detection Fe3+'s Using.
Further improvement of the present invention includes：
The application, its detection are limited to 1.68nM.
The preparation method of the carbon quantum dot is as follows：3.0g EDTA are added to after being completely dissolved in 30mL ultra-pure waters, will Solution is transferred in the autoclave of 50mL, when 180 DEG C of reactions 10 are small；Cooling, product centrifugation, crosses film, dry, you can to obtain carbon Quantum dot solid.
The preparation method of the 4- naphthyls -3- thiosemicarbazides is as follows：It is solvent to take isopropanol, at room temperature, the different sulphur of 1- naphthyls Cyanate and hydration hydrazine reaction, generate 4- naphthyl -3- thiosemicarbazides.
10mmol 1- naphthalenylisothiocyanates and 20mL isopropanols are added in 50mL round-bottomed flasks, until completely dissolved, 1.25mL hydrazine hydrates are added dropwise, at room temperature stirring reaction.TLC tracking reaction, 1 it is small when the reaction was complete.Filter, respectively with water and Isopropanol washs, and obtains product 4- naphthyl -3- thiosemicarbazides.
4- naphthyls -3- thiosemicarbazides modification carbon quantum dot the preparation process is as follows：Add in 20mL 0.2mg carbon quantum dots Enter 10mL 20mmol L-1EDC and 5mL 20mmol L-1NHS, vibration, 37 DEG C are reacted 30 minutes；Add 5mL50g L-1 4- Naphthyl -3- thiosemicarbazides, when reaction 2.5 is small；In product refrigerator overnight, dialysis, the product drying that will be dialysed, obtains modified carbon Quantum dot.
Carbon quantum dot detection Fe after the modification of 4- naphthyl -3- thiosemicarbazides3+The step of it is as follows：By 500 μ L 0.01g L- 1Carbon quantum dot and certain density Fe after the modification of NTSC-CDs, that is, 4- naphthyl -3- thiosemicarbazides3+Solution adds 1.5mL centrifuge tubes In, Tris-HCl buffer solutions are settled to 1.0mL, are sufficiently mixed, and react 25 minutes at room temperature, under the excitation wavelength of 340nm Detect its fluorescence intensity.
The present invention has synthesized carbon quantum dot first by using hydro-thermal method.Then, by being introduced in thiosemicarbazides molecule Big aromatic group-naphthyl, has synthesized 4- naphthyl -3- thiosemicarbazides (NTSC).With 4- naphthyl -3- thiosemicarbazides to carbon quantum dot Surface is modified, and is made to study the fluorescence property of carbon quantum dot and the identification to ion after 4- naphthyl -3- thiosemicarbazides is modified With.Found by studying, not only fluorescent emission is controllable for the carbon quantum dot after modification, but also good light stability, by pH value, ion Intensity and buffering are solution influenced small.Relative to other metal ions, the carbon quantum dot after modification is to Fe3+With special selectivity. , the reason for causing this phenomenon, can and the carbon quantum dot after thiosemicarbazides modification often has copper ion level identification effect Can be related with the naphthyl in 4- naphthyl -3- thiosemicarbazides molecules.Work as Fe3+When concentration is in the range of 0 to 0.20 μM, fluorescence intensity with Fe3+Concentration is in good correlation, and detection is limited to 1.68nM.It is worth noting that, the probe water solubility is fabulous, cytotoxicity is low, Biocompatibility and permeability of cell membrane are good, can also be to intracellular Fe3+It is detected analysis.
Brief description of the drawings
Fig. 1 a are original carbon quantum dot transmission electron microscope pictures.
Fig. 1 b are NTSC-CDs transmission electron microscope pictures.
Fig. 1 c are original carbon quantum dot grain size distributions.
Fig. 1 dNTSC-CDs grain size distributions.
Fig. 2 is infrared spectrogram, wherein (a) 4- naphthyl -3- thiosemicarbazides；(b) original carbon quantum dot and (c) NTSC- CDs。
Fig. 3 is the full spectrograms of XPS of NTSC-CDs.
Fig. 3 a are the high-resolution collection of illustrative plates of C1s.
Fig. 3 b are the high-resolution collection of illustrative plates of N1s.
Fig. 3 c are the high-resolution collection of illustrative plates of O1s.
Fig. 3 d are the high-resolution collection of illustrative plates of S2p.
Fig. 4 is CDs, 4- naphthyl -3- thiosemicarbazides and the UV-visible spectrum of NTSC-CDs.Wherein illustration is modification Fe is added in carbon quantum dot solution (left side) and modification carbon quantum dot3+Picture after (right side) under ultra violet lamp.
Fig. 5 is the emission spectrum of 4- naphthyl -3- thiosemicarbazides modification carbon quantum dot.
Fig. 6 is the excitation spectrum and emission spectrum of NTSC-CDs
Fig. 7 a are influences of the pH to NTSC-CDs luminescent properties.
Fig. 7 b are influence of the buffer system to NTSC-CDs luminescent properties.
Fig. 7 c are influence of the ionic strength to NTSC-CDs luminescent properties.
Influence of Fig. 8 probes dosage to fluorescence response.
The influence that Fig. 9 reaction time responds fluorescence probe.
Figure 10 a are that various concentrations Fe is added in probe solution3+Response.
The dependence of Figure 10 b fluorescence intensities and [Fe3+], wherein illustration be [Fe3+] at 0-0.20 μM, F/F0 and [Fe3 +] linear relationship.
Figure 11 a are the fluorescence responses of different metal ions.
Figure 11 b be under similarity condition Fe3+ exist and not in the presence of, interfering ion on fluorescence intensity influence.
Figure 12 is cytotoxicity experiment.
Embodiment
Elaborate with reference to embodiment to the present invention.
Embodiment 1
Experimental section
Primary drug
1- naphthalene isothiocyanate (97%) is purchased from Zhengzhou and wishes Parker Chemical Co., Ltd.；Isopropanol is purchased from Tianjin day power chemistry Reagent Company；Hydrazine hydrate, EDTA, glacial acetic acid, KCl, NaCl, ZnCl2、BaCl2、MnCl2、AlCl3、AgNO3、Mg(NO3)2、Cu (NO3)2、Ni(NO3)2、Co(NO3)2、Cd(NO3)2、Pb(NO3)2、Hg(NO3)2、Fe(NO3)3、FeSO4It is purchased from controlling interest of traditional Chinese medicines Learn reagent Co., Ltd.Medicine rank is that analysis is pure.Water used in experiment makes ultra-pure water by oneself for laboratory.
Instrument
PerkinElmer type FT-IR spectrometers (U.S.)；TU-1810 UV-vis spectrometers (the general analysis analytical instrument in Beijing Co., Ltd)；2100 TEM of JEOL (Japan)；PHI Quantera XPS (Japan)；Cary Eclipse Fluorescence Spectrometer (U.S.)；TCSSP5 Leica laser scanning co-focusing microscopes (Germany)；600,000,000 nuclear magnetic resonance chemical analysers of Bruker are (auspicious Scholar).
Experiment
(1) synthesis of carbon quantum dot
3.0g EDTA are added to after being completely dissolved in 30mL ultra-pure waters, solution are transferred in the autoclave of 50mL, When 180 DEG C of reactions 10 are small.Cooling, product centrifugation, crosses film, dry, you can to obtain carbon quantum dot solid.
(2) 4- naphthyls -3- thiosemicarbazides synthesizes
Synthetic route：Selection isopropanol is solvent, and at room temperature, 1- naphthalenylisothiocyanates and hydration hydrazine reaction, generate 4- Naphthyl -3- thiosemicarbazides.
Experiment
Step：
The first step：10mmol1- naphthalenylisothiocyanates and 20mL isopropanols are added in 50mL round-bottomed flasks, is treated completely After dissolving, 1.25mL hydrazine hydrates are added dropwise, at room temperature stirring reaction.TLC tracking reaction, 1 it is small when the reaction was complete.Filter, point Do not washed with water and isopropanol, obtain product.Vacuum drying, sets the chessman on the chessboard according to the chess manual.
4- naphthyl -3- thiosemicarbazides：Khaki powder, mp 172-173 DEG C；1H NMR(400MHz,DMSO):δ9.26 (s, 1H), 7.96-7.93 (m, 1H), 7.90-7.88 (m, 1H), 7.82-7.88 (d, J=12Hz, 1H), 7.67-7.65 (d, J =10.2Hz, 1H), 7.54-7.49 (m, 4H), 4.95 (br, 2H)；13C NMR(151MHz,DMSO):δ181.25,135.31, 133.69,129.74,128.09
(3) 4- naphthyls -3- thiosemicarbazides modification carbon quantum dot (NTSC-CDs)
10mL 20mmol L are added in 20mL 0.2mg carbon quantum dots-1EDC and 5mL 20mmol L-1NHS, vibration, 37 DEG C reaction 30 minutes.Add 5mL 50g L-14- naphthyl -3- thiosemicarbazides, when reaction 2.5 is small.In product refrigerator overnight, thoroughly Analysis, the product drying that will be dialysed, obtains modification carbon quantum dot.
(4)Fe3+Ion detection
By 500 μ L 0.01g L-1NTSC-CDs and certain density Fe3+Solution is added in 1.5mL centrifuge tubes, Tris- HCl buffer solutions are settled to 1.0mL, are sufficiently mixed, and react 25 minutes at room temperature, and it is glimmering to detect its under the excitation wavelength of 340nm Luminous intensity.
(5) toxicity test
By certain density HeLa cell inoculations on 96 orifice plates, when the 37 DEG C of cultures 24 of addition culture medium are small.Add not With content NTSC-CDs cultivate 24 it is small when after, add 20 μ L 5g L-1MTT continue cultivate 4 it is small when, PBS rinse after, add 150 μ L DMSO reacts 5 minutes, surveys absorbance.
(6) bio-imaging
Add a certain amount of NTSC-CDs solution in cultured HeLa cells, 37 DEG C cultivate 8 it is small when after PBS rinse, Imaging.Control group is the HeLa cells for not adding NTSC-CDs.
In the HeLa cells of NTSC-CDs cultures, Fe is continuously added3+When (30.0 μM) cultures 4 of solution are small, wash away and do not inhale The Fe of receipts3+, it is imaged under Laser Scanning Confocal Microscope.
Product characterizes
(1)TEM
The NTSC-CDs solution diluted is dropped on carbon film copper mesh, shape is observed under high-resolution-ration transmission electric-lens after dry Looks.
(2)XPS
Instrument makees excitaton source using Al K α, uses the element composition and existence form of XPS instrument analysis NTSC-CDs.
(3)FT-IR
A certain amount of KBr and NTSC-CDs is ground, film-making, the composition of infrared spectrum analysis NTSC-CDs.
(4) fluorescence spectrum
Using the luminescent properties of Fluorescence Spectrometer analysis NTSC-CDs.
(5) uv-visible absorption spectra
Blank is made with ultra-pure water, the optics of original carbon quantum dot, NTSC and NTSC-CDs are analyzed with ultraviolet-visible spectrometer Absorption characteristic.
Embodiment 2
4- naphthyl -3- thiosemicarbazides modifies the characterization of carbon quantum dot
Carbon quantum dot is synthesized first, is occurred using the carboxyl on carbon quantum dot surface and the amino of 4- naphthyl -3- thiosemicarbazides Condensation reaction, 4- naphthyl -3- thiosemicarbazides is modified to carbon quantum dot surface, carbon quantum dot is had certain identification function. Observed by high-resolution-ration transmission electric-lens, NTSC-CDs monodispersities in aqueous solution are good, shape almost spherical (Fig. 1 b), particle diameter collection In in the range of 1.6-4.4nm, average grain diameter is 3.1nm (Fig. 1 d).Original carbon quantum dot average grain diameter for 2.4nm (Fig. 1 a, C), the particle diameter of NTSC-CDs increases 0.7nm, shows that introducing 4- naphthyl -3- thiosemicarbazides on carbon quantum dot surface causes carbon The particle diameter increase of quantum dot.
Fourier's mid and far infrared spectrometer scanning result shows, original carbon quantum dot 3416cm-1And 1632cm-1The suction at place Peak is received, belongs to the stretching vibration peak of N-H/O-H and C=O, 3055cm-1The absworption peak at place belongs to O-H in carboxyl and stretches peak, explanation Contain a large amount of amino, carboxyl and hydroxyl (Fig. 2) in original carbon quantum dot surface.In carbon quantum dot after modification, from original carbon amounts The 3416cm of son point-1And 1632cm-1The absworption peak at place still suffers from, and 3055cm-1The O-H stretching vibration peaks at place, which are vanished from sight, (schemes 2), indicate the carboxyl in original carbon quantum dot and be converted to other groups, and amido link stretching vibration peak (1304cm-1) go out Now confirm this conjecture, illustrate 4- naphthyl -3- thiosemicarbazides amino and the carboxyl of carbon quantum dot there occurs amidation process. In addition, 4- naphthyl -3- thiosemicarbazides is in 1230cm-1And 1021cm-1The C=S bendings at place and stretching vibration peak and 786cm-1Place C-S stretching vibration peaks exist (Fig. 2) in NTSC-CDs.Confirm carbon quantum dot surface 4- naphthyl -3- thiosemicarbazides In the presence of.
In the full spectrograms of XPS of NTSC-CDs (Fig. 3), 284.9eV, 399.8eV, 531.1eV, 230.1eV and 161.7eV There are five peaks in place, corresponds respectively to C1s, N1s, O1s, S2s and S2p.High-resolution collection of illustrative plates (Fig. 3 a) display of wherein C1s, Five peaks at 288.2eV, 288.0eV, 286.4eV, 285.9eV and 284.6eV correspond respectively to C=O/C=S, C-S, C-O, C-N and C-C/C=C.And the peak at Fig. 3 b, 401.4eV and 399.6eV, correspond respectively to C-N and N-H.532.6eV and Peak at 531.2eV corresponds to C=O and O-H (Fig. 3 c).Peak at 163.0eV and 161.6eV corresponding to-C-S 2p1/2 and 2p3/2 (Fig. 3 d).This result is consistent with infrared results.And in material the content of C, N, O, S be respectively 62.5%, 13.4%, 22.2% and 1.9%.
Fig. 4 is that carbon quantum dot, 4- naphthyl -3- thiosemicarbazides and 4- naphthyl -3- thiosemicarbazides modify the ultraviolet of carbon quantum dot Spectrogram.Seen by figure, one and 4- naphthyl -3- thiosemicarbazides and original occurs at 281nm in the carbon quantum dot after modification The different absworption peak of carbon quantum dot, it was demonstrated that 4- naphthyl -3- thiosemicarbazides and original carbon quantum dot are successfully built together.
Excitation and the emission spectrum of modification carbon quantum dot are scanned with Fluorescence Spectrometer.Emission spectrum (Fig. 5-6) is analyzed, when sharp Wavelength is sent out in the range of 300nm to 330nm, the position of emission peak is almost unchanged with excitation wavelength increase, and fluorescence intensity is gradual Increase, 948 are risen to by 95.When excitation wavelength increases to 460nm by 340nm, emission peak is by 435nm red shifts to 515nm (Δs λ=81nm), fluorescence intensity is reduced to 130 by 948, and launch wavelength has dependence with excitation wavelength.Cause the original of dependence Cause, may come from the form difference of different functional groups on carbon quantum dot surface, cause so as to cause different emission sites.Modification The emission peak positions of carbon quantum dot are shown in Table 1-1 with excitation wavelength, the relation of fluorescence intensity afterwards.When excitation wavelength is 340nm, carbon Quantum dot fluorescence intensity reaches maximum (948), and emission peak is in 435nm, so as to obtain its optimal excitation and launch wavelength difference For 340nm and 435nm (Fig. 6), and the carbon quantum dot sends intense blue fluorescence under ultra violet lamp (Fig. 4 illustrations are right).
Table 1-1 compares excitation wavelength, emission peak and the fluorescence intensity of carbon quantum dot after modification
Embodiment 3
The stability of carbon quantum dot optical property after modification
It is well known that the existence of pH value influence carbon quantum dot surface functional group, and different functional groups and its presence State directly affects the optical property of carbon quantum dot again, for this reason, experimental selection pH investigates it to this materials optical for 3-10 The influence of energy, the result is shown in Fig. 7 a.As seen from the figure, when pH increases to 6 by 3, the fluorescence intensity of material slightly increases；PH is 6 to 8 When, fluorescence intensity is relatively stablized, and reaches maximum.Then as pH by 8 to 10, fluorescence intensity slightly reduces, on the whole pH value pair Its fluorescence intensity influences less, this may be related with the 4- naphthyl -3- thiosemicarbazides on carbon quantum dot surface, its pKa value is 10.4, influenced by pH small, therefore stability is preferable.It is excessive in view of pH value, Cu2+Hydrolysis and biologic applications reason, experimental selection 7.4 are used as optimal pH.In order to improve method sensitivity, and it have studied influence of the different buffer systems to material fluorescence intensity (pH=7.4).From Fig. 7 b, carbon nanomaterial fluorescence intensity in Tris-HCl buffer systems is maximum.Equally, ion is investigated Influence of the intensity to material emission performance shows (Fig. 7 c), strong in the fluorescence that concentration is NTSC-CDs in 0-1.0M NaCl solutions Degree is barely affected.This is detected in actual sample very useful with biologic applications for probe.
Embodiment 4
The optimization of experiment condition
Modification carbon quantum dot dosage, reaction time are optimized, obtain optimum experimental condition.Present invention measure Fe3+It is Fluorescence quenching effect mechanism based on probe, and the height of fluorescence probe intensity directly affects the sensitivity of method, fluorescence intensity It is again related with probe dosage, in order to improve method sensitivity, investigate influence of the probe dosage to fluorescence response.Take same concentrations (0.01g L-1) under the carbon quantum dots of different volumes add identical Fe to investigate3+When, the fluorescence intensity change of probe, as a result sends out It is existing, when NTSC-CDs dosages are 100 μ L, add Fe3+Fluorescence probe Strength Changes are smaller；Increase carbon quantum dot dosage, probe Fluorescence response also increase, when carbon quantum dot dosage is in the range of 400-800 μ L, fluorescence response is in almost a straight line, change Very little, shows Fe3+Combined with probe completely；When being further added by dosage to 900 μ L, fluorescence response increases again.At this moment fluorescence response Increase, it may be possible to due to adding the increase of carbon quantum dot concentration, result in aggregate themselves self-quenching and cause (Fig. 8).Carbon quantum dot Dosage is small, and probe response is low, so the dosage of carbon quantum dot elects 500 μ L as.Fig. 9 is shown in influence of the reaction time to probe response, When reaction starts 1 minute to 5 minutes, fluorescence probe intensity decreases quickly, continue thereafter with reduction；But it is 15 minutes between when reacted During by 30 minutes, the fluorescence intensity of probe is almost unchanged, illustrates that the reaction was complete, so it is 25 minutes that the reaction time is selected in experiment.
Embodiment 5
Sensitivity analysis of the probe to iron ion
Under optimum experimental condition, investigate and add Fe3+When concentration is 0-40.0 μ Μ, modified carbon quantum dot fluorescence intensity Change, with the sensitivity of Research Methods.Seen by result (Figure 10 a), with addition Fe3+Concentration increase, the fluorescence intensity of probe It is gradually reduced.As [Fe3+] in 0-0.20 μ Μ scopes (Figure 10 b), fluorescence intensity and concentration are in good correlation, its Stern- Volmer equations are F/F0=0.9970-0.4291 [Fe3+](R2=0.9984, F and F0Respectively Fe3+In the presence of with not in the presence of The fluorescence intensity of probe), detection is limited to 1.68nM, is significantly less than Fe in drinking water as defined in Environmental Protection in America part3+Maximum permit Perhaps concentration (18.3nM) and other Fe3+Fluorescence probe (table 1-2).
Table 1-2 differences fluorescent material based on Fe3+Probe
Embodiment 6
Probe selectively analyzes different metal ions
With same concentrations (30.0 μM) Na+、K+、Ca2+、Mg2+、Zn2+、Ba2+、Cu2+、Cd2+、Co2+、Mn2+、Pb2+、Ag+、 Fe2+、Fe3+、Al3+、Ni2+And Hg2+Come Research Methods selectivity, the result is shown in Figure 1 1a.Under equal conditions, add in probe solution Enter Fe3+Afterwards, the fluorescence intensity of probe significantly reduces, and 267 are dropped to by 968, and the amplitude that reduces is 72.4%.Relative to Fe3+, Fe2+With Cu2+The decline for causing fluorescence intensity is only 10.5% and 9.3%；And Na+、K+、Ca2+、Mg2+、Zn2+、Ba2+、Cd2+、Co2+、Mn2 +、Pb2+、Ag+、Al3+、Ni2+And Hg2+The decrease of fluorescence intensity is caused to be respectively less than 5.0%, probe is to Fe3+With selectivity.And incite somebody to action Same concentration Fe3+It is added in unmodified carbon quantum dot solution, its fluorescence intensity only reduces by 3.2%, this confirms fluorescence intensity Reduce related with the 4- naphthyl -3- thiosemicarbazides in carbon quantum dot.Contain three nitrogen-atoms, one sulphur atom in its molecule, Nitrogen, sulphur atom and metal ion, particularly heavy metal ion have a stronger complexing power, the oxygen-containing group in same carbon quantum dot Group is also effective coordination site of metal ion, but due to containing naphthyl in 4- naphthyl -3- thiosemicarbazides molecules, naphthyl can play one Fixed steric hindrance effect, and Fe3+Electronegativity is larger, ionic radius smaller, so that for other metal ions, Fe3+Binding ability between NTSC-CDs is more stronger, and inner filtering effect caused by the complexing between them has promoted carbon amounts The fluorescent quenching of son point.
Under similarity condition, work as Fe3+When being coexisted with above-mentioned metal ion, they are to Fe3+Quenching effect influence less (figure 11b).Research shows the probe to Fe above3+There are fine sensitivity and selectivity.
Embodiment 7
Intracellular iron ion detection
Using the cytotoxicity of MTT experiment research material.The NTSC-CDs solution of various concentrations is added in HeLa cells, 24h is cultivated, is found when the concentration of NTSC-CDs solution is 0.1g L-1When, the survival rate of cell is 96.2% (Figure 12).Increase dense Degree, when concentration is 1.0g L-1When, the survival rate of cell is still up to 87.5%.Continue to increase NTSC-CDs concentration, when concentration increases to 1.5g L-1With 2.0g L-1When, the survival rate of cell is respectively 85.0% and 84.1%, although survival rate is also reducing, drop Low amplitude is obviously reduced.Show NTSC-CDs small toxicities itself.
Equally, HeLa cells are selected to inquire into feasibilities of the NTSC-CDs as cell marker.Test NTSC-CDs It is added in HeLa cell culture mediums, when culture 8 is small, then fluorescence microscopy Microscopic observation is imaged.Seen by Figure 12, work as excitation wave During a length of 405nm and 488nm, HeLa cells have issued blueness and green fluorescence respectively, and the cell for not adding carbon quantum dot does not have There is fluorescence to send, show that NTSC-CDs can be used for biomarker, can also multi-color marking.In addition, it is also noted that in HeLa cells Fluorescence intensity is weaker in core, and intracytoplasmic fluorescence intensity is relatively strong, illustrates that carbon quantum dot is primarily present in cytoplasm, The application for being carbon quantum dot in cellular localization is laid certain basis by this.Continuously add Fe3+(30.0 μM) culture HeLa cells, The original fluorescence intensity of HeLa cells is substantially dimmed under fluorescence microscope, illustrates that NTSC-CDs can be additionally used in intracellular Fe3+Detection.
The present invention, has synthesized 4- naphthyl -3- thiosemicarbazides using straightforward procedure first.Then, with it to carbon quantum dot table Face is modified.Carbon quantum dot after modification can regulate and control fluorescent emission, obtain the fluorescence of different colours, good light stability, by pH value, Ionic strength and buffering are solution influenced small.Relative to other metal ions, Fe3+NTSC-CDs fluorescence, quenching effect can be effectively quenched Rate is up to 72.4%, and selectivity is good.And the sensitivity of method also increases substantially, test limit reaches 1.68nM, is not only far below Fe in the past3+The test limit of fluorescence probe, and the maximum acceptable concentration for the iron ions from drinking water formulated relative to the U.S. also drops It is nearly 10 times low.Carbon quantum dot cytotoxicity after modification is small, good biocompatibility, is imaged available for biological polychrome.It is and right Than in the endonuclear fluorescence intensities of HeLa, intracytoplasmic fluorescence intensity is relatively strong, shows that carbon quantum dot is primarily present in In cytoplasm, this has certain values for application of the carbon quantum dot in cellular localization.
The basic principles, main features and the advantages of the invention have been shown and described above.The technology of the industry Personnel are it should be appreciated that the present invention is not limited to the above embodiments, and the above embodiments and description only describe this The principle of invention, without departing from the spirit and scope of the present invention, various changes and modifications of the present invention are possible, these changes Change and improvement all fall within the protetion scope of the claimed invention.The claimed scope of the invention by appended claims and its Equivalent thereof.

Claims (8)

  1. Carbon quantum dot Fe in cell is detected after the modification of 1.4- naphthyl -3- thiosemicarbazides3+In application.
  2. Carbon quantum dot is in detection Fe after the modification of 2.4- naphthyl -3- thiosemicarbazides3+Application.
  3. 3. application according to claim 2, it is characterised in that its detection is limited to 1.68nM.
  4. 4. the application according to Claims 2 or 3, it is characterised in that the preparation method of the carbon quantum dot is as follows：Will 3.0gEDTA is added to be completely dissolved in 30mL ultra-pure waters after, solution is transferred in the autoclave of 50mL, 180 DEG C reaction 10 Hour；Cooling, product centrifugation, crosses film, dry, you can to obtain carbon quantum dot solid.
  5. 5. the application according to Claims 2 or 3, it is characterised in that the preparation method of the 4- naphthyls -3- thiosemicarbazides is such as Under：It is solvent to take isopropanol, and at room temperature, 1- naphthalenylisothiocyanates and hydration hydrazine reaction, generate 4- naphthyl -3- thiosemicarbazides.
  6. 6. application according to claim 5, it is characterised in that the different sulphur of 10mmol1- naphthyls is added in 50mL round-bottomed flasks Cyanate and 20mL isopropanols, until completely dissolved, are added dropwise 1.25mL hydrazine hydrates, at room temperature stirring reaction.TLC tracking is anti- Should, 1 it is small when the reaction was complete.Filter, washed respectively with water and isopropanol, obtain product 4- naphthyl -3- thiosemicarbazides.
  7. 7. the application according to Claims 2 or 3, it is characterised in that the 4- naphthyls -3- thiosemicarbazides modifies carbon quantum dot Preparation process is as follows：10mL 20mmol L are added in 20mL 0.2mg carbon quantum dots-1EDC and 5mL 20mmol L-1NHS, shakes Swing, 37 DEG C are reacted 30 minutes；Add 5mL 50g L-14- naphthyl -3- thiosemicarbazides, when reaction 2.5 is small；Mistake in product refrigerator At night, dialysis, the product drying that will be dialysed, obtains modification carbon quantum dot.
  8. 8. the application according to Claims 2 or 3, it is characterised in that carbon quantum dot is examined after the modification of 4- naphthyl -3- thiosemicarbazides Survey Fe3+The step of it is as follows：By 500 μ L 0.01g L-1NTSC-CDs, that is, 4- naphthyl -3- thiosemicarbazides modification after carbon quantum dot and Certain density Fe3+Solution is added in 1.5mL centrifuge tubes, and Tris-HCl buffer solutions are settled to 1.0mL, are sufficiently mixed, room temperature Lower reaction 25 minutes, detects its fluorescence intensity under the excitation wavelength of 340nm.
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