CN107902642A - A kind of preparation and application study of the graphene quantum dot being imaged for nuclei images and mitochondria - Google Patents
A kind of preparation and application study of the graphene quantum dot being imaged for nuclei images and mitochondria Download PDFInfo
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Abstract
The invention discloses a kind of graphene quantum dot for being used to target nuclei images and mitochondria imaging, it is by polyethyleneimine (PEI) and 1,3,6 trinitro- pyrenes react the graphene quantum dot (GQDs PEI) for forming PEI modifications under hydrothermal reaction condition, are imaged for cell nucleus targeting.On the basis of the GQDs PEI built, the surface construction that 4 carboxylic butyltriphenylphosphonium bromide phosphines (TPP) are grafted to GQDs PEI by amidation process is used for the GQDs TPP of Mitochondrially targeted imaging, and provides the preparation method and application of the two.Beneficial effects of the present invention are：The present invention applies nanometer technology, prepares GQDs PEI and GQDs TPP, is respectively used to cell nucleus targeting imaging and Mitochondrially targeted imaging, this method preparation is simple, and no especial equipment requirements are environmentally protective, of low cost, and can prepare on a large scale；GQDs PEI and GQDs TPP of the present invention have good biocompatibility and optical characteristics；Specific targeted imaging can be carried out to nucleus and mitochondria.
Description
Technical field
The present invention relates to biology medical material technical field, and in particular to one kind is used for nuclei images and mitochondria is imaged
Graphene quantum dot and its preparation method and application.
Background technology
Graphene quantum dot has a strong fluorescent brightness, good biocompatibility, dispersed and low light in high water
The characteristics such as bleaching, these characteristics make it be better than traditional fluorescent dye and inorganic-quantum-dot, in optical imagery, optical sensing, light
Volt battery and light emitting diode etc. are widely used.
In terms of fluorescence imaging, graphene quantum dot prepared by various synthetic methods is mainly used for various types of cells imaging, bag
Include tumour cell, nerve cell and stem cell etc..Since graphene quantum dot prepared at present is largely without targeting choosing
Selecting property, so can only be imaged to cytoplasm, lacks relevant research in nuclei images and mitochondria imaging etc..It is few
Number can carry out targeted imaging, but effect is all not satisfactory, and such as while some organelle is colored, surrounding other are thin
Born of the same parents' device may also be colored.Why nucleus and mitochondria are imagedIt is well known that nucleus is thin with mitochondria
Two important organelles of intracellular.One be inhereditary material control centre, one be energy source synthesis center, it is many
Genetic disease is related all with the dysfunction of the two organelles, thus be highly desirable to the forms of the two organelles and
Position is positioned.It is fluorescent dye to position at present common, but this kind of material has certain toxicity and light easily occurs
Bleaching phenomenon, these deficiencies limit its practical application.Compared with fluorescent dye, graphene quantum dot has good biofacies
Capacitive and low photobleaching Properties, therefore graphene quantum dot realization of the plan structure of the invention with targeting characteristic is important to two
The fluorescence imaging of organelle.
The generally use top-to-bottom method in terms of the preparation of graphene quantum dot at present, this method need the anti-of harshness
Condition, complicated processing step and low yield are answered, therefore improves the deficiency of existing preparation method, realizes graphene quantum dot
Extensive prepare is also innovation of the invention.
The content of the invention
The purpose of the present invention has toxicity and photobleaching aiming at the fluorescent dye of existing targeting nucleus and mitochondria
The deficiencies of, make full use of biocompatibility good existing for graphene quantum dot, high fluorescence intensity and low Bleachability etc.
Characteristic, develops a kind of extensive, environmentally protective preparation method, graphene quantum dot is can be achieved with to cell by simply modifying
The targeted imaging of core and mitochondria, while prepared graphene quantum dot has less toxic and good fluorescence characteristic.
To achieve these goals, technical solution provided by the invention is：One kind is used for specific to nucleus progress
The graphene quantum dot of targeted imaging, is by PEI and 1, and 3,6- trinitro- pyrenes realize 1,3 under high temperature, high pressure and alkaline condition,
Conversion of the 6- trinitro-s pyrene to graphene quantum dot and the table that PEI is grafted to graphene quantum dot by nucleophilic substitution
Face.Build on this basis it is a kind of for it is specific to nucleus carry out targeted imaging graphene quantum dot, be by 4- carboxylics
Butyltriphenylphosphonium bromide phosphine is grafted to the surface for the GQDs-PEI having been built up by amidation process.
Further, it is above-mentioned a kind of for the specific graphene quantum dot that targeted imaging is carried out to nucleus, institute
The weight ratio of the PEI stated and 1,3,6- trinitro-s are (0.5-2.5)：1.
Further, it is above-mentioned a kind of for the specific graphene quantum dot that targeted imaging is carried out to nucleus, be
The mixture of the two is placed in reaction kettle reacts 10-20h under the reaction condition of 180-220 degree.
Further, it is above-mentioned a kind of for the specific graphene quantum dot that targeted imaging is carried out to nucleus, be
Reaction solution is first passed through to 0.22 μm of miillpore filter to be filtered, large-sized carbon particle is removed, is then in molecular weight
Dialysis 3 days is carried out in 1000 bag filter.
Further, it is above-mentioned a kind of for the specific graphene quantum dot that targeted imaging is carried out to nucleus, its
Grain size is 1.75-6.25 nanometers, and average particle diameter size is 3.25 nanometers.Its excitation wavelength range is 369-450 nanometers, most
Big launch wavelength is 450 nanometers.
Further, it is above-mentioned a kind of for the specific graphene quantum dot that targeted imaging is carried out to nucleus, its
Concentration has relatively low cytotoxicity in the range of 18.75 μ g/mL-300 μ g/mL, can be specific in above range
Nucleus is imaged.
Constructed on the basis of GQDs-PEI a kind of for the specific graphene amount that targeted imaging is carried out to nucleus
It is sub-, it is the surface that 4- carboxylic butyltriphenylphosphonium bromide phosphines are grafted to the GQDs-PEI having been built up by amidation process.
Further, it is above-mentioned a kind of for the specific graphene quantum dot that targeted imaging is carried out to mitochondria, will
0.1-0.5g TPP are scattered in 100mL2- morpholino b acids, i.e. MES (0.1M, pH 4.5).
Further, it is above-mentioned a kind of for the specific graphene quantum dot that targeted imaging is carried out to mitochondria, fill
Nitrogen 30min, to remove the oxygen of dissolving.
Further, it is above-mentioned a kind of for the specific graphene quantum dot that targeted imaging is carried out to mitochondria, add
Enter condensing agent EDCI 0.06-0.33g, mix reaction 10min.
Further, it is above-mentioned a kind of for the specific graphene quantum dot that targeted imaging is carried out to mitochondria, add
Enter the GQDs-PEI of 1g synthesis, the reaction was continued 4-24h.
Further, it is above-mentioned a kind of for the specific graphene quantum dot that targeted imaging is carried out to mitochondria, be
Reaction solution is first passed through to 0.22 μm of miillpore filter to be filtered, large-sized carbon particle is removed, is then in molecular weight
Dialysis 3 days is carried out in 1000 bag filter.
Further, it is above-mentioned a kind of for the specific graphene quantum dot that targeted imaging is carried out to mitochondria, its
Grain size is 1.75-6.25 nanometers, and average particle diameter size is 3.25 nanometers.Its excitation wavelength range is 369-450 nanometers, most
Big launch wavelength is 450 nanometers.
Further, it is above-mentioned a kind of for the specific graphene quantum dot that targeted imaging is carried out to mitochondria, its
Concentration has relatively low cytotoxicity in the range of 18.75 μ g/mL-300 μ g/mL, can be specific in above range
Nucleus is imaged.
Second object of the present invention is：
Provide a kind of above-mentioned preparation side for the specific graphene quantum dot that targeted imaging is carried out to nucleus
Method, comprises the following steps：
1) by 0.1-0.5g PEI with being uniformly mixed after the 1,3,6- trinitro- pyrene ultrasounds 3-5h of 40mL 5mg/mL.Will be mixed
Close solution to be transferred in reaction kettle, react 10-20h in 180-220 degree, reaction kettle is naturally cooled into room temperature after the completion of reaction.
2) by the reaction solution that step 1) obtains in 0.22 μm of filtering with microporous membrane, remove large-size carbon particle and
Insoluble matter.
3) filtrate is placed in the bag filter that molecular weight is 1000 and dialysed, replaced a water every 6h, dialyse 3 days.
4) the GQDs-PEI solution that step 3) is prepared is freeze-dried, obtains GQDs-PEI powder.
Provide a kind of above-mentioned preparation side for the specific graphene quantum dot that targeted imaging is carried out to mitochondria
Method, comprises the following steps：
1) 0.1-0.5g TPP and 100mL 2-morpholine ethane sulfonic acid, i.e. MES (0.1M, pH 4.5) are uniformly mixed.
2) the solution inflated with nitrogen 30min for obtaining step 1).
3) EDCI 0.06-0.33g are added, after mixing reaction 10min, add in 1g claims 10 GQDs- prepared
PEI, reacts 4-24h under nitrogen protection after mixing.
4) by the reaction solution that step 1) obtains in 0.22 μm of filtering with microporous membrane, remove large-size carbon particle and
Insoluble matter.
5) filtrate is placed in the bag filter that molecular weight is 1000 and dialysed, a water, dialysis 3 are replaced when 6 is small
My god.
6) the GQDs-TPP solution that step 3) is prepared is freeze-dried, obtains GQDs-TPP powder.
Third object of the present invention is：
Provide a kind of graphene quantum dot and be used for the specific application that targeted imaging is carried out to nucleus.Further
, above-mentioned application, the valid density of the GQDs-PEI is 18.75 μ g-300 μ g/mL.
1.GQDs-PEI is used for the concrete scheme of cytotoxicity test：
(1) study subject：
Hela cells.
(2) test method：
Hela cell culture carries out cell culture in DMEM culture mediums, 10% hyclone of addition.The cell of culture is dilute
Concentration is interpreted into as 5 × 103100 μ L are added to 96 orifice plates, cultivate 24h by a/mL.The concentration for taking 100 μ L is respectively 18.75 μ g/
ML, 37.5 μ g/mL, 75 μ g/mL, 150 μ g/mL, the GQDs-PEI of 300 μ g/mL are added to 96 orifice plates, and control group only adds culture
Liquid.After cultivating 24h, 20 μ L MTT solution are added in the hole of 96 orifice plates, are further cultured for 4h, abandon upper solution, add 20 μ L
DMSO dissolves.The absorbance of the solution is recorded with microplate reader.Three parallel laboratory tests are carried out, data are subjected to statistical analysis.
The targeting nucleus fluorescence imaging concrete scheme of 2.GQDs-PEI：
(1) study subject：
Hela cells.
(2) test method：
Density is 3 × 103The Hela cell inoculations of a/mL are put into coverslip into six orifice plates, add 2mL culture mediums and cover
Lid coverslip carry out creep plate culture, incubator be incubated 24 it is small when after, add valid density be 18.75 μ g-300 μ g/mL GQDs-
PEI, carries out mitochondria dyeing and DAPI dyeing processing in incubation 2,4,8h respectively.
Step：
1) nutrient solution is sopped up, PBS is rinsed 3 times, each 5min, is added 4% paraformaldehydes of 1mL and is fixed 30min, is absorbed solid
Determine liquid, PBS is rinsed 3 times, each 5min；
2) 2 μ L are added under darkroomRed dyeing working solutions, incubation 30min, PBS flushing 3 times, every time
5min；
3) dye liquor is sopped up under darkroom, after PBS cleaning, DAPI dye liquors carry out core dye (cover coverslip just), put
Put 3-5min.Absorb DAPI, PBS cleaning 3 times, each 5min；
4) mounting, fixation, observe and take pictures under laser confocal microscope.
3.GQDs-TPP is used for the concrete scheme of cytotoxicity test with method 2.
4.GQDs-TPP is used for selectively targeted mitochondria fluorescence imaging concrete scheme with method 3.
For conventional fluorescent dyestuff there are easy photobleaching, prepared graphene quantum dot lack specific target tropism and
The problems such as preparation method is complicated, low yield, the present invention use simple hydro-thermal reaction and amidation process, are prepared on a large scale
The graphene quantum dot of PEI and TPP modifications.At the same time by carrying out simple chemical modification to quantum dot, realize to nucleus
And the selectively targeted imaging of mitochondria.Prepared quantum dot has good optical property and low cytotoxicity at the same time,
It disclosure satisfy that the requirement to nucleus and the imaging of mitochondria specificity fluorescent.
A kind of graphene quantum dot being used for nucleus and mitochondria specificity imaging provided by the invention and its preparation
Methods and applications, its advantage are as follows：
1st, the present invention uses simple hydro-thermal reaction and amidation process, is prepared for the graphene quantum of PEI and TPP modifications
Point, this method have the advantages that simple and easy to do, environmentally protective and yield is high.
2nd, the GQDs-PEI and GQDs-TPP that prepared by the present invention have good optical characteristics and low cytotoxicity.
3rd, GQDs-PEI and GQDs-TPP prepared by the present invention, is able to specific to nucleus and mitochondria progress
Targeted imaging.
Brief description of the drawings
The infrared spectrum of Fig. 1 PEI, GQDs-PEI, TPP and GQDs-TPP.
The XPS of Fig. 2 GQDs-PEI and GQDs-TPP are composed (A) entirely, GQDs-PEI and GQDs-TPP high-resolution C1s spectrums (B)
(C), GQDs-PEI and GQDs-TPP high-resolution N1s composes the high-resolution P2p spectrums of (D) (E) and GQDs-TPP.Fig. 3 XRD spectrums and
Raman spectrum.
The images of transmissive electron microscope (A) (B) of Fig. 4 GQDs-PEI and GQDs-TPP, illustration are distributed for the grain size of sample.
The atomic force microscope images (C) (D) of GQDs-PEI and GQDs-TPP, illustration are the height characteristic of sample.
The spectrum characteristic (A) (C) of Fig. 5 GQDs-PEI and GQDs-TPP and the fluorescence emission spectrum under different excitation wavelengths
(B)(D)。
The cytotoxicity that the GQDs-PEI and GQDs-TPP of Fig. 6 various concentrations co-culture 4 days with Hela cells detects.
The laser co-focusing picture of Fig. 7 GQDs-PEI specific cell nuclear imagings.DAPI dyes (A), GQDs-PEI dyeing
(B), Mito-Tracker dyes (C) and the picture (D) of different fluorescence superpositions.
The laser co-focusing picture of Fig. 8 GQDs-TPP specific mitochondrials imaging.DAPI dyes (A), GQDs-PEI dyeing
(B), Mito-Tracker dyes (C) and the picture (D) of different fluorescence superpositions.
Embodiment
Embodiment one
A kind of above-mentioned preparation method for being used for specificity and the graphene quantum dot of targeted imaging being carried out to nucleus is provided,
Comprise the following steps：
1) by 0.1g PEI with being uniformly mixed after the 1,3,6- trinitro- pyrene ultrasounds 3-5h of 40mL 5mg/mL.It will mix molten
Liquid is transferred in reaction kettle, reacts 10h in 180 degree, reaction kettle is naturally cooled to room temperature after the completion of reaction.
2) by the reaction solution that step 1) obtains in 0.22 μm of filtering with microporous membrane, remove large-size carbon particle and
Insoluble matter.
3) filtrate is placed in the bag filter that molecular weight is 1000 and dialysed, a water, dialysis 3 are replaced when 6 is small
My god.
4) the GQDs-PEI solution that step 3) is prepared is freeze-dried, obtains GQDs-PEI powder.
Provide a kind of above-mentioned preparation side for the specific graphene quantum dot that targeted imaging is carried out to mitochondria
Method, comprises the following steps：
1) 0.1g TPP and 100mL MES (0.1M, pH 4.5) are uniformly mixed.
2) the solution inflated with nitrogen 30min for obtaining step 1).
3) EDCI 0.06g are added, after mixing reaction 10min, the GQDs-PEI prepared is added in 1g claims 10, mixes
4h is reacted after even under nitrogen protection.
4) by the reaction solution that step 1) obtains in 0.22 μm of filtering with microporous membrane, remove large-size carbon particle and
Insoluble matter.
5) filtrate is placed in the bag filter that molecular weight is 1000 and dialysed, replaced a water every 6h, dialyse 3 days.
6) GQDs-TPP solution prepared by step 3) is freeze-dried, obtains GQDs-TPP powder.
Embodiment two
Provide a kind of above-mentioned preparation side for the specific graphene quantum dot that targeted imaging is carried out to nucleus
Method, comprises the following steps：
1) by 0.1g PEI with being uniformly mixed after the 1,3,6- trinitro- pyrene ultrasounds 3-5h of 40mL 5mg/mL.It will mix molten
Liquid is transferred in reaction kettle, reacts 10h in 200 degree, reaction kettle is naturally cooled to room temperature after the completion of reaction.
2) by the reaction solution that step 1) obtains in 0.22 μm of filtering with microporous membrane, remove large-size carbon particle and
Insoluble matter.
3) filtrate is placed in the bag filter that molecular weight is 1000 and dialysed, replaced a water every 6h, dialyse 3 days.
4) the GQDs-PEI solution that step 3) is prepared is freeze-dried, obtains GQDs-PEI powder.
Provide a kind of above-mentioned preparation side for the specific graphene quantum dot that targeted imaging is carried out to mitochondria
Method, comprises the following steps：
1) 0.1g TPP and 100mL MES (0.1M, pH 4.5) are uniformly mixed.
2) the solution inflated with nitrogen 30min for obtaining step 1).
3) EDCI 0.06g are added, after mixing reaction 10min, the GQDs-PEI prepared is added in 1g claims 10, mixes
4h is reacted after even under nitrogen protection.
4) by the reaction solution that step 1) obtains in 0.22 μm of filtering with microporous membrane, remove large-size carbon particle and
Insoluble matter.
5) filtrate is placed in the bag filter that molecular weight is 1000 and dialysed, replaced a water every 6h, dialyse 3 days.
6) the GQDs-TPP solution that step 3) is prepared is freeze-dried, obtains GQDs-TPP powder.
Embodiment three
Provide a kind of above-mentioned preparation side for the specific graphene quantum dot that targeted imaging is carried out to nucleus
Method, comprises the following steps：
1) will be uniformly mixed after during the 0.1g PEI and 1,3,6- trinitro- pyrene ultrasounds 3-5 of 40mL 5mg/mL small.Will mixing
Solution is transferred in reaction kettle, and when 200 degree of reactions 20 are small, reaction kettle is naturally cooled to room temperature after the completion of reaction.
2) by the reaction solution that step 1) obtains in 0.22 μm of filtering with microporous membrane, remove large-size carbon particle and
Insoluble matter.
3) filtrate is placed in the bag filter that molecular weight is 1000 and dialysed, replaced a water every 6h, dialyse 3 days.
4) the GQDs-PEI solution that step 3) is prepared is freeze-dried, obtains GQDs-PEI powder.
Provide a kind of above-mentioned preparation side for the specific graphene quantum dot that targeted imaging is carried out to mitochondria
Method, comprises the following steps：
1) 0.1g TPP and 100mL MES (0.1M, pH 4.5) are uniformly mixed.
2) the solution inflated with nitrogen 30min for obtaining step 1).
3) EDCI 0.06g are added, mix reaction after ten minutes, add in 1g claims 10 GQDs-PEI prepared, are mixed
4h is reacted after even under nitrogen protection.
4) by the reaction solution that step 1) obtains in 0.22 μm of filtering with microporous membrane, remove large-size carbon particle and
Insoluble matter.
5) filtrate is placed in the bag filter that molecular weight is 1000 and dialysed, replaced a water every 6h, dialyse 3 days.
6) the GQDs-TPP solution that step 3) is prepared is freeze-dried, obtains GQDs-TPP powder.
Example IV
Provide a kind of above-mentioned preparation side for the specific graphene quantum dot that targeted imaging is carried out to nucleus
Method, comprises the following steps：
1) by 0.5g PEI with being uniformly mixed after the 1,3,6- trinitro- pyrene ultrasounds 3-5h of 40mL 5mg/mL.It will mix molten
Liquid is transferred in reaction kettle, reacts 10h in 200 degree, reaction kettle is naturally cooled to room temperature after the completion of reaction.
2) by the reaction solution that step 1) obtains in 0.22 μm of filtering with microporous membrane, remove large-size carbon particle and
Insoluble matter.
3) filtrate is placed in the bag filter that molecular weight is 1000 and dialysed, replaced a water every 6h, dialyse 3 days.
4) the GQDs-PEI solution that step 3) is prepared is freeze-dried, obtains GQDs-PEI powder.
Provide a kind of above-mentioned preparation side for the specific graphene quantum dot that targeted imaging is carried out to mitochondria
Method, comprises the following steps：
1) 0.5g TPP and 100mL MES (0.1M, pH 4.5) are uniformly mixed.
2) the solution inflated with nitrogen 30min for obtaining step 1).
3) EDCI 0.33g are added, after mixing reaction 10min, the GQDs-PEI prepared is added in 1g claims 10, mixes
4h is reacted after even under nitrogen protection.
4) by the reaction solution that step 1) obtains in 0.22 μm of filtering with microporous membrane, remove large-size carbon particle and
Insoluble matter.
5) filtrate is placed in the bag filter that molecular weight is 1000 and dialysed, replaced a water every 6h, dialyse 3 days.
6) the GQDs-TPP solution that step 3) is prepared is freeze-dried, obtains GQDs-TPP powder.
As a result：
1. Fig. 1 is the infrared spectrum of PEI, GQDs-PEI, TPP and GQDs-TPP.
After PEI is grafted to GQDs, PEI is located at 1568cm-1Primary amine peak die down, and GQDs-PEI is located at 1660cm-1It is secondary
Amine peak becomes strong, shows that nucleophilic substitution occurs between the two.When TPP is grafted to GQDs-PEI by amido link, in 1656cm-1Place
There is the peak of I key of acid amides.
2. the XPS that Fig. 2 is GQDs-PEI and GQDs-TPP is composed (A) entirely, GQDs-PEI and GQDs-TPP high-resolution C1s spectrums
(B) the high-resolution P2p spectrums of (C), GQDs-PEI and GQDs-TPP high-resolution N1s spectrums (D) (E) and GQDs-TPP.
GQDs-PEI contains carbon, nitrogen and oxygen element, after forming GQDs-TPP, in addition to carbon, nitrogen and oxygen element, also has new appearance
P elements.The C1s high-resolution spectrogram of GQDs-PEI can be fitted to three peaks, respectively positioned at 284.7,286.1 and
287.9eV, corresponding to C=C, C-N and C-O keys.After forming GQDs-TPP, except C=C, outside C-N keys, there are C=O keys, demonstrate,prove
The formation of bright amido link.
3. Fig. 3 is XRD spectrum and Raman spectrum.
There is a non-structure cell 20.1 degree of position in PEI, after it forms GQDs-PEI, graphene occurs at 25.8 degree
Characteristic peak.There are the characteristic peak of TPP in the XRD spectrum of GQDs-TPP, it was demonstrated that TPP is grafted on GQDs.
4. Fig. 4 is the images of transmissive electron microscope (A) (B) of GQDs-PEI and GQDs-TPP, illustration divides for the grain size of sample
Cloth.The atomic force microscope images (C) (D) of GQDs-PEI and GQDs-TPP, illustration are the height characteristic of sample.
Transmission electron microscope shows that the particle size range of GQDs-PEI and GQDs-TPP arrives 6.25nm for 1.75, and wherein GQDs-PEI is put down
Equal particle diameter is 3.75nm, and the average particle diameter size of GQDs-TPP is 3.25nm.Atomic force microscope shows the height of GQDs-PEI
It is highly 4.6nm to spend for 3.5nm, GQDs-TPP.
5. the spectrum characteristic (A) (C) of Fig. 5 GQDs-PEI and GQDs-TPP and the fluorescence emission under different excitation wavelengths
Compose (B) (D).
Strong green fluorescence occurs under the irradiation of ultraviolet light for GQDs-PEI and GQDs-TPP.The optical absorption peak of GQDs-PEI
It is located at 230nm and 369nm respectively positioned at 250nm and 369nm, GQDs-TPP optical absorption peak respectively.The emission spectrum of the two is presented
Exciting light dependence, with the increase of excitation wavelength, occurs Red Shift Phenomena.
Embodiment 6：
GQDs-PEI is used for the concrete scheme of cytotoxicity test：
(1) study subject：
Hela cells.
(2) test method：
Hela cell culture carries out cell culture in DMEM culture mediums, 10% hyclone of addition.The cell of culture is dilute
Concentration is interpreted into as 5 × 103100 μ L are added to 96 orifice plates, cultivate 24h by a/mL.The concentration for taking 100 μ L is respectively 18.75 μ g/
ML, 37.5 μ g/mL, 75 μ g/mL, 150 μ g/mL, the GQDs-PEI of 300 μ g/mL are added to 96 orifice plates, and control group only adds culture
Liquid.After cultivating 24h, 20 μ L MTT solution are added in the hole of 96 orifice plates, are further cultured for 4h, abandon upper solution, add 20 μ L
DMSO dissolves.The absorbance of the solution is recorded with microplate reader.Three parallel laboratory tests are carried out, data are subjected to statistical analysis.
The glimmering nucleus fluorescence imaging concrete scheme of targeting of GQDs-PEI：
(1) study subject：
Hela cells.
(2) test method：
Density is 3 × 103The Hela cell inoculations of/mL are put into coverslip into six orifice plates, add the covering of 2mL culture mediums
Coverslip carries out creep plate culture, after incubator is incubated 24h, the GQDs-PEI that valid density is 18.75 μ g/mL is added, after being incubated 2h
Carry out mitochondria dyeing and DAPI dyeing processing.
Step：
1) nutrient solution is sopped up, PBS is rinsed 3 times, each 5min, is added 4% paraformaldehydes of 1mL and is fixed 30min, is absorbed solid
Determine liquid, PBS is rinsed 3 times, each 5min；
2) 2 μ L are added under darkroomRed dyeing working solutions, incubation 30min, PBS flushing 3 times, every time
5min；
3) dye liquor is sopped up under darkroom, after PBS cleaning, DAPI dye liquors carry out core dye (cover coverslip just),
Place 3-5min.Absorb DAPI, PBS cleaning 3 times, each 5min；
4) mounting, fixation, observe and take pictures under laser confocal microscope.
GQDs-TPP is used for the concrete scheme of cytotoxicity test with method 2.
GQDs-TPP is used for selectively targeted mitochondria fluorescence imaging concrete scheme with method 3.
Embodiment 7：
GQDs-PEI is used for the concrete scheme of cytotoxicity test：
(1) study subject：
Hela cells.
(2) test method：
Hela cell culture carries out cell culture in DMEM culture mediums, 10% hyclone of addition.The cell of culture is dilute
Concentration is interpreted into as 5 × 103100 μ L are added to 96 orifice plates, cultivate 24h by a/mL.The concentration for taking 100 μ L is respectively 18.75 μ g/
ML, 37.5 μ g/mL, 75 μ g/mL, 150 μ g/mL, the GQDs-PEI of 300 μ g/mL are added to 96 orifice plates, and control group only adds culture
Liquid.After cultivating 24h, 20 μ L MTT solution are added in the hole of 96 orifice plates, are further cultured for 4h, abandon upper solution, add 20 μ L
DMSO dissolves.The absorbance of the solution is recorded with microplate reader.Three parallel laboratory tests are carried out, data are subjected to statistical analysis.
The glimmering nucleus fluorescence imaging concrete scheme of targeting of GQDs-PEI：
(1) study subject：
Hela cells.
(2) test method：
Density is 3 × 103The Hela cell inoculations of/mL are put into coverslip into six orifice plates, add the covering of 2mL culture mediums
Coverslip carries out creep plate culture, after incubator is incubated 24h, the GQDs-PEI that valid density is 18.75 μ g/mL is added, after being incubated 4h
Carry out mitochondria dyeing and DAPI dyeing processing.
Step：
1) nutrient solution is sopped up, PBS is rinsed 3 times, each 5min, is added 4% paraformaldehydes of 1mL and is fixed 30min, is absorbed solid
Determine liquid, PBS is rinsed 3 times, each 5min；
2) 2 μ L are added under darkroomRed dyeing working solutions, incubation 30min, PBS flushing 3 times, every time
5min；
3) dye liquor is sopped up under darkroom, after PBS cleaning, DAPI dye liquors carry out core dye (cover coverslip just),
Place 3-5min.Absorb DAPI, PBS cleaning 3 times, each 5min；
4) mounting, fixation, observe and take pictures under laser confocal microscope.
GQDs-TPP is used for the concrete scheme of cytotoxicity test with method 2.
GQDs-TPP is used for selectively targeted mitochondria fluorescence imaging concrete scheme with method 3.
Embodiment 8：
GQDs-PEI is used for the concrete scheme of cytotoxicity test：
(1) study subject：
Hela cells.
(2) test method：
Hela cell culture carries out cell culture in DMEM culture mediums, 10% hyclone of addition.The cell of culture is dilute
Concentration is interpreted into as 5 × 103100 μ L are added to 96 orifice plates, cultivate 24h by a/mL.The concentration for taking 100 μ L is respectively 18.75 μ g/
ML, 37.5 μ g/mL, 75 μ g/mL, 150 μ g/mL, the GQDs-PEI of 300 μ g/mL are added to 96 orifice plates, and control group only adds culture
Liquid.After cultivating 24h, 20 μ L MTT solution are added in the hole of 96 orifice plates, are further cultured for 4h, abandon upper solution, add 20 μ L
DMSO dissolves.The absorbance of the solution is recorded with microplate reader.Three parallel laboratory tests are carried out, data are subjected to statistical analysis.
The glimmering nucleus fluorescence imaging concrete scheme of targeting of GQDs-PEI：
(1) study subject：
Hela cells.
(2) test method：
Density is 3 × 103The Hela cell inoculations of/mL are put into coverslip into six orifice plates, add the covering of 2mL culture mediums
Coverslip carries out creep plate culture, after incubator is incubated 24h, the GQDs-PEI that valid density is 18.75 μ g/mL is added, after being incubated 8h
Carry out mitochondria dyeing and DAPI dyeing processing.
Step：
1) nutrient solution is sopped up, PBS is rinsed 3 times, each 5min, is added 4% paraformaldehydes of 1mL and is fixed 30min, is absorbed solid
Determine liquid, PBS is rinsed 3 times, each 5min；
2) 2 μ L are added under darkroomRed dyeing working solutions, incubation 30min, PBS flushing 3 times, every time
5min；
3) dye liquor is sopped up under darkroom, after PBS cleaning, DAPI dye liquors carry out core dye (cover coverslip just),
Place 3-5min.Absorb DAPI, PBS cleaning 3 times, each 5min；
4) mounting, fixation, observe and take pictures under laser confocal microscope.
GQDs-TPP is used for the concrete scheme of cytotoxicity test with method 2.
GQDs-TPP is used for selectively targeted mitochondria fluorescence imaging concrete scheme with method 3.
Embodiment 9：
GQDs-PEI is used for the concrete scheme of cytotoxicity test：
(1) study subject：
Hela cells.
(2) test method：
Hela cell culture carries out cell culture in DMEM culture mediums, 10% hyclone of addition.The cell of culture is dilute
Concentration is interpreted into as 5 × 103100 μ L are added to 96 orifice plates, cultivate 24h by a/mL.The concentration for taking 100 μ L is respectively 18.75 μ g/
ML, 37.5 μ g/mL, 75 μ g/mL, 150 μ g/mL, the GQDs-PEI of 300 μ g/mL are added to 96 orifice plates, and control group only adds culture
Liquid.After cultivating 24h, 20 μ L MTT solution are added in the hole of 96 orifice plates, are further cultured for 4h, abandon upper solution, add 20 μ L
DMSO dissolves.The absorbance of the solution is recorded with microplate reader.Three parallel laboratory tests are carried out, data are subjected to statistical analysis.
The glimmering nucleus fluorescence imaging concrete scheme of targeting of GQDs-PEI：
(1) study subject：
Hela cells.
(2) test method：
Density is 3 × 103The Hela cell inoculations of/mL are put into coverslip into six orifice plates, add the covering of 2mL culture mediums
Coverslip carries out creep plate culture, after incubator is incubated 24h, adds the GQDs-PEI that valid density is 150 μ g/mL, it is laggard to be incubated 4h
Line plastochondria dyes and DAPI dyeing processing.
Step：
1) nutrient solution is sopped up, PBS is rinsed 3 times, each 5min, is added 4% paraformaldehydes of 1mL and is fixed 30min, is absorbed solid
Determine liquid, PBS is rinsed 3 times, each 5min；
2) 2 μ L are added under darkroomRed dyeing working solutions, incubation 30min, PBS flushing 3 times, every time
5min；
3) dye liquor is sopped up under darkroom, after PBS cleaning, DAPI dye liquors carry out core dye (cover coverslip just),
Place 3-5min.Absorb DAPI, PBS cleaning 3 times, each 5min；
4) mounting, fixation, observe and take pictures under laser confocal microscope.
GQDs-TPP is used for the concrete scheme of cytotoxicity test with method 2.
GQDs-TPP is used for selectively targeted mitochondria fluorescence imaging concrete scheme with method 3.
Embodiment 10：
GQDs-PEI is used for the concrete scheme of cytotoxicity test：
(1) study subject：
Hela cells.
(2) test method：
Hela cell culture carries out cell culture in DMEM culture mediums, 10% hyclone of addition.The cell of culture is dilute
Concentration is interpreted into as 5 × 103100 μ L are added to 96 orifice plates, cultivate 24h by a/mL.The concentration for taking 100 μ L is respectively 18.75 μ g/
ML, 37.5 μ g/mL, 75 μ g/mL, 150 μ g/mL, the GQDs-PEI of 300 μ g/mL are added to 96 orifice plates, and control group only adds culture
Liquid.After cultivating 24h, 20 μ L MTT solution are added in the hole of 96 orifice plates, are further cultured for 4h, abandon upper solution, add 20 μ L
DMSO dissolves.The absorbance of the solution is recorded with microplate reader.Three parallel laboratory tests are carried out, data are subjected to statistical analysis.
The glimmering nucleus fluorescence imaging concrete scheme of targeting of GQDs-PEI：
(1) study subject：
Hela cells.
(2) test method：
Density is 3 × 103The Hela cell inoculations of/mL are put into coverslip into six orifice plates, add the covering of 2mL culture mediums
Coverslip carries out creep plate culture, after incubator is incubated 24h, adds the GQDs-PEI that valid density is 300 μ g/mL, it is laggard to be incubated 4h
Line plastochondria dyes and DAPI dyeing processing.
Step：
1) nutrient solution is sopped up, PBS is rinsed 3 times, each 5min, is added 4% paraformaldehydes of 1mL and is fixed 30min, is absorbed solid
Determine liquid, PBS is rinsed 3 times, each 5min；
2) 2 μ L are added under darkroomRed dyeing working solutions, incubation 30min, PBS flushing 3 times, every time
5min；
3) dye liquor is sopped up under darkroom, after PBS cleaning, DAPI dye liquors carry out core dye (cover coverslip just),
Place 3-5min.Absorb DAPI, PBS cleaning 3 times, each 5min；
4) mounting, fixation, observe and take pictures under laser confocal microscope.
GQDs-TPP is used for the concrete scheme of cytotoxicity test with method 2.
GQDs-TPP is used for selectively targeted mitochondria fluorescence imaging concrete scheme with method 3.
As a result：
Fig. 6 is the cytotoxicity detection that the GQDs-PEI and GQDs-TPP of various concentrations and Hela cells co-culture 4 days.
PEI has higher cytotoxicity, and after forming GQDs-PEI, toxicity substantially reduces.TPP and GQDs-TPP are hardly
With cytotoxicity.
Fig. 7 is the laser co-focusing picture of GQDs-PEI specific cell nuclear imagings.DAPI dyes (A), GQDs-PEI dyes
The picture (D) of color (B), Mito-Tracker dyeing (C) and different fluorescence superpositions.
Laser confocal imaging shows：GQDs-PEI can selectively targeted nuclei images, make nuclei dyeing into green,
And it is completely superposed with the cell nuclear location of DAPI dyeing.Common location effect is further detected with by Image J softwares, is sent out
It is completely superposed both existing in the intensity at peak and the position at peak.
The laser co-focusing picture of Fig. 8 GQDs-TPP specific mitochondrials imaging.DAPI dyes (A), GQDs-PEI dyeing
(B), Mito-Tracker dyes (C) and the picture (D) of different fluorescence superpositions.
Laser confocal imaging shows：GQDs-TPP can the imaging of selectively targeted mitochondria, mitochondria is dyed green,
And it is completely superposed with the mitochondria position of Mito-Tracker dyeing.With by Image J softwares to common location effect into one
Step detection, it is found that the position at the two peak is completely superposed, the intensity somewhat difference at peak.
Finally it should be noted that：The foregoing is only a preferred embodiment of the present invention, is not intended to limit the invention,
Although the present invention is described in detail with reference to the foregoing embodiments, for those skilled in the art, it still may be used
To modify to the technical solution described in foregoing embodiments, or equivalent substitution is carried out to which part technical characteristic.
Within the spirit and principles of the invention, any modification, equivalent replacement, improvement and so on, should be included in the present invention's
Within protection domain.
Claims (10)
1. a kind of for the specific graphene quantum dot that targeted imaging is carried out to nucleus and mitochondria, i.e. GQDs, it is special
Sign is, is by polyethyleneimine, i.e. PEI, and 1,3,6- trinitro- pyrenes form the graphite of PEI modifications under hydrothermal reaction condition
Alkene quantum dot, i.e. GQDs-PEI, and by amidation process by 4- carboxylic butyltriphenylphosphonium bromide phosphines, i.e. TPP, is grafted to GQDs-
The GQDs-TPP of the surface construction of PEI.
2. the preparation method according to claim 1 for the specific GQDs that targeted imaging is carried out to nucleus, its
It is characterized in that, comprises the following steps：
1) by PEI with being uniformly mixed after 1,3,6- trinitro- pyrene ultrasounds 3-5h.Mixed solution is transferred in reaction kettle, in 180-
220 degree of reaction 10-20h, room temperature is naturally cooled to after the completion of reaction by reaction kettle；
2) by the reaction solution that step 1) obtains in filtering with microporous membrane, the carbon particle and insoluble matter of large-size are removed；
3) filtrate is placed in the bag filter that molecular weight is 1000 and dialysed, replaced a water every 6h, dialyse 3 days；
4) the GQDs-PEI solution that step 3) is prepared is freeze-dried, obtains GQDs-PEI powder；
5) the GQDs-PEI powder that step 4) is prepared is diluted in PBS, the GQDs-PEI of various concentrations is made, together
Hela cells co-culture, and detect its cytotoxicity；
6) it is a kind of for the specific graphene amount that targeted imaging is carried out to nucleus according to claim 1-5 is any
Son point carries out the application of specific cell nuclear imaging.
3. the preparation according to claim 1 for the specific graphene quantum dot that targeted imaging is carried out to nucleus
Method, it is characterised in that comprise the following steps：
1) by TPP and 0.1M, the 2-morpholine ethane sulfonic acid of pH 4.5, i.e. MES, are uniformly mixed；
2) the solution inflated with nitrogen 30min for obtaining step 1)；
3) EDCI is added, after mixing reaction 10min, the GQDs-PEI prepared is added in 1g claims 10, in nitrogen after mixing
The lower reaction 4-24h of protection；
4) by the reaction solution that step 1) obtains in 0.22 μm of miillpore filter, the carbon particle and insoluble matter of large-size are removed；
5) filtrate is placed in the bag filter that molecular weight is 1000 and dialysed, a water is replaced when 6 is small, dialysed 3 days；
6) the GQDs-TPP solution that step 3) is prepared is freeze-dried, obtains GQDs-TPP powder；
7) the GQDs-TPP powder that step 6) is prepared is diluted in PBS, the GQDs-TPP of various concentrations is made, together
Hela cells co-culture, and detect its cytotoxicity.
4. according to claim 1 a kind of for the specific GQDs that targeted imaging is carried out to nucleus, its feature exists
In PEI and 1 in the reaction system, the weight ratio of 3,6- trinitro- pyrenes is (0.5-2.5)：1.
5. according to claim 1 a kind of for the specific GQDs that targeted imaging is carried out to mitochondria, its feature exists
In TPP and GQDs-PEI weight ratios are (0.5-7.5) in the reaction system：1.
6. according to claim 2 a kind of for the specific GQDs that targeted imaging is carried out to nucleus, its feature exists
In, be by PEI and 1,3,6- trinitro- pyrenes reacted under 180-220 degree hydrothermal reaction conditions 10-20 it is small when, utilize nucleophilic displacement of fluorine
PEI is grafted to the surface of GQDs by reaction.
7. according to claim 3 a kind of for the specific GQDs that targeted imaging is carried out to mitochondria, its feature exists
In, be by TPP and GQDs-PEI condensing agent 1- (3- dimethylamino-propyls) -3- ethyl carbodiimides (EDCI) effect under will contain
The surface construction GQDs-TPP that TPP is grafted to GQDs-PEI by amidation process occurs for the amino of the TPP and GQDs-PEI of carboxyl.
The weight ratio 1.5 of TPP and EDCI：1.
8. according to claim 4 a kind of for the specific GQDs that targeted imaging is carried out to nucleus, its feature exists
In its grain size is 1.75-6.25 nanometers, and average particle diameter size is 3.75 nanometers.Its excitation wavelength range is received for 369-450
Rice, maximum emission wavelength are 475 nanometers.
9. according to claim 5 a kind of for the specific GQDs that targeted imaging is carried out to mitochondria, its feature exists
In its grain size is 1.75-6.25 nanometers, and average particle diameter size is 3.25 nanometers.Its excitation wavelength range is received for 369-450
Rice, maximum emission wavelength are 450 nanometers.
10. it is according to claim 1 it is a kind of for it is specific to nucleus carry out targeted imaging GQDs-PEI or
GQDs-TPP, it has relatively low cytotoxicity in the range of 18.75 μ g/mL-300 μ g/mL, can be special in above range
Property is imaged nucleus.
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