CN104804722B - A kind of temperature sensitive intelligent fluorescent nano probe and preparation method thereof - Google Patents
A kind of temperature sensitive intelligent fluorescent nano probe and preparation method thereof Download PDFInfo
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- CN104804722B CN104804722B CN201510239011.XA CN201510239011A CN104804722B CN 104804722 B CN104804722 B CN 104804722B CN 201510239011 A CN201510239011 A CN 201510239011A CN 104804722 B CN104804722 B CN 104804722B
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Abstract
A kind of temperature sensitive intelligent fluorescent nano probe, it is characterised in that it is assembled using the quantum dot of temperature sensing polymer modification as donor, organic fluorescence molecule as acceptor, and emission spectrum and the absorption spectrum of organic fluorescence molecule of the quantum dot have overlapping.The invention provides a kind of thermally sensitive novel intelligent fluorescent nano probe, Thermo-sensitive and fluorescence are combined, there is Thermo-sensitive and fluorescence double-response, by the photoluminescent property of macroscopic visual, reach the purpose monitored in real time.
Description
Technical field
The present invention relates to a kind of temperature sensitive intelligent fluorescent nano probe and preparation method thereof, belong to biochemistry.
Background technology
As life science deepens continuously research, the conventional method of some research cellular environments is in use, can not accomplish
Dynamic monitoring is carried out to intracellular microenvironment in real time under living cells physiological condition.FRET technology (FRET)
Technology successfully solves this problem.The technology is applied to each quasi-molecule of living cells and fixed cell, sensitivity and resolution ratio
Height, and energy blur-free imaging, it can intuitively provide positioning and the quantitative information of protein interaction.Protein sensor conduct
The means of a kind of protein and the intermolecular forces of other molecules increasingly show its advantage.In FRET, selection is suitable
Donor Acceptor pair can ensure efficient energy transfer.Semiconductor-quantum-point (Qdots) is due to its tunable transmitted wave
It is long, narrow and symmetrical emission peak, wide excitation wave, anti-light Bleachability and the advantages that multiple dye molecules can be connected simultaneously, make
It turns into the donor of FRET more more preferable than organic fluorescence molecule.
Controllable assembly is carried out to Qdots and organic fluorescence molecule using functional polymer, fluorescence resonance energy can be achieved and turn
Move (FRET), by the high molecular molecular structure of adjusting function and aggregated structure, by the stimulation of environment, different pH value,
Change the spacing of Qdots and organic fluorescence molecule under the environment such as temperature, solvent, FRET efficiency occurs to change accordingly therewith.
Maltose-binding protein (MBP) that Medintz I L, Clapp A R etc. modify quantum dot and dye marker
Donor, acceptor pair of the beta-schardinger dextrin (β-CD) as FRET, it is proposed that the maltose bio-sensing of maltose attachment proteins (MBP)
The design of device.They also using multiple maltose attachment proteinses (MBP) mark light sensitivity 1,3,3- trimethyls-spiral shell [2,
2- indoline 2H-1- chromenes] (abbreviation BIPS) carry out the emission spectrum of reverse quantum point, and the efficiency of regulation can pass through
The quantity for changing BIPS is controlled, and the device or biology sensor of light sensitivity switch are realized with this.Medintz I L,
Anti- TNT specific antibodies fragment is connected on hydrophilic fluorescence quantum by Trammell S A etc. by metal adsorption effect, complete
Into the assembling of solution nanoscale sensor.Wei Q D, Lee M etc. using quantum dot as a kind of new fluorescence probe,
The monoclonal antibody of erss (ER- β) is marked, more grams of ER- β are marked with organic fluorescent dye Alexa Fluor (AF)
Grand antibody, then pass through the distance between the FRET detection probes between quantum dot and organic fluorescent dye.But at present, for inciting somebody to action
The research that temperature sensing polymer prepares temperature sensitive intelligent fluorescent nano probe with quantum dot progress Controllable assembly is also rarely reported.
The content of the invention
The technical problems to be solved by the invention are to provide a kind of Thermo-sensitive for above-mentioned the shortcomings of the prior art
Intelligent fluorescent nano-probe and preparation method thereof, there is Thermo-sensitive and fluorescence double-response, pass through the fluorescence of macroscopic visual
Matter, reach the purpose monitored in real time.
The present invention is to solve the problems, such as that used technical scheme set forth above is：
A kind of temperature sensitive intelligent fluorescent nano probe, it is used as donor, organic using the quantum dot of temperature sensing polymer modification
Fluorescence molecule assembles as acceptor, and emission spectrum and the absorption spectrum of organic fluorescence molecule of the quantum dot have overlapping.
By such scheme, the quantum dot be ZnS monokaryons quantum dot, CdS monokaryons quantum dot, CdSe monokaryons quantum dot,
CdSe/ZnS core shell quantum dots, CdSe/CdS/ZnS core shells/shell quantum dot, CdxZn1-xS quantum dots, CdSe/CdxZn1-xS cores/
Any one in alloy shell quantum dot etc..
By such scheme, the temperature sensing polymer is Thermo-sensitive double-end amino polymer or one end band amino one end band mercapto
The polymer of base, molecular weight is below 3000.Wherein, the Thermo-sensitive double-end amino polymer can be selected from the poly- second of double-end amino
Glycol (AT-PEG), double-end amino azobenzene polymer, amine terminated polyether, poly-o-phenylenediamine, double amidourethane (such as double-end aminos
Formic acid esters) etc. polymer；Described one end is selected from mercaptoaniline polymer, amino-poly- second with polymer of the amino one end with sulfydryl
The polymer such as glycol-sulfydryl.Such temperature sensing polymer has certain biocompatibility and particular functional group, the end of its strand
Hold away from surrounding environment pH value, solvent, temperature change and change, the distance change scope of molecule chain end is 1-20nm, pH
The amplitude of variation of value is 4-10, and temperature and solvent are with the different and different of molecular structure.
By such scheme, the organic fluorescence molecule is carboxylic organic fluorescence molecule, can be selected from carboxylic sieve
It is a kind of in red bright B, fluorescein isothiocynate and Fluoresceincarboxylic acid (such as 5-FAM, 6-FAM), dihydro fluorescein diacetate.Due to this
Quantum dot in invention is modified through temperature sensing polymer, and the quantum dot of temperature sensing polymer modification contains amino or sulfydryl, so
Organic fluorescence molecule selects carboxylic organic fluorescence molecule.
The preparation method of above-mentioned temperature sensitive intelligent fluorescent nano probe is：First quantum dot is repaiied with temperature sensing polymer
Decorations, the quantum dot after then temperature sensing polymer is modified carries out Controllable assembly with organic fluorescence molecule, so as to which Thermo-sensitive be made
Novel intelligent fluorescent nano material.
By such scheme, method that the temperature sensing polymer is modified quantum dot specifically comprises the following steps：
1) quantum dot is dissolved in chloroform, and adds temperature sensing polymer, until completely dissolved the ultrasound 2 at 20~45 DEG C
~8h；
2) cushioning liquid that pH is 8~10 is added dropwise into step 1) resulting solution, continues 12~24h of ultrasound, then stands
Layering, isolate the upper strata aqueous solution and with chloroform to remove organic impurities, then through dialysis, that is, obtain temperature sensing polymer and repair
The quantum dot aqueous solution after decorations.
In such scheme, temperature sensitive polymer is excessive abundant to ensure to modify relative to quantum dot in the step 1)
Carry out, the mol ratio of temperature sensing polymer and quantum dot is more than 80 in the present invention, wherein excessive temperature sensitive polymer can be rear
Removed in continuous dialysis step.
In such scheme, the pH scopes that buffer solution described in the step 2) is are 8~10, can select PBS
Or Tris-HCl buffer solutions etc..Wherein PBS, it is the mixed solution of potassium dihydrogen phosphate and disodium hydrogen phosphate；Tris-
The pH scopes of HCl buffer solutions are also 8~10, are the mixed liquors of tris solution and hydrochloric acid.
In such scheme, the volume of the buffer solution in the step 2) is advisable for 1~2 times of step 1) resulting solution.
In such scheme, the time of dialysis is 24~72h in the step 2).
By such scheme, the method for quantum dot and organic fluorescence molecule Controllable assembly after temperature sensing polymer modification, tool
Body comprises the following steps：
A) it is organic fluorescence molecule, 1- ethyls -3- (3- dimethylaminos)-carbodiimide (EDC) and N- hydroxysuccinimidyls acyl is sub-
Amine (NHS) stirs 2~4h in water, the quantum dot aqueous solution then added after temperature sensing polymer modification, stirs 1~4h；
B) plus alkali regulating step a) resulting solutions pH is 8~10, and stirs 30min~2h；Then be added dropwise pH be 6.8~
8.2 cushioning liquid and 12~24h of ultrasound, then through dialysing, being dried in vacuo, that is, obtain obtaining temperature sensitive intelligent fluorescent nano probe.
In such scheme, organic fluorescence molecule, 1- ethyls -3- (3- dimethylaminos)-carbodiimide in the step a)
(EDC) and n-hydroxysuccinimide (NHS) molar ratio range be 1：1~1.2：1~1.2.Wherein, step a) reclaimed waters plus
Entering 200-400 times that amount is organic fluorescence molecular mass is advisable, and organic fluorescence small molecule is about 10-30 times of quantum point mass.
In such scheme, the alkali in the step b) is TMAH (TMAOH).
In such scheme, pH of buffer scope is 6.8~8.2 in the step b), can select HEPES cushioning liquid,
It is 4- hydroxyethyl piperazineethanesulfonic acids solution and the mixed liquor of NaOH solution.
In such scheme, the time of dialysis is 24~72h in the step b), and the vacuum drying time is 24~48h.
Compared with prior art, the beneficial effects of the invention are as follows：
First, temperature sensitive intelligent fluorescent nano probe of the present invention has Thermo-sensitive and fluorescence dual responsiveness,
By environmental stimulus, transferring efficiency of fluorescence resonance energy changes therewith, can reach what is monitored in real time by macroscopical fluorescence
Purpose；
Second, temperature sensitive intelligent fluorescent nano probe transferring efficiency of fluorescence resonance energy of the present invention is high, and with
Environmental change, transferring efficiency of fluorescence resonance energy changes greatly, that is, is quick on the draw.
Brief description of the drawings
Fig. 1 is CdSe/CdxZn1-xThe fluorescence emission spectrum of S quantum dots and the absorption spectra of rhodamine B.
Fig. 2~5 are respectively that embodiment 1,2,3,4 prepares fluorescence resonance of the temperature sensitive intelligent fluorescent nano probe at 25 DEG C
Energy transfer collection of illustrative plates, transferring efficiency of fluorescence resonance energy press E=1-FDA/FDRelational expression calculates, wherein FDAIn the presence of acceptor
The fluorescence intensity of donor, FDThe fluorescence intensity of donor when being not present for acceptor.
Fig. 6 prepares temperature sensitive intelligent fluorescent nano probe for embodiment 4 and obtains fluorescence resonance energy turn at different temperatures
Move collection of illustrative plates.
Wherein, QDs represents quantum dot, and RhB represents rhodamine B, and FRET-1, FRET-2, FRET-3, FRET-4 are corresponded to respectively
Embodiment 1,2,3,4 prepares temperature sensitive intelligent fluorescent nano probe.
Fig. 7 is CdSe/Cd used in embodiment 1~4xZn1-xTransmission electron microscope (TEM) image of S quantum dots.
Fig. 8 is Cd used in embodiment 5~6xZn1-xThe TEM image of S quantum dots.
Embodiment
For a better understanding of the present invention, with reference to the embodiment content that the present invention is furture elucidated, but the present invention is not
It is limited only to the following examples.
CdSe/Cd used in embodiment 1~4xZn1-xThe preparation of S quantum dots：
Accurately weigh 4mmol zinc acetates (ZnAc2), 0.2mmol CdO, 5ml oleic acid and 15ml 1- octadecylenes (ODE)
It is put into 100ml three-necked flask, after 150 DEG C or so vacuumize 30min repeatedly, nitrogen is passed through as protective gas, by temperature
Degree is increased to 300 DEG C, to obtain clear transparent solutions, that is, Zn and Cd precursor mixed solution is made；Weigh 4mmol sulphur powders and
0.1mol selenium powders are put into reaction tube, and 0.5ml tri-n-octyl phosphines (TOP) are rapidly injected into reaction tube after vacuumizing repeatedly, 60 DEG C
Lower stirring 3h is completely dissolved Se, S, produces Se, S presoma；Se, S presoma are rapidly injected Zn's and Cd at 300 DEG C
In precursor mixed solution, mixed liquor is down to room temperature after 10min is stirred vigorously at 300 DEG C, will then react obtained mixing
It is 3 that liquid, which adds volume ratio,:1 acetone and chloroform centrifuges for several times, the CdSe/Cd that will finally obtainxZn1-xS nanocrystals exist
Dried at 60 DEG C, it is stand-by.
Cd used in embodiment 5~6xZn1-xThe preparation of S quantum dots：
The accurate ZnAc for weighing 10mmol2, 1mmol CdO, 5ml oleic acid and 15ml ODE be put into 100mL three mouthfuls of burnings
In bottle, after 150 DEG C or so vacuumize 30min repeatedly, nitrogen is passed through as protective gas, temperature is increased to 300 DEG C, to obtain
To clear transparent solutions, i.e., obtained Zn and Cd precursor mixed solution；Weigh 10mmol sulphur powders to be put into reaction tube, take out repeatedly
1mmol TOP is rapidly injected in reaction tube after vacuum, 3h is stirred at 60 DEG C is completely dissolved S, produces S presomas；
S presomas are rapidly injected in Zn, Cd precursor mixed solution at 300 DEG C, mixed liquor is stirred vigorously at 300 DEG C
Room temperature is down to after 10min, it is 3 that the mixed liquor for then obtaining reaction, which adds volume ratio,:1 acetone and chloroform centrifugation number
CdSe/Cd that is secondary, will finally obtainingxZn1-xS nanocrystals are dried at 60 DEG C, stand-by.
Embodiment 1
A kind of temperature sensitive intelligent fluorescent nano probe, its CdSe/Cd modified with temperature sensing polymerxZn1-xS quantum dots
Assemble as donor, organic fluorescence molecule rhodamine B as acceptor, the CdSe/CdxZn1-xS quantum dot emissions peak exists
At 500~600nm, the maximum absorption band of rhodamine B is at 470~600nm, CdSe/CdxZn1-xThe emission spectrum of S quantum dots
It is overlapping with the absorption spectrum of rhodamine B.Wherein, temperature sensing polymer use molecular weight for 400 amino-end peg AT-
PEG400。
The preparation method of above-mentioned temperature sensitive intelligent fluorescent nano probe, specifically include following two step：
The first step：Temperature sensing polymer is modified quantum dot
1) 10mg CdSe/Cd is takenxZn1-xS quantum dots are dissolved in 30ml chloroforms, are configured to the chloroformic solution of quantum dot；Take
The chloroformic solution for the quantum dot that 5ml is configured, by the amino-end peg AT- that the 2.5g molecular weight of excess is 400
PEG400 is added thereto, and at 20 DEG C in Ultrasound Instrument ultrasonic 8h；
2) the Tris-HCl solution 5ml that pH is 8 are added dropwise into step 1) resulting solution, continue ultrasonic 24h, then stands and divides
Layer, isolate the upper strata aqueous solution and with chloroform more than 3 times to remove organic impurities, then obtained aqueous solution is dialysed 48h, i.e.,
Obtain the quantum dot aqueous solution after pure temperature sensing polymer modification.
Second step：Quantum dot and organic fluorescence molecule Controllable assembly after temperature sensing polymer modification
A) it is about 1 by mol ratio:1:1.2 34mg rhodamine Bs, 14mg EDC and 9.5mg NHS are stirred in 10ml water
2h, the quantum dot aqueous solution then added after the above-mentioned obtained temperature sensing polymer modifications of 3ml continue to stir 1h；
B) 25wt% TMAH is added dropwise until pH is adjusted to 8, and stirs 30min in step a) resulting solutions；
Then the HEPES cushioning liquid that 15ml pH are 6.8 and ultrasonic 12h is added dropwise；Mixture after ultrasound is dialysed in distilled water
24h, 24h is dried in vacuo, that is, obtains temperature sensitive intelligent fluorescent nano probe.
Data test
1st, CdSe/Cd is observed with RF-5301PC type sepectrophotofluorometers (excitation wavelength 380nm)xZn1-xS quantum dots
Fluorescence emission spectrum, with Shimadzu UV-2550 types ultraviolet-visible spectrophotometer (UV-vis) observe rhodamine B ultraviolet suction
Spectrum is received, as shown in Figure 1.As shown in Figure 1：The maximum absorption band of rhodamine B is in 555nm or so, CdSe/CdxZn1-xS quantum dots
Emission peak at 544nm, the emission spectra of the quantum dot has overlapping to a certain degree with the absorption spectra of rhodamine B, thus is prepared into
To the temperature sensitive intelligent fluorescent nano probe meet FRET occur condition.
2nd, the Intelligent fluorescent nano-probe being prepared in embodiment is configured to certain density solution to carry out fluorescence strong
Degree test, as shown in Figure 2：The fluorescent nano probe obtained in embodiment 1, the fluorescence intensity at 544nm is relative to quantum dot
Fluorescence intensity weaken, fluorescence intensity 578nm at increases relative to the fluorescence intensity of rhodamine B, illustrates that there occurs fluorescence
Resonance energy transfer, it is 49.39% in 25 DEG C of transferring efficiency of fluorescence resonance energy to be calculated.
Embodiment 2
A kind of temperature sensitive intelligent fluorescent nano probe, its CdSe/Cd modified with temperature sensing polymerxZn1-xS quantum dots
Assemble as donor, organic fluorescence molecule rhodamine B as acceptor, the CdSe/CdxZn1-xThe emission peak of S quantum dots
At 500~600nm, the maximum absorption band of rhodamine B is at 470~600nm, CdSe/CdxZn1-xThe absorption light of S quantum dots
Spectrum is overlapping with the absorption spectrum of rhodamine B.Wherein, temperature sensing polymer uses poly-o-phenylenediamine, and molecular weight is about 2000.
The preparation method of above-mentioned temperature sensitive intelligent fluorescent nano probe, specifically include following two step：
The first step：Temperature sensing polymer is modified quantum dot
1) 10mg CdSe/Cd is takenxZn1-xS quantum dots are dissolved in 30ml chloroforms, are configured to the chloroformic solution of quantum dot；Take
The chloroformic solution for the quantum dot that 5ml is configured, the 2.5g poly-o-phenylenediamines of excess are added thereto, and in Ultrasound Instrument at 45 DEG C
Middle ultrasonic 2h；
2) the Tris-HCl solution 8ml that pH is 10 are added dropwise into step 1) resulting solution, continues ultrasonic 12h, then stands
Layering, isolate the upper strata aqueous solution and with chloroform more than 3 times to remove organic impurities, then obtained aqueous solution dialysed 48h,
Obtain the quantum dot aqueous solution after pure temperature sensing polymer modification.
Second step：Quantum dot and organic fluorescence molecule Controllable assembly after temperature sensing polymer modification
A) it is about 1 by mol ratio:1:1.2 34mg rhodamine Bs, 14mgEDC and 9.5mg NHS are stirred in 10ml water
2h, the quantum dot aqueous solution then added after the above-mentioned obtained temperature sensing polymer modifications of 3ml continue to stir 4h；
B) 25wt% TMAH is added dropwise until pH is adjusted to 10, and stirs 2h in step a) resulting solutions；So
The HEPES cushioning liquid that 15ml pH are 8.2 and ultrasonic 24h are added dropwise afterwards；Mixture after ultrasound is dialysed 72h in distilled water,
48h is dried in vacuo, that is, obtains temperature sensitive intelligent fluorescent nano probe.
From the figure 3, it may be seen that Thermo-sensitive Intelligent fluorescent nano-probe is at 25 DEG C in embodiment 2, FRET effect
Rate is respectively 52.84%.
Embodiment 3
A kind of temperature sensitive intelligent fluorescent nano probe, its CdSe/Cd modified with temperature sensing polymerxZn1-xS quantum dots
Assemble as donor, organic fluorescence molecule rhodamine B as acceptor, the CdSe/CdxZn1-xThe emission peak of S quantum dots
At 500~600nm, the maximum absorption band of rhodamine B is at 470~600nm, CdSe/CdxZn1-xThe absorption light of S quantum dots
Spectrum is overlapping with the absorption spectrum of rhodamine B.Wherein, temperature sensing polymer use molecular weight for 800 Amino End Group PPOX
Ether.
The preparation method of above-mentioned temperature sensitive intelligent fluorescent nano probe, specifically include following two step：
The first step：Temperature sensing polymer is modified quantum dot
1) 10mg CdSe/Cd is takenxZn1-xS quantum dots are dissolved in 30ml chloroforms, are configured to the chloroformic solution of quantum dot；Take
The chloroformic solution for the quantum dot that 5ml is configured, the 2.5g Amino End Group PPOX ethers of excess are added thereto, and at 30 DEG C
The ultrasonic 6h in Ultrasound Instrument；
2) the Tris-HCl solution 10ml that pH is 8 are added dropwise into step 1) resulting solution, continues ultrasonic 18h, then stands
Layering, isolate the upper strata aqueous solution and with chloroform more than 3 times to remove organic impurities, then obtained aqueous solution dialysed 72h,
Obtain the quantum dot aqueous solution after pure temperature sensing polymer modification.
Second step：Quantum dot and organic fluorescence molecule Controllable assembly after temperature sensing polymer modification
A) it is about 1 by mol ratio:1.2:1.2 34mg rhodamine Bs, 16mgEDC and 9.5mg NHS are stirred in 10ml water
2h, the quantum dot aqueous solution then added after the above-mentioned obtained temperature sensing polymer modifications of 3ml continue to stir 2h；
B) 25wt% TMAH is added dropwise until pH is adjusted to 9, and stirs 2h in step a) resulting solutions；Then
The HEPES cushioning liquid that 15ml pH are 6.8 and ultrasonic 18h is added dropwise；Mixture after ultrasound is dialysed 72h in distilled water, very
Sky dries 48h, that is, obtains temperature sensitive intelligent fluorescent nano probe.
Thermo-sensitive Intelligent fluorescent nano-probe is at 25 DEG C in embodiment 3 as shown in Figure 4, transferring efficiency of fluorescence resonance energy
Respectively 55.70%.
Embodiment 4
A kind of temperature sensitive intelligent fluorescent nano probe, its CdSe/Cd modified with temperature sensing polymerxZn1-xS quantum dots
Assemble as donor, organic fluorescence molecule rhodamine B as acceptor, the CdSe/CdxZn1-xThe emission peak of S quantum dots
At 500~600nm, the maximum absorption band of rhodamine B is at 470~600nm, CdSe/CdxZn1-xThe absorption light of S quantum dots
Spectrum is overlapping with the absorption spectrum of rhodamine B.Wherein, temperature sensing polymer use molecular weight for 600 Amino End Group polyethylene glycol oxide
Ether.
The preparation method of above-mentioned temperature sensitive intelligent fluorescent nano probe, specifically include following two step：
The first step：Temperature sensing polymer is modified quantum dot
1) 10mg CdSe/Cd is takenxZn1-xS quantum dots are dissolved in 30ml chloroforms, are configured to the chloroformic solution of quantum dot；Take
The chloroformic solution for the quantum dot that 5ml is configured, the 2.5g Amino End Group polyethylene glycol oxide ethers of excess are added thereto, and at 30 DEG C
The ultrasonic 4h in Ultrasound Instrument；
2) the PBS solution 10ml that pH is 8 is added dropwise into step 1) resulting solution, continues ultrasonic 12h, then stratification,
Isolate the upper strata aqueous solution and with chloroform more than 3 times to remove organic impurities, then obtained aqueous solution is dialysed 24h, produce
The quantum dot aqueous solution to after pure temperature sensing polymer modification.
Second step：Quantum dot and organic fluorescence molecule Controllable assembly after temperature sensing polymer modification
A) it is about 1 by mol ratio:1.2.:1.2 34mg rhodamine Bs, 16mgEDC and 9.5mg NHS are stirred in 10ml water
3h is mixed, the quantum dot aqueous solution then added after the above-mentioned obtained temperature sensing polymer modifications of 3ml continues to stir 2h；
B) 25wt% TMAH is added dropwise until pH is adjusted to 9, and stirs 30min in step a) resulting solutions；
Then the HEPES cushioning liquid that 15ml pH are 6.8 and ultrasonic 12h is added dropwise；Mixture after ultrasound is dialysed in distilled water
24h, 24h is dried in vacuo, that is, obtains temperature sensitive intelligent fluorescent nano probe.
Thermo-sensitive Intelligent fluorescent nano-probe is at 25 DEG C in embodiment 4 as shown in Figure 5, transferring efficiency of fluorescence resonance energy
Respectively 58.99%.
It will be appreciated from fig. 6 that when temperature is respectively 15 DEG C, 25 DEG C, 35 DEG C, the fluorescence of temperature sensitive intelligent fluorescent nano probe is total to
The energy transfer efficiency that shakes is respectively 73.98%, 58.99% and 42.43%, it is seen that：With the change of temperature, fluorescence resonance energy
The efficiency of transfer gradually reduces, and the fluorescent nano probe for illustrating to be prepared has sensitive response to environmental change.
Embodiment 5
A kind of temperature sensitive intelligent fluorescent nano probe, its Cd modified with temperature sensing polymerxZn1-xS quantum dots are as confession
Body, organic fluorescence molecule assemble as dihydro fluorescein diacetate acceptor, the CdxZn1-xS quantum dot emissions peak 450~
At 530nm, the absworption peak of dihydro fluorescein diacetate is at 460~520nm, CdxZn1-xThe emission spectrum of S quantum dots and dihydro vinegar
The absorption spectrum of sour fluorescein is overlapping.Wherein, temperature sensing polymer use molecular weight for 400 amino-end peg AT-
PEG400。
The preparation method of above-mentioned temperature sensitive intelligent fluorescent nano probe, specifically include following two step：
The first step：Temperature sensing polymer is modified quantum dot
1) 10mg Cd is takenxZn1-xS quantum dots are dissolved in 30ml chloroforms, are configured to the chloroformic solution of quantum dot；Take 5ml institutes
The chloroformic solution of the quantum dot of configuration, the amino-end peg AT-PEG400 that 2.5g molecular weight is 400 is added thereto,
And at 20 DEG C in Ultrasound Instrument ultrasonic 8h；
2) the Tris-HCl solution 5ml that pH is 8 are added dropwise into step 1) resulting solution, continue ultrasonic 24h, then stands and divides
Layer, isolate the upper strata aqueous solution and with chloroform more than 3 times to remove organic impurities, then obtained aqueous solution is dialysed 48h, i.e.,
Obtain the quantum dot aqueous solution after pure temperature sensing polymer modification.
Second step：Quantum dot and organic fluorescence molecule Controllable assembly after temperature sensing polymer modification
A) it is about 1 by mol ratio:1:1.2 30mg dihydros fluorescein diacetate, 14mgEDC and 9.5mg NHS are in 10ml water
Middle stirring 2h, the quantum dot aqueous solution then added after the above-mentioned obtained temperature sensing polymer modifications of 3ml continue to stir 1h；
B) 25wt% TMAH is added dropwise until pH is adjusted to 8, and stirs 30min in step a) resulting solutions；
Then the HEPES cushioning liquid that 15ml pH are 6.8 and ultrasonic 12h is added dropwise；Mixture after ultrasound is dialysed in distilled water
24h, 24h is dried in vacuo, that is, obtains temperature sensitive intelligent fluorescent nano probe.
Embodiment 6
A kind of temperature sensitive intelligent fluorescent nano probe, its Cd modified with temperature sensing polymerxZn1-xS quantum dots are as confession
Body, organic fluorescence molecule assemble as dihydro fluorescein diacetate acceptor, the CdxZn1-xS quantum dot emissions peak 450~
At 530nm, the absworption peak of dihydro fluorescein diacetate is at 460~520nm, CdxZn1-xThe emission spectrum of S quantum dots and dihydro vinegar
The absorption spectrum of sour fluorescein is overlapping.Wherein, temperature sensing polymer use molecular weight for 600 Amino End Group polyethylene glycol oxide ether.
The preparation method of above-mentioned temperature sensitive intelligent fluorescent nano probe, specifically include following two step：
The first step：Temperature sensing polymer is modified quantum dot
1) 10mg Cd is takenxZn1-xS quantum dots are dissolved in 30ml chloroforms, are configured to the chloroformic solution of quantum dot；Take 5ml institutes
The chloroformic solution of the quantum dot of configuration, the Amino End Group polyethylene glycol oxide ether that the 2.5g molecular weight of excess is 600 is added thereto, and
At 20 DEG C in Ultrasound Instrument ultrasonic 4h；
2) the PBS solution 5ml that pH is 10 is added dropwise into step 1) resulting solution, continues ultrasonic 18h, then stratification,
Isolate the upper strata aqueous solution and with chloroform more than 3 times to remove organic impurities, then obtained aqueous solution is dialysed 24h, produce
The quantum dot aqueous solution to after pure temperature sensing polymer modification.
Second step：Quantum dot and organic fluorescence molecule Controllable assembly after temperature sensing polymer modification
A) it is about 1 by mol ratio:1:1.2 30mg dihydros fluorescein diacetate, 14mgEDC and 9.5mg NHS are in 10ml water
Middle stirring 2h, the quantum dot aqueous solution then added after the above-mentioned obtained temperature sensing polymer modifications of 3ml continue to stir 1h；
B) 25wt% TMAH is added dropwise until pH is adjusted to 8, and stirs 30min in step a) resulting solutions；
Then the HEPES cushioning liquid that 15ml pH are 6.8 and ultrasonic 12h is added dropwise；Mixture after ultrasound is dialysed in distilled water
24h, 24h is dried in vacuo, that is, obtains temperature sensitive intelligent fluorescent nano probe.
Described above is only the preferred embodiment of the present invention, it is noted that is come for one of ordinary skill in the art
Say, without departing from the concept of the premise of the invention, some modifications and variations can also be made, these belong to the present invention's
Protection domain.
Claims (8)
1. a kind of temperature sensitive intelligent fluorescent nano probe, it is characterised in that it is used as confession using the quantum dot of temperature sensing polymer modification
Body, organic fluorescence molecule assemble as acceptor, the emission spectrum of the quantum dot and the absorption spectrum of organic fluorescence molecule
Have overlapping；
The temperature sensing polymer is Thermo-sensitive double-end amino polymer or one end with polymer of the amino one end with sulfydryl, its point
Son amount is below 3000；The quantum dot is ZnS monokaryons quantum dot, CdS monokaryons quantum dot, CdSe monokaryons quantum dot, CdSe/
ZnS core/shell quantum dot, CdSe/CdS/ZnS core shells/shell quantum dot, CdxZn1-xS quantum dots, CdSe/CdxZn1-xS cores/alloy
Any one in shell quantum dot.
A kind of 2. temperature sensitive intelligent fluorescent nano probe according to claim 1, it is characterised in that the organic fluorescence point
Son is carboxylic organic fluorescence molecule.
3. a kind of preparation method of temperature sensitive intelligent fluorescent nano probe, it is characterised in that it is first with temperature sensing polymer to amount
Son point is modified, and the quantum dot after then temperature sensing polymer is modified carries out Controllable assembly with organic fluorescence molecule, so as to
Temperature sensitive intelligent fluorescent nano probe is made；
The temperature sensitive intelligent fluorescent nano probe is using the quantum dot that temperature sensing polymer is modified as donor, organic fluorescence point
Son assembles as acceptor, and emission spectrum and the absorption spectrum of organic fluorescence molecule of the quantum dot have overlapping；The temperature
Sensitive polymer be Thermo-sensitive double-end amino polymer or one end with polymer of the amino one end with sulfydryl, its molecular weight is 3000
Below；The quantum dot is ZnS monokaryons quantum dot, CdS monokaryons quantum dot, CdSe monokaryons quantum dot, CdSe/ZnS core shell quantum
Point, CdSe/CdS/ZnS core shells/shell quantum dot, CdxZn1-xS quantum dots, CdSe/CdxZn1-xIn S cores/alloy shell quantum dot
Any one.
4. the preparation method of a kind of temperature sensitive intelligent fluorescent nano probe according to claim 3, it is characterised in that described
The method that temperature sensing polymer is modified quantum dot, specifically comprises the following steps：
1）Quantum dot is dissolved in chloroform, and adds temperature sensing polymer, until completely dissolved the ultrasound 2 ~ 8 at 20~45 DEG C
h；
2）To step 1）The cushioning liquid that pH is 8 ~ 10 is added dropwise in resulting solution, continues 12 ~ 24 h of ultrasound, then stratification,
Isolate the upper strata aqueous solution and with chloroform to remove organic impurities, then through dialysis, that is, after obtaining temperature sensing polymer modification
The quantum dot aqueous solution.
5. the preparation method of a kind of temperature sensitive intelligent fluorescent nano probe according to claim 4, it is characterised in that described
Step 1）Middle temperature sensing polymer is excessive relative to quantum dot.
6. the preparation method of a kind of temperature sensitive intelligent fluorescent nano probe according to claim 3, it is characterised in that temperature sensitive
Property it is polymer-modified after quantum dot and organic fluorescence molecule Controllable assembly method, specifically comprise the following steps：
a）By organic fluorescence molecule, 1- ethyls -3-（3- dimethylaminos）- carbodiimide and n-hydroxysuccinimide stir in water
2~4 h are mixed, the quantum dot aqueous solution then added after temperature sensing polymer modification, stir 1~4 h；
b）Add alkali regulating step a）Resulting solution pH is 8~10, and stirs the h of 30 min~2；Then it is 6.8 ~ 8.2 that pH, which is added dropwise,
Cushioning liquid and 12~24 h of ultrasound, then through dialysing, being dried in vacuo, that is, obtain obtaining temperature sensitive intelligent fluorescent nano probe.
7. the preparation method of a kind of temperature sensitive intelligent fluorescent nano probe according to claim 6, it is characterised in that described
Step a）Middle organic fluorescence molecule, 1- ethyls -3-（3- dimethylaminos）The mol ratio of-carbodiimide and n-hydroxysuccinimide
Scope is 1：1~1.2：1~1.2.
8. the preparation method of a kind of temperature sensitive intelligent fluorescent nano probe according to claim 6, it is characterised in that described
Step b）In alkali be TMAH.
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