CN108424766A - A kind of preparation and application with multiple response polymer quantum dot TPE-PDEAEAM - Google Patents

A kind of preparation and application with multiple response polymer quantum dot TPE-PDEAEAM Download PDF

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CN108424766A
CN108424766A CN201810283173.7A CN201810283173A CN108424766A CN 108424766 A CN108424766 A CN 108424766A CN 201810283173 A CN201810283173 A CN 201810283173A CN 108424766 A CN108424766 A CN 108424766A
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tpe
pdeaeam
quantum dot
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CN108424766B (en
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关晓琳
李志飞
来守军
孟丽
王林
王凯龙
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Northwest Normal University
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    • C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
    • C09K11/00—Luminescent, e.g. electroluminescent, chemiluminescent materials
    • C09K11/06—Luminescent, e.g. electroluminescent, chemiluminescent materials containing organic luminescent materials
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
    • C08F120/00—Homopolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and only one being terminated by only one carboxyl radical or a salt, anhydride, ester, amide, imide or nitrile thereof
    • C08F120/02—Monocarboxylic acids having less than ten carbon atoms; Derivatives thereof
    • C08F120/52—Amides or imides
    • C08F120/54—Amides, e.g. N,N-dimethylacrylamide or N-isopropylacrylamide
    • C08F120/60—Amides, e.g. N,N-dimethylacrylamide or N-isopropylacrylamide containing nitrogen in addition to the carbonamido nitrogen
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    • 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
    • G01N21/6486—Measuring fluorescence of biological material, e.g. DNA, RNA, cells
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    • C09K2211/00—Chemical nature of organic luminescent or tenebrescent compounds
    • C09K2211/18—Metal complexes
    • C09K2211/188—Metal complexes of other metals not provided for in one of the previous groups

Abstract

The invention discloses one kind, and there is multiple response polymer quantum dot TPE PDEAEAM to be formed using TPE BMP as initiator by atom transfer radical polymerization.Due to containing lignocaine and acrylamide group in TPE PDEAEAM polymer quantum dots, TPE PDEAEAM polymer quantum dots are to temperature, pH and CO2Stimulation all make corresponding response.Fluorescent polymer shows unique aggregation inducing transmitting（AIE）Behavior easily by HeLa cell dissociations and can track cell up to nine generations, therefore this may be used as the long-term observation fluorescer for cell imaging.

Description

A kind of preparation and application with multiple response polymer quantum dot TPE-PDEAEAM
The present invention relates to a kind of preparation of polymer fluorescent quantum dot more particularly to a kind of TPE- with multiple response The preparation of PDEAEAM polymer quantum dots, the polymer quantum dot have the characteristics that AIE fluorescence, can be used as the fluorescence of cell imaging Agent.
Background technology
In recent years, a noninvasive technology of the fluorescence imaging as life science is sensitive due to its substance Spend relatively high, easy to operate, the excellent feature such as of low cost, cause scientific research field extensive concern.To being at present Only, various fluorescent moleculars have been synthesized and have been used as bio-imaging reagent.In these numerous reagents, long-term observation cell It is raw that the exploitation of the Long-term cell tracer of process monitors biological study person generation, development, invasion and transfer of cancer cell etc. Object process has a very important significance.
As well-known fluorescent, inorganic nanoparticles, quantum dot（QDs）, due to the high brightness of their unique light It is used as the fluorecyte tracer of various biotechnology applications with the stability of light.For example, CdSe/ZnS quantum dots are It is imaged by the Long-term cell as Cellular tracking agent for living cells, has and compare high emission and excellent photostability.But no Good fortune, the heavy metal component of QD（Such as Cd 2+）With higher cytotoxicity, these weakness limit they biology at Many applications as in.In addition, the fluorescin of green（GFP）And other other families, a high fluorescent molecular are wide It is used as long-term in vitro Cellular tracking agent generally.However, due to some disadvantages of GFP itself, as proteolytic enzyme is sensitive, stoke The variation of this displacement, the function etc. that the stability of light is poor, fluorescin infection can interfere many normal cells, hinder More practical applications of GFP probes.
Assemble light emitting-type polymer quantum dot（AIE）Quantum dot relative to inorganic semiconductor（QDs）With organic dyestuff one A little small molecules, advantage are apparent.As half-peak breadth is relatively narrow, emission peak is relatively more symmetrical, cytotoxicity is smaller, biocompatibility It is good.It is set to have good application prospect in fields such as optics, biology.Aggregation is prepared by different synthetic methods at present Light emitting-type small molecule and polymer, AIE types polymer are gradually synthesized as cell probe.TPE（Tetraphenyl ethylene） It is a kind of AIE of classics（Aggregation Induced Emission aggregation-induced emissions）Molecule does not have in the solution Fluorescence, but but there is stronger fluorescence in solid, with conventional aggregation quencher molecule just on the contrary, therefore in organic photoelectrical material etc. Field has important application, also more and more causes the concern of people.For example, Tang Benzhong et al. is by by a large amount of four benzene second Alkene（TPE）It is connected to chitosan（CS）On chain, then allows its polymer to track the cell developing agent of living cells as long-time again, connect It and reports a kind of novel fluorescence probe.Et al. it is poly- from tetraphenyl ethylene base（N-isopropyl acrylamide）（TPE-PNIPAM） A kind of thermally sensitive organic polymer nano-particles of ATRP have been synthesized, the Cellular tracking agent of Long-term cell tracer is can be used as, It can carry out passing on up to the cell in seven generations in HeLa cells.However, still about this report based on AIE type polymer Relatively seldom.Therefore, the polymer based on AIE is designed and synthesizes as the long-term observation agent right and wrong of biologic applications often with there is challenge The task of property.
The stimulating responsive polymer that or interior change relatively small to changes in environmental conditions can make a response has very More values or wider array of biological developing application.Thermo-responsive polymer is in lower critical solution temperature（LCST）Lower display can Molten-insoluble phase transformation.However pH sensitive polymers are by changing the size of pH value, and then change the size of polymer come around responding Media variations.There is document report poly- [N- [2- (lignocaine) ethyl] acrylamide]（PDEAEAM）Temperature and pH/CO2 The homopolymer of response（Song, Z.; Wang, K.; Gao, C.; Wang, S.; Zhang, W. A New Thermo-, pH-, and CO2-Responsive Homopolymer of Poly[N-[2-(diethylamino)ethyl] acrylamide]:Is the Diethylamino Group Underestimated Macromolecules. 2016,49, 162-171.）, pH/CO2It is adjustable.But only a kind of fluorescence polymer material of stimulation can't reach biology at present The requirement of medical application.More stimulus responsive polymers based on AIE types combine aggregation and shine（AIE）And stimulus responsive polymers The advantages of, good water-soluble, biocompatibility and the aggregation characteristics of luminescence are shown, and show to tie in outside stimulus Structure and the corresponding variation that shines.These the more stimulating responsive polymers combined with AIE type fluorogens may be used as overcoming long-term Main problem existing for cell tracker.Therefore, exploitation have multiple response fluorescent polymer be very it is necessary to.Wherein, hot Quick property, pH and photosynthesis-carbon dioxide response polymer are most popular in the design and synthesis of novel Irr. M.
Invention content
The preparation side of the object of the present invention is to provide a kind of TPE-PDEAEAM polymer quantum dots with multiple response Method；
It is another object of the present invention to the structure for the above-mentioned TPE-PDEAEAM polymer quantum dots with multiple response, AIE Characteristic and the application being imaged for the Long-term cell of living cells as Cellular tracking agent are analyzed.
One, the preparation of TPE-PDEAEAM polymer quantum dot
（1）The synthesis of 4- hydroxyphenyl ethylene
With tetrahydrofuran（THF）For solvent, zinc powder, TiCl4（Zn is reducing agent, TiCl4For catalyst, mixing reproducibility catalysis Agent）, under protection of argon gas, 4- dihydroxy benaophenonels and benzophenone are with 1:1~1:1.2 molar ratio reflux is for 24 hours；After answering It is cooled to room temperature, K is added2CO3Solution quenching reaction, then filters, is extracted with ethyl acetate, and column chromatography for separation purification obtains yellowish Four ethylene of color solid 4- hydroxyls（TPE）.
The addition of zinc powder be 4- dihydroxy benaophenonels, 3 ~ 4 times of benzophenone integral molar quantity；TiCl4Addition be 2 ~ 3 times of 4- dihydroxy benaophenonels, benzophenone integral molar quantity.
（2）4- (12- hydroxydodecyls) tetraphenyl ethylene（TPE-OH）Synthesis
By four ethylene of 4- hydroxyls, the bromo- DODECANOL, 1-s of 12- and K2CO3It is dissolved in anhydrous acetonitrile, flows back 20 under protection of argon gas ~24 h；It is cooled to room temperature after reaction, filtering reacting liquid simultaneously evaporates organic layer, and product is purified through column chromatography for separation, is obtained yellowish Color solid 4- (12- hydroxydodecyls) tetraphenyl ethylene.
The effect of the bromo- DODECANOL, 1-s of 12- is to extend carbochain, enhances hydrophobicity, the bromo- 1- of four ethylene of 4- hydroxyls and 12- ten The molar ratio of dialkanol is 1:1~1:1.2.
K2CO3Effect be to provide alkalinity and water imbibition.Four ethylene of 4- hydroxyls and K2CO3Molar ratio be 1:1~1:1.2.
（3）The synthesis of TPE-BPM
By 4- (12- hydroxydodecyls) tetraphenyl ethylene（TPE-OH）, triethylamine and the bromo- 2- methyl propionyl bromides of 2- be added to it is anhydrous Tetrahydrofuran（THF）In, reaction is stirred at room temperature 20 ~ 24 hours；After reaction, filtering reacting liquid；Concentration filtrate obtains slightly Product, crude product are purified through column chromatography for separation, and it is TPE-BMP to obtain faint yellow solid product.
The effect of triethylamine is to provide alkalinity.The molar ratio of TPE-OH and triethylamine is 1:1~1:1.25.
The effect of the bromo- 2- methyl propionyl bromides of 2- is synthesis initiator, the molar ratio of TPE-OH and the bromo- 2- methyl propionyl bromides of 2- It is 1:1~1:1.25.
（4）The synthesis of TPE-PDEAEAM
By N (2- (diethylamino) ethyl) acrylamide（DEAEAM）It is dissolved in Water-Methanol Mixtures, is protected in argon gas Under sequentially add three (2- dimethylaminoethyls) amine（Me6TREN）, CuBr and TPE-BMP, 20 ~ 24 h are stirred at room temperature, so Reaction stoste is dialysed 70 ~ 72 hours with ultra-pure water afterwards, then dialyzate is freeze-dried 70 ~ 72 hours, obtains faint yellow solid Powder, as TPE-PDEAEAM.
The volume ratio of the Water-Methanol Mixtures, water and methanol is 1.5:1~2:1.
Three (2- dimethylaminoethyls) amine（Me6TREN）Effect be Cu2+Ligand；Its addition is DEAEAM mass 19%~20%。
The effect of CuBr is catalyst.Its addition is the 0.5% ~ 1% of DEAEAM mass.
The mass ratio of PDEAEAM and TPE-BMP is 60:1~65:1.
Two, the structure and characteristic of TPE-PDEAEAM polymer quantum dot
1, nucleus magnetic hydrogen spectrum
The nucleus magnetic hydrogen spectrum of Fig. 1,2 respectively TPE-BPM, TPE-PDEAEAM.There is aromatic ring at the places 6.9ppm or so in chemical shift The signal peak of proton hydrogen occurs the signal peak of methyl hydrogen at 1.0ppm, is respectively TPE- at 2.6 ppm and 3.2 ppm There is the methylene signals peak on TPE-PDEAEAM in the hydrogen peak of imino group above PDEAEAM, 1.5ppm.This shows TPE points Son and PDEAEAM are successfully covalently bound together.
2, infrared spectrum
Fig. 3 is the infrared spectrum of TPE-PDEAEAM.In 1658 cm-1The characteristic peak of amide can clearly be observed by locating us, In 1553cm-1There is the vibration of N-H keys at place.Further demonstrate polymer quantum dot structure designed with us it is consistent.
3, GPC is tested
Table 1 is the GPC data of TPE-PDEAEAM.
The structure of TPE-PDEAEAM, number-average molecular weight are further demonstrated by GPC tests（Mn）And weight average molecular weight （Mw）Respectively 1.1 × 104With 2.1 × 104。
4, the pattern of polymer TPE-PDEAEAM polymer quantum dot self assembly in aqueous solution
Fig. 4 is Tyndall effect of the TPE-PDEAEAM polymer quantum dots in tetrahydrofuran solution and aqueous solution.Work as light beam Line penetrates colloid, and one occurred in colloid bright " access ", this phenomenon are observed that from the vertical direction of incident light Be Tyndall phenomenon, by this phenomenon, we tentatively judge TPE-PDEAEAM polymer quantum dot in aqueous solution from group It fills relatively good.
Fig. 5 is that TPE-PDEAEAM polymer quantum dots are dissolved in aqueous solution, the fluid dynamics of a concentration of 2mg/mL Size shows that the hydrodynamics size of quantum dot is held in 250nm or so.The grain size of polymer quantum dot is 250 or so.
Fig. 6 is the pattern of TPE-PDEAEAM polymer quantum dot self assembly in aqueous solution（SEM）.Prove TPE- PDEAEAM polymer nano-particles are the relatively regular spherical shapes of shape, and the size of nano-particle is relatively uniform, and grain size is about 250nm。
5, the AIE characteristics of TPE-PDEAEAM polymer quantum dot
THF and H2Two kinds of solvents of O can dissolve TPE-PDEAEAM polymer segments well.Polymer quantum dot TPE- PDEAEAM in water can all dissolve to form transparent solution in certain temperature range.Its aqueous solution is in ultraviolet lamp（365nm） Under be still luminous, and it is almost invisible light in pure THF solution.This phenomenon and TPE small molecules AIE（Aggregation shines）Property is very similar.
We investigate influence of the concentration variation to polymer fluorescent, as a result, it has been found that, when its polymer TPE-PDEAEAM is water-soluble Liquid concentration is by 0.001 mgmL-1Increase to 1 mg mL-1When, then it can show the increase fluorescence with polymer concentration It can enhance therewith, excitation and launch wavelength are respectively 337 nm and 475 nm.With the increase of concentration, TPE-PNIPAM molecules must Surely certain aggregation can occur, and fluorescence significantly increases and illustrates its AIE characteristic.
TPE-PDEAEAM is observed in the change in fluorescence of water/tetrahydrofuran in the mixed solvent of different proportion, is further demonstrate,proved The AIE characteristics of the polymer are illustrated：With H2The increase of O volume fractions, the fluorescence intensity of TPE-PDEAEAM is with nonlinear side Formula weakens.H2The volume fraction of O is more than after 80%, and fluorescence intensity sharply increases, this is glimmering with the aggregation inducing of the report in document Light enhancing is much like.It is main reasons is that with H2The increase of O volume fractions, polymer chain are expanded so that TPE is sent out Raw aggregation, the aggregation of TPE molecules increase so that emitting fluorescence.
6, influence of the temperature to TPE-PDEAEAM polymer quantum dot fluorescence intensities
The temperature sensitivity energy of TPE-PDEAEAM polymer quantum dots can be easily by higher than LCST（Phase transition temperature Degree）At a temperature of heat the aqueous solutions of TPE-PDEAEAM polymer quantum dots, then be cooled below LCST come it is true It is fixed.The aqueous solution of TPE-PDEAEAM polymer quantum dots is transparent at room temperature.The solution after being heated above LCST Become muddy opaque, upon cooling to room temperature, becomes muddy and further become transparent, which demonstrate the TPE- at LCST The reversible phase transformation of PDEAEAM.Fig. 7 show when a concentration of 2g/L TPE-PDEAEAM polymer quantum dot aqueous solutions with temperature Spend the light transmittance of variation.When polymer concentration is 2 g/L, LCSTs of the TPE-PDEAEAM in LCST aqueous solutions is 60 DEG C.
In addition, influence of the research temperature to TPE-PDEAEAM polymer quantum dot photoluminescent properties.Fig. 8 is as TPE- PDEAEAM polymer quantum dot solution from 25 DEG C to 66 DEG C in fluorescence variation：TPE-PDEAEAM polymer quantum dots are with temperature The raising fluorescence intensity of degree reduces instead.This is because the polymer segment of PDEAEAM is Thermo-sensitive material.
7, the pH sensitivity characteristics of TPE-PDEAEAM polymer quantum dot
Investigate influence of the pH value change to TPE-PDEAEAM polymer quantum dot fluorescence intensities.It is well known that hydrochloric acid or hydrogen is added Sodium oxide molybdena is easily destroyed intermolecular hydrogen bonding, and the original coherent condition of TPE-PDEAEAM polymer quantum dots is caused to change.By In the variation of state of aggregation, the variation of the fluorescence intensity of the polymer with AIEgen will occur.Fig. 9 polymerize for TPE-PDEAEAM Object quantum dot fluorescence spectrum in different pH solution（It is left）And the relative intensity of fluorescence under difference pH in aqueous solution（I / I0） Variation（It is right）（TPE-PDEAEAM] = 2.0g L-1）, show the Fluorescence Increasing of the increase polymer quantum dot with pH value. Therefore, the fluorescence TPE-PDEAEAM of pH sensitivities may be used as promising optical sensor, be measured for low or high ph-values.
8, the CO of TPE-PDEAEAM polymer quantum dot2Sensitivity characteristic
Photosynthesis-carbon dioxide response polymer and carbon dioxide capture material have been a great concern in recent years, because of carbon dioxide With good biocompatibility, excellent membrane permeability, abundant availability, non-toxic and good invertibity etc. is many excellent Point.Carbon dioxide can easily be reacted with reactive group such as amine or amidine, generate hydrophilic compounds and amidino groups polymer, Constitute maximum one kind in photosynthesis-carbon dioxide response polymer.Therefore, the bearing amidino groups of TPE-PDEAEAM polymer quantum dot Group, can react to form electrically charged amidine bicarbonate with carbon dioxide and water, will have photosynthesis-carbon dioxide response.Figure 10 is TPE-PDEAEAM polymer quantum dot is in different CO2The fluorescence spectrum of bulk solution, illustration are the linear of relative intensity of fluorescence （I/I0）With the relationship of CO2 volumes.As shown in Figure 10, work as CO2Volume from 0.0 increase to 0.4mL when, TPE-PDEAEAM polymerization The fluorescence intensity of object quantum dot drastically declines, along with small blue shift.Illustration shows blank fluorescence intensity and quenching fluorescence intensity The ratio between（I0/I）With the CO of 0.0 ~ 0.4 mL2Addition is linear, illustrates that TPE-PDEAEAM polymer quantum dots can be used as CO2Fluorescent optical sensor.Reason is attributed in TPE-tetraPDEAEAM the interaction between carbon dioxide and tertiary amine group, Which confirms tertiary amine groups can be protonated to form electrically charged ammonium hydrogen carbonate by carbon dioxide.
Two, toxicity evaluations of the TPE-PDEAEAM to Hela cells
For biomedical applications, the cytotoxicity of assessment material is vital.
（1）Plating cells：The Hela cells of logarithmic growth phase, 0.25% pancreatin（Containing EDTA）It is digested, haemocyte Tally is counted, and it is 1 × 10 so that cell is obtained density4/ hole.（Edge hole is populated with sterile PBS）.It will be every with liquid-transfering gun 100 μ L cell liquid of hole is inoculated in 96 well culture plates, and incubator temperature is 37 DEG C, CO2For in 5% saturated humidity incubator Culture.Every group needs that 3 multiple holes are arranged.
（2）Cell administration：CO2Incubator（Temperature is 37 DEG C, 5% CO2Content）Middle culture for 24 hours, is then respectively adding The TPE-PDEAEAM of a concentration of 50 μ g/mL, 100 μ g/mL, 200 μ g/mL, 300 μ g/mL, 400 μ g/mL.
（3）MTT is added：After the Hela cells of dosing cultivate 24 hours in the incubator, the culture medium having originally is outwelled, The MTT solution for adding 100mL in every hole again places into incubator after cultivating 4h and stops being further cultured for.The operating process of experiment needs It is protected from light as possible.
（4）Dissolve Hela cells：The culture medium of 96 well culture plates weight is sucked out, the DMSO that 150mL is added per hole is incubated 40min makes precipitation be completely dissolved, and can be detected（Operating process does not touch the bottom of 96 well culture plates in order to avoid influencing detection Experimental result）.
（5）Microplate reader measures the OD values in each hole of 96 well culture plates：The Hela cells shaken are carried out with microplate reader It measures, we need to open computer first, allow computer and microplate reader to be attached, then open the software of measurement again, to 96 holes Plate measures, and the result of measurement has carried out data processing with Excel.
（6）The survival rate of Hela cells：OD values are measured, the Hela containing TPE-PDEAEAM is calculated by following formula Cell survival rate（%）.Then with a concentration of abscissa of TPE-PAA solution, with the Hela cell survivals containing TPE-PDEAEAM Rate is ordinate.
Cell survival rate (%)=(OD test group-OD zeroings group)/（OD blank group-OD zeroing groups）× 100%
HeLa cytotoxicity tests after being handled 48 hours with the TPE-PNIPAM of various concentration, the result is shown in Figure 11.It has been shown that, 95% with On Hela cell survivals, hatched with the TPE-PNIPAM of various concentration（50~400μg mL-1）48h.Illustrate TPE-PDEAEAM Toxicity it is smaller, be expected to be used for the biologic applications of cell developing or more.
Three, TPE-PDEAEAM is to Hela cell imaging applications
The characteristics of based on AIE fluorescence, TPE-PDEAEAM polymer quantum dots are used as the fluorescer of cell imaging.Selection HeLa cells prove ability of the TPE-PDEAEAM polymer quantum dots for cell in vitro tracking application as body.It will HeLa cells are seeded in the density of 10000 cells/wells in 96 well culture plates.After incubator culture 24 hours, cell is used The TPE-PDEAEAM processing of various concentration.Hela cells are washed with the PBS prepared, then by MTT solution（5mg/mL, 10 μ L） With Hela cell culture mediums（90μL）It is added in the cell in each hole.By 96 well culture plates at 37 DEG C（Containing CO25%） It is incubated 4 hours in incubator.The culture medium containing MTT is removed, dimethyl sulfoxide is added（DMSO, 100 μ L）Dissolving is by living cells shape At first a ceremonial jade-ladle, used in libation crystal.Absorbance is measured at 492nm using RT-6100 microplate reader.
It first, will be on Hela cells in advance 12 well culture plate cell sheets of inoculation.Culture medium is containing 1% streptomysin and 10% 1640 solution of fetal calf serum.Then 12 orifice plates are placed in in the humidified incubator of CO2 and 37 DEG C of temperature of 5% concentration 24 small When.Hela cells are handled with 100 μ gmL-1TPE-PAA, after 24 hours, take out cell sheet, with phosphate buffer solution by tablet It flushes three times.Cell imaging picture is shot under confocal fluorescence microscopy.Use Olympus FV1000 Laser Scanning Confocal Microscopes （Olympus Tokyo Japan）It excites in 332nm and emits in 460 ~ 490nm.
In order to assess whether TPE-PDEAEAM polymer quantum dot can track cell for a long time as probe, TPE- is used The cell repetition in every 48 hours of PDEAEAM polymer quantum dots processing is passed on, and is measured per generation TPE- by Laser Scanning Confocal Microscope The intracellular Fluorescence of PDEAEAM polymer quantum dots.Hela cells and 100 μ gmL-1TPE-PDEAEAM polymer quantum dots incubate It educates 24 hours（The first generation）Afterwards, the signal strength of TPE-PDEAEAM polymer quantum dot in HeLa cells is observed（Figure 12）.Carefully The relatively weak region of intracellular fluorescence intensity may be the position of nucleus, illustrate that polymer quantum dot has successfully been escaped completely The endosomal compartment of aggregation and the cytosolic domain for selectively having dyed HeLa cells.The TPE-PDEAEAM intracellular from HeLa Polymer quantum dot monitors strong signal, even if with 100 μ gmL-1After concentration is incubated 48 hours, still it can observe strong glimmering Optical signal.Cell after dyeing is up in 20 days time（It was up to for 9 generations）It remains to keep stronger fluorescence intensity.This shows Fluorescent polymer can be used as fluorescent bio-probes to be tracked for Long-term cell.
In conclusion having synthesized tetraphenyl ethylene by ATRP polymerization is grafted poly- [N- [2- (diethylamino)-ethyl] third Acrylamide]（TPE-PDEAEAM polymer quantum dots）More stimulus responsive polymers.Due to TPE-PDEAEAM polymer quantum Contain lignocaine and acrylamide group in point, TPE-PDEAEAM polymer quantum dot is to temperature, pH and CO2Stimulation all Make corresponding response.As a concentration of 2.0 g L of polymer quantum dot-1When, TPE-PDEAEAM polymer quantum dots are water-soluble LCST in liquid is 60 DEG C, shows that its thermal transition temperature changes possibly through solution concentration is changed.Meanwhile TPE- The fluorescent emission intensity of PDEAEAM polymer quantum dots is with the raising of temperature and fluorescence drops to 66 DEG C from 25 DEG C, shows it The certain thermo-responsive of the fluorescence of polymer quantum dot.The salting-out effect caused by pH adjusting agent, and then cause TPE- The variation of the fluorescence intensity of PDEAEAM polymer quantum dots.Further, since the tertiary amine in TPE-PDEAEAM polymer quantum dots Group can be by CO2The ammonium hydrogen carbonate of protonation and formation electrification, the fluorescence intensity of polymer is with addition CO2The increase of volume and Significant reduction, and I/I0Ratio and addition carbon dioxide volume from 0.4 to 1.2 milliliter, polymer quantum dot it is glimmering Intensity variation is not apparent.It is furthermore interesting that fluorescent polymer shows unique aggregation inducing transmitting（AIE）Behavior, It may be used as the long-term observation fluorescer for cell imaging.
Description of the drawings
Fig. 1 is the nucleus magnetic hydrogen spectrum of TPE-BPM.
Fig. 2 is the nucleus magnetic hydrogen spectrum of TPE-PDEAEAM.
Fig. 3 is the infrared spectrum of TPE-PDEAEAM.
Fig. 4 is Tyndall effect of the TPE-PDEAEAM polymer quantum dots in tetrahydrofuran solution and aqueous solution.
Fig. 5 is the hydrodynamic size that TPE-PDEAEAM polymer quantum dots are dissolved in a concentration of 2mg/mL in water.
Fig. 6 is the pattern of TPE-PDEAEAM polymer quantum dot self assembly in aqueous solution（SEM）.
Fig. 7 is the light transmittance of the TPE-PDEAEAM polymer quantum dot aqueous solutions of a concentration of 2g/L varied with temperature.
Fig. 8 as TPE-PDEAEAM polymer quantum dot solution from 25 DEG C to 66 DEG C in fluorescence variation
Fig. 9 is TPE-PDEAEAM polymer quantum dot fluorescence spectrum in different pH solution（It is left）And in aqueous solution under difference pH In relative intensity of fluorescence（I / I0）Variation（It is right）（TPE-PDEAEAM] = 2.0g L-1）.
Figure 10 is TPE-PDEAEAM polymer quantum dot in different CO2The fluorescence spectrum of bulk solution.Illustration：Relatively Fluorescence intensity it is linear（I/I0）With CO2The relationship of volume.
Figure 11 is the cytotoxicity test of TPE-PDEAEAM polymer quantum dots.
Figure 12 is the confocal images of TPE-PDEAEAM polymer quantum Vertex Coloring HeLa cell difference algebraically.
Specific implementation mode
The synthesis of TPE-PDEAEAM polymer quantum dot of the present invention and application are done into one below by specific embodiment Walk explanation.
1, the synthesis of TPE-PDEAEAM polymer quantum dot
（1）Monomer N (2-(diethylamino) ethyl) acrylamide（DEAEAM）Synthesis：N is synthesized according to literature procedure [ 2 -（Diethylamino）Ethyl] acrylamide（DEAEAM）：Under magnetic stirring, by N, N- diethyl ethylenediamines （75mmol, 8.72g）And CHCl3（75mL）It is added in the single necked round bottom flask of 250mL, flask is then placed in ice-water bath.With Afterwards, it is slowly added dropwise in 1 hour into being dissolved in CHCl 3（50mL）In acryloyl chloride（90mmol, 7.2mL）.It is being added completely into third Alkene acyl chlorides and then 1h is reacted at room temperature.Reaction solution NaOH aqueous solutions（100mL, 1mol/L）And water（100mL）It washes It washs, finally with anhydrous MgSO4Dry, rotary evaporation removes CHCl3Solvent, crude product is used as eluant, eluent by ethyl acetate, obtains To DEAEAM（10.3g, yield 85%）.
（2）The synthesis of hydroxyphenyl ethylene：By Zn powder（20g, 0.31mol）, 4- dihydroxy benaophenonels（9.5g, 0.05mol） And benzophenone（8.7g, 0.05mol）It is dissolved in 200mL THF, TiCl is added under argon gas protective condition4（30.0 mL, 0.27 mol）And 24 h that flow back.After reaction, reaction mixture is cooled to room temperature, the K of 150 mL 10% is added2CO3It is molten Liquid is simultaneously vigorously stirred, and then filters mixed liquor, and organic layer is extracted with ethyl acetate to obtain, and finally uses column chromatography point to product Carry out separating-purifying（Eluent：VEthyl acetate:VPetroleum ether=1:10）, 6.1 g faint yellow solid 4- hydroxyls, four ethylene is finally obtained, is produced Rate is about 61 %.
1H NMR (400 MHz, CDCl3) δ 7.13 – 6.97 (m, 15H), 6.89 (d, J = 8.6 Hz, 2H), 6.55 (d, J = 8.6 Hz, 2H), 4.60 (s, 1H)。
13C NMR (151 MHz, CDCl3) δ 153.96 (s), 144.28 – 143.62 (m), 140.29 (d, J = 33.3 Hz), 136.37 (s), 132.71 (s), 131.52 – 131.16 (m), 127.63 (d, J = 15.1 Hz), 126.24 (s), 114.57 (s).
ESI-MS：M = 348.48.
（3）4- (12- hydroxydodecyls) tetraphenyl ethylene（TPE-OH）Synthesis：By four ethylene of 4- hydroxyls（3.48g 0.01mol）, the bromo- DODECANOL, 1-s of 12-（0.28mL, 0.012mol）And K2CO3（1.66g 0.012mol）Be dissolved in 100mL without In water-acetonitrile, flow back 24 h under argon gas protective condition.After reaction, after reaction solution is cooled to room temperature, by reaction solution mistake Filter, organic layer is evaporated, and finally column chromatography for separation method is used to carry out separating-purifying to product（Eluent：VEthyl acetate:VPetroleum ether=1: 5）, 2.6 g faint yellow solids 4- (12- hydroxydodecyls) tetraphenyl ethylenes are finally obtained, yield is about 61%.
1H NMR (600 MHz, CDCl3) δ 7.16-7.05 (m, 15H), 6.98 (d, J = 8.8 Hz, 2H), 6.67 (d, J = 8.8 Hz, 2H), 3.92-3.85 (m, 2H), 3.65 (t, J = 6.7 Hz, 2H), 3.45- 3.37 (m, 1H), 2.03 -1.82 (m, 2H), 1.81 -1.75 (m, 2H), 1.67 – 1.54 (m, 4H), 1.47 (dd, J = 14.2, 9.0 Hz, 4H), 1.35 (s, 8H)。
13C NMR (151 MHz, CDCl3) δ 153.96 (s), 144.28 - 143.62 (m), 140.29 (d, J = 33.3 Hz), 136.37 (s), 132.71 (s), 131.52 -131.16 (m), 127.63 (d, J = 15.1 Hz), 126.24 (s), 114.57 (s)。
（4）The synthesis of TPE-BPM：Take TPE-OH（5.26g 0.01mol）, triethylamine（1.75mL 0.0125mol）And 2- Bromo- 2- methyl propionyl bromide（1.5mL, 0.0125mol）, it is added and fills anhydrous THF（150ml）Three-necked flask in（250mL）. 24 hours, after reaction, reaction solution filtering are stirred at room temperature in the mixture；Filtrate concentrates, and crude product is using column chromatography point Separating-purifying is carried out to product from method（Eluent：VEthyl acetate:VPetroleum ether=1:10）, finally obtain 7 g faint yellow solid products TPE- BMP, yield are about 65%.
1H NMR (600 MHz, H2O) δ 8.89 – 7.06 (m, 15H), 7.06 – 6.92 (m, 2H), 6.85 – 6.49 (m, 2H), 4.39 – 4.16 (m, 2H), 3.93 – 3.70 (m, 2H), 2.14 – 1.91 (m, 7H), 1.93 – 0.80 (m, 24H).
The characterize data of TPE-BPM such as Fig. 1.
（5）The synthesis of TPE-PDEAEAM：By DEAEAM（3g）It is dissolved in 10 mL water and 5mL methanol (VWater: VMethanol= 2: 1) in the mixed solvent and 20 min of stirring, then sequentially add three under argon gas protective condition（2- dimethylaminoethyls）Amine Me6TREN (0.6g), CuBr (0.0282g, 0.1960mmol), TPE-BMP (0.05 g, 0.05 mmol), at room temperature After stirring 24 h, reaction stoste ultra-pure water is dialysed 72 hours, dialyzate is freeze-dried 72 hours, obtains 2.3g pale yellow colored solids Body powder, as TPE-PDEAEAM.The characterize data of TPE-PDEAEAM such as Fig. 2 and Fig. 3.
1HNMR (600 MHz, H2O): δ 7.01-6.25 (m), 3.20 (m), 2.51(m), 1.90-1.30 (m), 0.93(m)。
2, TPE-PDEAEAM is to Hela cell imaging applications
It first, will be on Hela cells in advance 12 well culture plate cell sheets of inoculation.Culture medium is containing 1% streptomysin and 10% tire ox 1640 solution of serum.Then 12 orifice plates are placed in the CO of 5% concentration2With 24 hours in the humidified incubator of 37 DEG C of temperature.With 100 μ gmL-1TPE-PAA handle Hela cells, after 24 hours, take out cell sheet, and tablet is rinsed three with phosphate buffer solution It is secondary.Cell imaging picture is shot under confocal fluorescence microscopy.Use Olympus FV1000 Laser Scanning Confocal Microscopes（Olympus Tokyo Japan）It excites in 332nm and emits in 460 ~ 490nm.
Figure 12 is the Laser Scanning Confocal Microscope figure of TPE-PDEAEAM polymer quantum Vertex Coloring HeLa cell difference algebraically Picture, engineer's scale are 25 microns.Figure 12 shows, Hela cells and 100 μ gmL-1TPE-PDEAEAM polymer quantum dots be incubated 24 Hour（The first generation）Afterwards, the signal strength of TPE-PDEAEAM polymer quantum dot in HeLa cells is observed.Intracellular Fluorescence is strong The position that relatively weak region may be nucleus is spent, illustrates that polymer quantum dot has successfully escaped the inner body of complete aggregation Compartment and the cytosolic domain for selectively having dyed HeLa cells.In addition, from TPE-PDEAEAM polymer intracellular HeLa Quantum dot monitors strong signal, even if with 100 μ gmL-1Concentration is incubated 48 hours（Generation）Afterwards, we still can observe by force Fluorescence signal.The cell handled with TPE-PDEAEAM polymer quantum dots repetition in every 48 hours is passed on, burnt micro- by being copolymerized Mirror measures the intracellular Fluorescence per generation TPE-PDEAEAM polymer quantum dots.The result shows that the cell after dyeing is up to 20 In it time（It was up to for 9 generations）It remains to keep stronger fluorescence intensity.Thus TPE-PDEAEAM polymer quantum dots may be One ideal long-term fluorecyte tracer.

Claims (10)

1. a kind of preparation method with multiple response polymer quantum dot TPE-PDEAEAM, comprises the following steps that：
（1）The synthesis of 4- hydroxyphenyl ethylene：Using tetrahydrofuran as solvent, zinc powder is reducing agent, TiCl4For catalyst, protected in argon gas Under shield, 4- dihydroxy benaophenonels and benzophenone are with 1:1~1:1.2 molar ratio reflux 22 ~ for 24 hours；Room is cooled to after answering K is added in temperature2CO3Solution quenching reaction, then filters, is extracted with ethyl acetate, product is purified through column chromatography for separation, is obtained faint yellow Four ethylene TPE of solid 4- hydroxyls；
（2）The synthesis of 4- (12- hydroxydodecyls) tetraphenyl ethylene：By the bromo- DODECANOL, 1- of 4- hydroxyls four ethylene TPE, 12- and K2CO3It is dissolved in anhydrous acetonitrile, flow back 20 ~ 24 h under protection of argon gas；It is cooled to room temperature after reaction, filtering reacting liquid And organic layer is evaporated, product is purified through column chromatography for separation, obtains faint yellow solid 4- (12- hydroxydodecyls) tetraphenyl ethylene TPE- OH；
（3）The synthesis of TPE-BPM：By 4- (12- hydroxydodecyls) tetraphenyl ethylene TPE-OH, triethylamine and the bromo- 2- methyl of 2- Propionyl bromide is added in anhydrous tetrahydro furan, reaction is stirred at room temperature 20 ~ 24 hours；After reaction, filtering reacting liquid is dense Contracting filtrate obtains crude product, and crude product is purified through column chromatography for separation, and it is TPE-BMP to obtain faint yellow solid product；
（4）The synthesis of TPE-PDEAEAM：N (2- (diethylamino) ethyl) acrylamide is dissolved in Water-Methanol Mixtures In, three (2- dimethylaminoethyls) amine, CuBr and TPE-BMP are sequentially added under protection of argon gas, are stirred at room temperature 20 ~ 24 Then reaction stoste is dialysed 70 ~ 72 hours with ultra-pure water, then dialyzate is freeze-dried 70 ~ 72 hours by h, obtain faint yellow Solid powder, as target product TPE-PDEAEAM.
2. a kind of preparation method with multiple response polymer quantum dot TPE-PDEAEAM as described in claim 1, feature It is：Step（1）In, the addition of zinc powder be 4- dihydroxy benaophenonels, 3 ~ 4 times of benzophenone integral molar quantity；TiCl4Plus Enter amount be 4- dihydroxy benaophenonels, 2 ~ 3 times of benzophenone integral molar quantity.
3. a kind of preparation method with multiple response polymer quantum dot TPE-PDEAEAM as described in claim 1, feature It is：Step（2）In, the molar ratio of four ethylene of 4- hydroxyls and the bromo- DODECANOL, 1-s of 12- is 1:1~1:1.2.
4. a kind of preparation method with multiple response polymer quantum dot TPE-PDEAEAM as described in claim 1, feature It is：Step（2）In, four ethylene of 4- hydroxyls and K2CO3Molar ratio be 1:1~1:1.2.
5. a kind of preparation method with multiple response polymer quantum dot TPE-PDEAEAM as described in claim 1, feature It is：Step（3）In, the molar ratio of TPE-OH and triethylamine is 1:1~1:1.25.
6. a kind of preparation method with multiple response polymer quantum dot TPE-PDEAEAM as described in claim 1, feature It is：Step（3）In, the molar ratio of TPE-OH and the bromo- 2- methyl propionyl bromides of 2- is 1:1~1:1.25.
7. a kind of preparation method with multiple response polymer quantum dot TPE-PDEAEAM as described in claim 1, feature It is：Step（4）In, the volume ratio of the Water-Methanol Mixtures, water and methanol is 1.5:1~2:1.
8. a kind of preparation method with multiple response polymer quantum dot TPE-PDEAEAM as described in claim 1, feature It is：Step（4）In, three (2- dimethylaminoethyls) amine（Me6TREN）Addition be DEAEAM mass 19% ~ 20%； The addition of CuBr is the 0.5% ~ 1% of DEAEAM mass.
9. a kind of preparation method with multiple response polymer quantum dot TPE-PDEAEAM as described in claim 1, feature It is：Step（4）In, the mass ratio of DEAEAM and TPE-BMP are 60:1~65:1.
10. one kind prepared by method as described in claim 1 has multiple response polymer quantum dot TPE-PDEAEAM as length The application of phase fluorecyte tracer.
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CN114957656A (en) * 2022-07-01 2022-08-30 济南大学 Preparation method of multi-stimulus-responsive fluorescent polyethyleneimine with LCST and UCST

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CN109385269B (en) * 2018-11-08 2021-09-24 西北师范大学 Preparation and application of pH-sensitive dual-fluorescent polymer quantum dot
CN114957656A (en) * 2022-07-01 2022-08-30 济南大学 Preparation method of multi-stimulus-responsive fluorescent polyethyleneimine with LCST and UCST

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