CN109575240B - Red light polymer with high fluorescence quantum efficiency, quantum dot solution and application - Google Patents

Red light polymer with high fluorescence quantum efficiency, quantum dot solution and application Download PDF

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CN109575240B
CN109575240B CN201710908468.4A CN201710908468A CN109575240B CN 109575240 B CN109575240 B CN 109575240B CN 201710908468 A CN201710908468 A CN 201710908468A CN 109575240 B CN109575240 B CN 109575240B
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陈于蓝
严春梅
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Abstract

The invention discloses a red light polymer with high fluorescence quantum efficiency, a quantum dot solution and application, wherein the structural formula of the red light polymer with high fluorescence quantum efficiency is as follows:

Description

Red light polymer with high fluorescence quantum efficiency, quantum dot solution and application
Technical Field
The invention belongs to the field of polymer preparation, and particularly relates to a red light polymer with high fluorescence quantum efficiency, a quantum dot solution and application thereof.
Background
Organic polymers have received a great deal of attention from many researchers as a hotspot in the field of emerging materials. This is mainly because the skeleton formed by the long repeating units of the polymer can realize good energy transfer, so that the polymer has strong light capture capability and optical signal amplification effect. Compared with most of small molecular compounds, the organic polymer has the characteristics of better optical performance, cell compatibility, low toxicity, easiness in modification and the like, so that the organic polymer is widely applied to the fields of luminescent materials, chemical and biological sensing, imaging and the like.
In the study of cell biology, the most common method for observing structures and molecules of interest is to fluorescently label them. However, conventional fluorescent probes have many limitations in the field of biomarkers. The near-infrared fluorescent probe is used as a non-invasive fluorescent imaging technology and has important significance for exploring a life process. Compared with the traditional fluorescent dye, the semiconductor polymer quantum dot has the excellent characteristics of high luminous brightness, high radiation rate, good light stability, good biocompatibility and the like, and has wide application in the field of biological fluorescence imaging. However, most of the reported quantum dots of semiconductor polymers have short fluorescence emission wavelength, and have great damage to tissue cells when applied to living body imaging; the fluorescence quantum efficiency is low, the signal-to-noise ratio is small, and the accuracy is poor when the fluorescence quantum efficiency is applied to fluorescence imaging.
The semiconductor polymer quantum dot emitting near infrared fluorescence has the advantages of small damage to biological tissues, high signal-to-noise ratio and high imaging accuracy due to long fluorescence emission wavelength, and is always a hotspot of research. However, because the structure design process of the semiconductor polymer quantum dots emitting near infrared fluorescence is complex, the synthesis and preparation are difficult, and few reports are reported in recent years, particularly the semiconductor polymer quantum dots with the emission wavelength positioned in the first optical window (650-900nm) of biological tissues. Therefore, it is necessary to design a polymer having a new structure and a fluorescence emission peak at 650-900nm, which is easy to prepare.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provide a red-light polymer with high fluorescence quantum efficiency.
The second purpose of the invention is to provide a preparation method of the red-light polymer with high fluorescence quantum efficiency.
The third purpose of the invention is to provide a red light polymer quantum dot solution with high fluorescence quantum efficiency.
The fourth purpose of the invention is to provide the application of the red-light polymer quantum dot solution with high fluorescence quantum efficiency in preparing the cell imaging agent.
The fifth purpose of the invention is to provide the application of the red-light polymer quantum dot solution with high fluorescence quantum efficiency in preparing the tubulin labeling agent.
The technical scheme of the invention is summarized as follows:
a red light polymer with high fluorescence quantum efficiency, wherein the polymer has a structural formula:
Figure BDA0001424328880000021
the weight average molecular weight of the polymer was 35 kg/mol.
The preparation method of the red light polymer with high fluorescence quantum efficiency comprises the following steps: mixing the compounds M1, M2, M3 and NaHCO3Adding redistilled tetrahydrofuran and deionized water into a reaction vessel, degassing, introducing nitrogen, and adding catalyst Pd (PPh)3)4Degassing, filling nitrogen, heating to reflux, reacting, and cooling to room temperature to obtain the red light polymer with high fluorescence quantum efficiency;
the reaction formula is as follows:
Figure BDA0001424328880000022
the weight average molecular weight of the polymer was 35 kg/mol.
Preferably: m1, M2, M3 and NaHCO3And Pd (PPh)3)4At a molar ratio of 9:1:10:234:0.19, and the ratio of M1 to redistilled tetrahydrofuran and deionized water was 36.6mg:6mL:3 mL.
A red light polymer quantum dot solution with high fluorescence quantum efficiency is prepared by the following steps: weighing a red light polymer which is shown in a formula (I) and has the weight-average molecular weight of 35kg/mol and high fluorescence quantum efficiency, dissolving the red light polymer in tetrahydrofuran to prepare a solution with the concentration of 0.5mg/mL, wherein the solution is a solution I; weighing polystyrene-maleic anhydride with the number average molecular weight of 1.7kg/mol, dissolving in tetrahydrofuran to prepare a solution with the concentration of 0.5mg/mL to be a solution II; mixing the first solution and the second solution according to the volume ratio of 5:1 to obtain a precursor solution, and performing ultrasonic treatment on the precursor solution according to the volume ratio of 3: 10 the precursor solution is injected into deionized water; and maintaining for 2-5min at N2Heating to 90-95 deg.C under protection, evaporating to remove tetrahydrofuran, and stopping N2And stopping heating, cooling to room temperature, and passing through a 220nm water system filter head to obtain a filtrate which is a red light polymer quantum dot solution with high fluorescence quantum efficiency.
The application of the red light polymer quantum dot solution with high fluorescence quantum efficiency in preparing a cell imaging agent.
The application of the red-light polymer quantum dot solution with high fluorescence quantum efficiency in preparing the tubulin labeling agent.
The invention has the advantages that:
the red light polymer with high fluorescence quantum efficiency has fluorescence emission in deep red and near infrared regions (650nm-900 nm); the red-light polymer quantum dots with the particle size of less than 20nm and high fluorescence quantum efficiency are prepared from the polymer by a nano reprecipitation method, and effective marking of a cell tubulin structure is successfully realized. Cytotoxicity tests prove that the red light polymer quantum dots with high fluorescence quantum efficiency have small influence on cell activity and good biocompatibility, and are near-infrared fluorescent probes for living body imaging.
Drawings
FIG. 1 is a liquid nuclear magnetic hydrogen spectrum of a red-light polymer prepared in example 1.
FIG. 2 is a liquid NMR spectrum of a red polymer prepared in example 1.
FIG. 3 is a UV absorption spectrum (a) and a fluorescence emission spectrum (b) of the red-emitting polymer prepared in example 1.
Fig. 4 is an ultraviolet absorption spectrum (a) and a fluorescence emission spectrum (b) of the red polymer quantum dot with high fluorescence quantum efficiency prepared in example 2.
Fig. 5 is an atomic force microscope image (a) and a dynamic light scattering image (b) of the red-light polymer quantum dot with high fluorescence quantum efficiency prepared in example 2.
Fig. 6 is a cell microtubule imaging graph (a) and a cytotoxicity test graph (b) of the red-light polymer quantum dot with high fluorescence quantum efficiency prepared in example 2.
Detailed Description
The present invention is further described with reference to specific examples, which are provided to enable those skilled in the art to better understand the present invention, but are not intended to limit the present invention in any way.
Example 1
A preparation method of a red light polymer with high fluorescence quantum efficiency comprises the following steps:
1. preparation of monomer M1:
(1) a1(2g,6.8mmol), thiophene borate (3.57g,17mmol), NaHCO3(5.72g,68mmol), tris (dibenzylideneacetone) dipalladium (0.31g,0.34mmol), and tris (o-methylphenyl) phosphine (0.41g,1.36mmol) were added to a mixed solution of tetrahydrofuran (80mL) and water (30mL), and degassing was repeated in the presence of N2Raising the temperature to 90 ℃ under the atmosphere and reacting for 72 h.
The reaction solution was cooled to room temperature, extracted with chloroform and saturated brine, the organic phase was collected, dried over anhydrous magnesium sulfate, and the solvent was spin-dried. After passing through a silica gel column, the crude product was recrystallized from a mixed solvent of methylene chloride and petroleum ether to give B1(1.2g, 60%) as an orange-red solid.
(2) B1(130mg,0.43mmol) and N-bromosuccinimide (231.5mg,13.9mmol) were added to a solution of chloroform (15mL) and stirred at room temperature for 12 h. The reaction solution was filtered with suction to give M1(177mg, 89%) as an orange-red solid.
Figure BDA0001424328880000041
2. Preparation of monomer M2:
(1) a2(16.79mmol,4.5g), tert-butyryl chloride (31.45mmol,4.23g), and triethylamine (4mL) were added to 100mL of a tetrahydrofuran solution, and the mixture was stirred at room temperature for 12 hours. The solvent was spun off under reduced pressure, dichloromethane and water were added for extraction washing, and then dried over anhydrous magnesium sulfate, and finally spun dry. Purifying by column chromatography, wherein an eluent is dichloromethane: petroleum ether is 2: 3 to give compound B2(6.17g, 84%) as a white solid.
(2) Mixing B2(2.29mmol,1g), thiophene borate (5.28mmol,1.11g) and NaHCO3(95.22mmol,8g), tetrakis (triphenylphosphine) palladium (0.07mmol,0.08g) were added to a 250mL two-necked flask, degassed three times with suction, added to a mixed solution of tetrahydrofuran (75mL) and water (30mL), degassed three times with suction, and finally heated to 70 ℃ under nitrogen for 24 hours under reflux. Cooling to room temperature, removing the solvent under reduced pressure, adding dichloromethane and water for extraction washing, drying with anhydrous magnesium sulfate, and finally spin-drying. Performing column chromatography purification, wherein an eluent is dichloromethane: petroleum ether is 1:1, to give Compound C2(0.61g, 61%) as a white solid.
(3) C2(1.13mmol,0.5g), phosphorus oxychloride (15mL), phosphorus pentoxide (23.25mmol,3.3g) was charged into a 50mL single neck round bottom flask, heated to 116 deg.C and refluxed for 30 hours. After cooling to room temperature, the mixture was slowly poured into ice water, and the pH was adjusted to 10 with potassium hydroxide, followed by cooling. Dichloromethane was added for extraction, followed by drying over anhydrous magnesium sulfate and finally spin-drying. After passing through a neutral alumina column, the mixture was finally recrystallized from dichloro and petroleum ether to give compound D2(0.32g, 71%) as a pale yellow solid.
(4) Adding diisopropylamine (1mL) into tetrahydrofuran (20mL), freezing to remove oxygen, slowly adding n-butyllithium (2.5mL) at-78 deg.C, maintaining the temperature for 1 hr, taking out, stirring at room temperature for 1 hr to obtain yellow solution; then, D2(0.25mmol,0.1g) was added at-78 deg.C, the temperature was maintained at low for 1 hour, and the mixture was taken out and stirred at room temperature for 1 hour until the solution became green; then, carbon tetrabromide (2.6mmol,0.86g) was added at-78 ℃ and the solution was brown overnight with natural warming. The reaction was quenched by addition of water, the solvent was decanted, dichloromethane was added for extraction, dried over anhydrous magnesium sulfate and finally spun dried. After passing through a neutral alumina column, the mixture was finally recrystallized from dichloro-benzene and petroleum ether to obtain pure white solid compound M2(0.12g, 84%).
Figure BDA0001424328880000051
Wherein R is1Is a tert-butyl group.
3. Preparation of monomer M3:
(1) a3(12.0g,37mmol), 1-bromo-n-octane (15.7g,81.5mmol) and tetrabutylammonium bromide (0.5g,1.6mmol) were added to an aqueous NaOH solution (50 wt%, 30mL), and the mixture was stirred at 70 ℃ for 24 hours. The reaction solution was cooled to room temperature, and then saturated brine was poured into the reaction solution, and the organic phase was collected after extraction with dichloromethane, washed with deionized water, dried over anhydrous magnesium sulfate, and the solvent was spin-dried. After passing through a silica gel column, the crude product was recrystallized from ethanol to give white needle-like crystals B3(16g, 80%).
(2) Mixing B3(1g,1.8mmol), bis pinacolato borate (1.4g,5.4mmol) and PdCl2(dppf) (0.13g,0.18mmol) and potassium acetate (1.37g,13.96mmol) were added to 1, 4-dioxane (30mL), degassed repeatedly, and then charged under N2Raising the temperature to 80 ℃ in the atmosphere and reacting for 12 h. Cooling the reaction liquid to room temperature, heating to remove 1, 4-dioxane, extracting with dichloromethane and saturated saline water, collecting the organic phase, adding anhydrous magnesium sulfate, drying, and spin-drying the solvent. After passing through a silica gel column, the crude product was recrystallized from n-hexane to give M3(0.8g, 69%) as a white solid.
Figure BDA0001424328880000052
4. Preparation of a red-light polymer with high fluorescence quantum efficiency:
compounds M1(36.6mg, 80.1. mu. mol), M2(5mg, 8.9. mu. mol), M3(57.1mg, 89. mu. mol), NaHCO were added3(175mg, 2080. mu. mol), redistilled tetrahydrofuran (6mL), deionized water (3mL) were added to the reactor, and after repeated degassing, the freshly prepared catalyst Pd (PPh) was added rapidly under nitrogen blanket3)4(2mg, 1.7. mu. mol), degassing several times, heating to reflux the reaction mixture under nitrogen atmosphere, reacting for 72 hours, cooling to room temperature, adding saturated saline, and adding 90mL of CHCl3The extraction was carried out three times, and the organic layers were combined and dried over anhydrous magnesium sulfate. The solvent was dried by spinning, the crude product was precipitated with methanol, the precipitate was filtered and then dried under vacuum to give the polymer as a purple solid (90mg, 91%).
FIGS. 1 and 2 are a liquid nuclear magnetic hydrogen spectrum and a carbon spectrum of the polymer obtained in this example, and the test was carried out on a 400MHZ BrukeraV 400 nuclear magnetic spectrometer.
Polymer in chloroform solution (concentration 1X 10)-5mol/L-1) The ultraviolet absorption spectrum of (A) is shown in FIG. 3(a), and the test is performed on a Perkinelmer Lambda 750UV/VIS/NIR ultraviolet spectrometer. The ultraviolet absorption of the polymer is mainly concentrated in the wavelength range of 350nm-650nm, and two remarkable absorption peaks are shown, wherein the two absorption peaks are respectively at 397nm (molar absorption coefficient is 7.82x 10)4M-1cm-1) And 552nm (molar absorption coefficient 7.91x 10)4M-1cm-1) To (3). These two absorption peaks demonstrate the presence of two blocks within the polymer I molecule. Polymer I molecules have UV absorption at (420-600nm), demonstrating that they can be excited by conventional bio-friendly lasers (e.g., 488nm laser or 522nm laser).
Polymer in chloroform solution (concentration 1X 10)-5mol/L-1) The fluorescence emission spectrum (397nm excitation) in (1) is shown in FIG. 3(b), and the test was performed on a Hitachi F-7000 fluorescence spectrometer. The two blocks present within the polymer I generate energyThe maximum emission wavelength of the polymer I molecule reaches 653nm, the maximum emission wavelength has larger Stokes shift (101nm) compared with the ultraviolet absorption spectrum, the fluorescence quantum efficiency is up to 72.6 percent (by taking rhodamine B as a reference), and the test proves that the fluorescence emission of the polymer I molecule extends to the near infrared region (600-800 nm).
And (3) proving that: the structure of the polymer is shown in a formula I,
Figure BDA0001424328880000061
the weight average molecular weight of the polymer was 35kg/mol as determined by an Agilent technologies 1200 gel permeation chromatography instrument.
The reaction formula is as follows:
Figure BDA0001424328880000062
example 2
A red light polymer quantum dot solution with high fluorescence quantum efficiency is prepared by the following steps: weighing a red light polymer with high fluorescence quantum efficiency shown in a formula I, dissolving the red light polymer in tetrahydrofuran to prepare a solution with the concentration of 0.5mg/mL as a solution I; weighing polystyrene-maleic anhydride with the number average molecular weight of 1.7kg/mol, dissolving in tetrahydrofuran to prepare a solution with the concentration of 0.5mg/mL to be a solution II; mixing the first solution and the second solution according to the volume ratio of 5:1 to obtain a precursor solution, and performing ultrasonic treatment on the precursor solution according to the volume ratio of 3: 10 the precursor solution is injected into deionized water; and keeping for 2min (experiments prove that the keeping time can be 2-5 min), and keeping in N2Heating to 90 deg.C under protection (experiment proves that the temperature can be 90-95 deg.C), evaporating to remove tetrahydrofuran, and stopping N2And stopping heating, cooling to room temperature, and passing through a 220nm water system filter head to obtain a filtrate which is a red light polymer quantum dot solution with high fluorescence quantum efficiency.
The ultraviolet absorption spectrum of the red-light polymer quantum dot with high fluorescence quantum efficiency in aqueous solution is shown in fig. 4 (a). The ultraviolet absorption of the red-light polymer quantum dots with high fluorescence quantum efficiency is mainly concentrated in the wavelength range of 350nm-650nm, and two remarkable absorption peaks are presented, wherein the two absorption peaks are respectively at 369nm and 515 nm.
The fluorescence emission spectrum (515nm excitation) of the red-light polymer quantum dot with high fluorescence quantum efficiency in aqueous solution is shown in fig. 4 (b). The red light polymer quantum dot with high fluorescence quantum efficiency has obvious emission in deep red and near infrared regions (580-850nm), the maximum emission wavelength is 677nm, and the absolute fluorescence quantum efficiency is as high as 6.2 percent, so that the red light polymer quantum dot with high fluorescence quantum efficiency, which is obtained by the invention, is proved to be the red light polymer quantum dot with high fluorescence quantum efficiency.
An atomic force microscope image of red polymer quantum dots with high fluorescence quantum efficiency is shown in fig. 5(a), and the test was performed on an atomic force microscope of Bruker mutimede 8. The dynamic light scattering pattern of the red polymer quantum dots with high fluorescence quantum efficiency is shown in fig. 5(b), and the test is performed on a dynamic light scattering instrument of Malvern Zetasizer NanoS. FIG. 5 shows that the red-light polymer with high fluorescence quantum efficiency can obtain spherical quantum dots with uniform and compact particle size by a nano-reprecipitation method, and the average particle size is 13 nm.
Example 3
The red light polymer quantum dots with high fluorescence quantum efficiency are biological.
mu.L of a 5% weight/volume aqueous polyethylene glycol solution (weight average molecular weight: 3.35kg/mol) and 20. mu.L of HEPES (hydroxyethylpiperazine ethanethiosulfonic acid) buffer (1M, pH: 7.2) were added to 1mL of a 50. mu.g/mL aqueous red-light polymer quantum dot solution with high fluorescence quantum efficiency, and streptavidin (1mg/mL, 60. mu.L) was added and mixed. 20 μ L of a freshly prepared solution of EDC (1-ethyl 3- [ 3-dimethylaminopropyl ] carbodiimide hydrochloride) (5mg/mL in Milli-Q water) was added, mixed, and the mixture was stirred at room temperature for 4 hours. And (4) centrifuging by using an ultrafiltration centrifugal tube (molecular weight cut-off is 100,000) to obtain the streptavidin-biologized quantum dots of the red-light-emitting polymer.
Example 4 microtubule labeling of Red-emitting Polymer Quantum dot streptavidin with high fluorescence Quantum efficiency
5% CO at 37 ℃2In (1), the composition is prepared by adding 10% bovine serum albumin (FBS) and 1% PenStrep (5000 units/mL Pannichi)Forest G, 50. mu.g/mL streptavidin sulfate in 0.85% NaCl) in DMEM complete medium (a medium containing various amino acids and glucose) to culture human cervical cancer cells (Hela cells). Cells were pre-cultured before the experiment until aggregation was reached.
1.5mL of an extract (PIPES (1, 4-piperazinediethanesulfonic acid) having a concentration of 0.1M, EGTA (ethyleneglycol bis (2-aminoethyl ether) tetraacetic acid) having a concentration of 1mM, MgCl was added2Was immersed in HeLa cells at a concentration of 1mM and Triton X-100 ((polyethylene glycol octylphenyl ether-100) volume fraction of 0.2%), extracted for 5min, and then the HeLa cells obtained by the extraction were washed three times with PBS (phosphate buffered saline). Then, HeLa cells were soaked with 1.5mL of a PBS solution (paraformaldehyde, volume fraction of 4%, glutaraldehyde, volume fraction of 0.1%) at room temperature for 15 minutes, fixed, washed three times with the PBS solution, and then permeabilized with 1.5mL of a LTriton-X100 solution (volume fraction of 0.5%). Cell-free buffer solution (bovine serum albumin, 5% by volume, Triton-X100, 0.1% by volume) was added to the cells for 30 minutes. The streptavidin-linked tubulin antibody was diluted to 5. mu.g/mL with a blocking buffer solution, added to a HeLa cell culture dish, cultured at room temperature for 1 hour, and washed with PBS to remove excess antibody. mu.L of streptavidin-biotinylated red-light polymer in water (50. mu.g/mL) was added, incubated at room temperature for 1 hour, and the cells were washed three times with PBS in water. Finally, the cell microtubule imaging was observed with a total internal reflection fluorescence microscope (excited with 405nm laser).
An image of microtubule of a cell with high fluorescence quantum efficiency of red-light polymer quantum dots is shown in fig. 6(a), and the test is performed on a total internal reflection fluorescence microscope of Olympus IX71, which demonstrates that the high fluorescence quantum efficiency of red-light polymer quantum dots can realize effective labeling of tubulin of the cell.
Example 5
Cytotoxicity analysis of red-light polymer quantum dots with high fluorescence quantum efficiency
Toxicity tests were performed using the MTT assay, growth medium solutions ( concentrations 0,10,20,40,80,100 μ g/mL, respectively) containing red-emitting polymer quantum dots with high fluorescence quantum efficiency were prepared, and the number of surviving cells was observed after HeLa cells were cultured with the growth medium solutions in a 96-well plate for 24 hours.
The cytotoxicity test chart of the red-light polymer quantum dot with high fluorescence quantum efficiency is shown in fig. 6(b), the test is carried out on an imaging reader of a BioTek Instruments Inc, and the chart proves that when the concentration of the red-light polymer quantum dot with high fluorescence quantum efficiency is 10-100ppm, the red-light polymer quantum dot with high fluorescence quantum efficiency basically has no toxic effect on cells and shows better biocompatibility.

Claims (6)

1. A red light polymer with high fluorescence quantum efficiency is characterized in that the structural formula of the polymer is as follows:
Figure FDA0002613224320000011
the weight average molecular weight of the polymer was 35 kg/mol.
2. The method for preparing a red-light polymer with high fluorescence quantum efficiency according to claim 1, comprising the steps of: mixing the compounds M1, M2, M3 and NaHCO3Adding redistilled tetrahydrofuran and deionized water into a reaction vessel, degassing, introducing nitrogen, and adding catalyst Pd (PPh)3)4Degassing, filling nitrogen, heating to reflux, reacting, and cooling to room temperature to obtain the red light polymer with high fluorescence quantum efficiency;
the reaction formula is as follows:
Figure FDA0002613224320000012
the weight-average molecular weight of the polymer (I) was 35 kg/mol.
3. The method as set forth in claim 2, wherein said M1, M2, M3 and NaHCO are used as raw materials3And Pd (PPh)3)4In a molar ratio of 9:1:10:234:0.19, of said M1 with redistilled tetrahydrofuran and deionized waterThe ratio was 36.6mg to 6mL to 3 mL.
4. A red light polymer quantum dot solution with high fluorescence quantum efficiency is characterized by being prepared by the following method: weighing the red light polymer with high fluorescence quantum efficiency according to claim 1, wherein the weight average molecular weight of the red light polymer is 35kg/mol, and dissolving the red light polymer in tetrahydrofuran to prepare a solution with the concentration of 0.5mg/mL to obtain a solution I; weighing polystyrene-maleic anhydride with the number average molecular weight of 1.7kg/mol, dissolving in tetrahydrofuran to prepare a solution with the concentration of 0.5mg/mL to be a solution II; mixing the first solution and the second solution according to the volume ratio of 5:1 to obtain a precursor solution, and performing ultrasonic treatment on the precursor solution according to the volume ratio of 3: 10 the precursor solution is injected into deionized water; and maintaining for 2-5min at N2Heating to 90-95 deg.C under protection, evaporating to remove tetrahydrofuran, and stopping N2And stopping heating, cooling to room temperature, and passing through a 220nm water system filter head to obtain a filtrate which is a red light polymer quantum dot solution with high fluorescence quantum efficiency.
5. Use of the high fluorescence quantum efficiency red-emitting polymer quantum dot solution of claim 4 in the preparation of a cell imaging agent.
6. Use of the red-emitting polymer quantum dot solution with high fluorescence quantum efficiency according to claim 4 for preparing tubulin labeling agent.
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