Aqueous quantum dots with high fluorescence, colloidal stability and biocompatibility encapsulated by an amphiphilic fluorine copolymer

Polymer 179 (2019) 121706

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Aqueous quantum dots with high fluorescence, colloidal stability and biocompatibility encapsulated by an amphiphilic fluorine copolymer

T

Kai Yanga, Guanzhou Luoa, Xinjuan Zenga, Mengyi Xub,∗∗, Pihui Pia, Shouping Xua, Xiufang Wena,∗ a

School of Chemistry and Chemical Engineering, Guangdong Provincial Key Lab of Green Chemical Product Technology, South China University of Technology, Guangzhou, 510640, China b School of Chemical Engineering and Technology, Guangdong Industry Polytechnic, Guangzhou, 510300, China

H I GH L IG H T S

amphiphilic fluorine copolymers O A F were synthesized. • Novel CdSe encapsulated by O A F (O A F @QDs) was successfully transfered from CHCl • Oleylamine-capped hydrodynamic diameters of O A F @QDs were tunable. • The were colloidally stable in aqueous solution. • OO AA FF @QDs @QDs exhibited low levels of nonspecific binding to proteins. • m

n p

m

m

m

n p

m

n p

n p

m

n p

3

to water.

n p

A R T I C LE I N FO

A B S T R A C T

Keywords: Amphiphilic fluorine copolymers Stable Nonspecific binding Fluorescent probes

Quantum dots (QDs) encapsulated by amphiphilic fluorine copolymers have shown great promise as molecular probes for bio-imaging. Here, a series of amphiphilic fluorine copolymers, methoxypolyethylene glycols-blockpoly (2-(diethylamino)ethyl methacrylate)-block-poly (2,2,3,4,4,4-hexafluorobutyl methacrylate) (OmAnFp), were successfully coated on the surface of semi conductive quantum dots (QDs) to overcome their stability issues in aqueous solution. The hydrodynamic diameters (Dh,DLS) of these OmAnFp encapsulated QDs (OmAnFp@QDs) can be tuned between 40 nm and 61 nm by choosing amphiphilic fluorine copolymers with different hydrophobic portions. The obtained OmAnFp@QDs colloids are found stable over a wide pH range (4.0–12.0) with no sign of aggregation. In the pH range of 4.0–7.0, the repulsion of block O and cationic block A on OmAnFp@QDs contributes to the colloids’ stability in aqueous solution. The protonation of block A also leads to the decrease of Dh,DLS from 61 to 51 nm and the enhancement of the transmittance of O113A11F19@QDs solution from 78% to 92%. In the pH range from 8.0 to 12.0, the repulsion between the strong hydrated block O makes OmAnFp@QDs nanoparticles stable with deprotonated hydrophobic block A. The fluorescent quantum yield properties of QDs are well preserved in OmAnFp@QDs aqueous solution due to the protection of block F wrapping tightly around the QDs cores. The photoluminescence quantum yield (PL QY) is found reserved better under basic conditions due to the co-encapsulation of hydrophobic block A. The PL intensity of OmAnFp@QDs in 400 mM NaCl solution changes only slightly. Block O and F in the polymer offer OmAnFp@QDs with good biocompatibility and low levels of nonspecific binding to bovine serum albumin proteins (BSA). These advantages render OmAnFp@QDs an attractive candidate as ideal fluorescent probes in advanced biomedical imaging studies.

1. Introduction Quantum dots (QDs) have drawn tremendous attention in medical imaging due to their superior optical properties [1,2] such as bright

∗

fluorescence, broad excitation spectra, high absorption coefficients, and tunable optoelectronic properties [3–8]. Colloidal QDs obtained by high-temperature synthesis in coordinating organic solvents are highly crystalline and monodispersed [9,10]. However, the hydrophobic

Corresponding author. Corresponding author. E-mail addresses: [email protected] (M. Xu), [email protected] (X. Wen).

∗∗

https://doi.org/10.1016/j.polymer.2019.121706 Received 16 April 2019; Received in revised form 14 July 2019; Accepted 12 August 2019 Available online 13 August 2019 0032-3861/ © 2019 Elsevier Ltd. All rights reserved.

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(termed as F) to encapsulate QDs (OmAnFp@QDs) (Scheme 1). Block O on the polymer endows OmAnFp@QDs with good biocompatibility and blocks A and F provide those colloids stability in aqueous solution. By varying lengths of the F cores, the hydrodynamic diameters of the obtained OmAnFp@QDs can be adjusted from 61 nm to 40 nm. By circumventing any direct ligand exchange at the QD surface, the obtained OmAnFp@QDs maintained the original photoluminescence in deionized water. Its photoluminescence (PL) intensity only changes slightly when it is in NaCl solution with the concentration range from 0 to 400 mM. It also exhibits long-term stability across wide pH range (pH = 4.0 to 12.0) and shows no sign of aggregation after being stored up for 4 h. Moreover, OmAnFp@QDs shows negligible nonspecific binding to BSA. With all these properties important as biological imaging probes, the introduction of this amphiphilic fluorine copolymer coating advances an important step for the use of these important bio-imaging nanomaterials in biomedical practice.

ligands grafted on QDs repel water, making them stable only in organic solvent, which greatly limits their primary biological applications in aqueous environments [11–14]. Displacing organophilic surface species with soft and flexible hydrophilic ligands is an effective way to allow QDs to present in aqueous solutions. Yet the coating on the surface of QDs is too thin to avoid aggregate formation [15–18]. The outer ligand shell of QDs coated with proteins through hydrophobic or ionic interactions had been explored to improve their colloidal stability [19]. But nonspecific adsorption cannot be effectively avoided in this way and the modified QDs are stable just in a solution under certain pH value [18]. Wrapped by polyethylene glycol modified polymers endow those nanostructures with low nonspecific adsorption [20–22]. However, the loose di-block copolymer matrix on the shell of QDs, such as poly(N-isopropyl acrylamide)-block-poly(acrylic acid) and polystyrene-b-poly(acrylic acid), allows small molecules diffusion and consequent QDs oxidation, which causes their QY drop dramatically [23–26]. Water-soluble polymercoated QDs with good photoluminescence response, excellent colloidal stability, and low nonspecific adsorption are highly desired. In this work, we designed and synthesized a series of amphiphilic fluorine copolymer (OmAnFp) composed of hydrophilic methoxypolyethylene glycols (termed as O), middle hydrophobic poly (2(diethylamino)ethyl methacrylate) bridging block (termed as A), and fluorinanted poly (2,2,3,4,4,4-hexafluorobutyl methacrylate block

2. Materials and methods 2.1. Materials Methoxypolyethylene glycols (average Mn = 5000 g/mol), 2-bromoisobutyryl bromide, 3-butyn-1-ol, and sodium azide (NaN3), 2-(diethylamino)ethyl methacrylate (DEAEMA), 2,2,3,4,4,4-hexafluorobutyl

Scheme1. Synthetic pathway towards amphiphilic fluorine copolymer OmAnFp (top) and schematic illustration on preparing water-dispersed OmAnFp@QDs (bottom). 2

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step. IR (KBr): 2887, 2137, 1740 (vs, C=O), 1625, 1471, 1110, 1107, 961, 840 cm−1. GPC (CHCl3): Mn, GPC = 7050 g/mol, Mw/Mn = 1.23. Synthesis of OmAnFp via click chemistry. The procedure for the synthesis of copolymers was proceeded with a feed ratio of O113A10-N3/ F5-alkyne/CuBr/PMDETA = 1/1/1/1. O113A10-N3 (1.00 g, 0.2 mmol), F5-alkyne (0.04 g, 0.2 mmol) and DMF (5 ml) were added to a 25 ml Schlenk flask and the mixture was degassed by three freeze-pump-thaw cycles. The flask was then filled with argon. PMDETA and CuBr were quickly added to the flask while the reaction mixture was kept frozen in liquid nitrogen. After sealing the flask, it was evacuated and purged with argon three times. The flask was then heated to 100 °C and maintained at that temperature for 24 h. The reaction was then stopped by adding THF and exposing the reaction mixture to air. The solvent was removed under reduced pressure to leave blue product that was further purified by column chromatography on silica gel using chloroform. The solution was then concentrated and precipitated in hexane. For further purification, the product was dissolved in DMF and dialysis against water with dialysis bag (7000 Da) for 12 h. The final product was dried in a vacuum oven for 24 h. IR (KBr): 2985, 1740 (vs, C=O), 1625, 1471, 1402, 1197, 1110, 961, 840, 617 cm−1. GPC (CHCl3): Mn,GPC = 8609 g/mol, Mw/Mn = 1.16.

methacrylate (HFBMA), N,N,N′,N’’,N’’-pentamethyldiethylenetriamine (PMDETA), cuprous bromide (CuBr) were purchased from SigmaAldrich and used as received. All solvents were of analytical grade. Dialysis bags with the specific molecular weight cut-off (2000 Da and 7000 Da) were purchased from Roth and washed with deionized water before use. Oleylamine-capped CdSe nanoparticles (8 nm core diameter) (Fig. S4) dispersed in CHCl3 (10 mg/ml) were purchased from Xingzi (Shanghai) New-Material Technology Development co., LTD and were diluted to 0.1 mg/ml in CHCl3 for further use. Note that the concentration (0.1 mg/ml) and amount (1 ml) of oleylamine-coated CdSe used in all samples remain unchanged in the following experiments. The QDs mentioned below denoted oleylamine-capped CdSe except where noted. Citrate buffer (CB, pH = 5.0), phosphate-buffered saline (PBS, pH = 7.4), carbonate-bicarbonate buffer tablets (CBB, pH = 9.4), fluorescein isothiocynate labeled bovine serum albumin (FITC-BSA, CBSA = 5 mg/ml, CFITC = 75 μg/ml) and Pierce BCA Protein Assay Kit were purchased from Thermo Fisher Scientific. 2.2. Methods 2.2.1. Synthesis of amphiphilic fluorine copolymer OmAnFp Synthesis of alkyne terminated Fp-alkyne. Initiator BBiB (0.20 g, 0.09 mmol) was synthesized as described in supplementary material, HFBMA (0.18 g, 0.72 mmol) and butanone (5 ml) were added to a 25 ml Schlenk flask equipped with a magnetic stir bar and the mixture was degassed by three freeze-pump-thaw cycles. The flask was then filled with argon gas and had PMDETA (0.16 g 0.09 mmol) and CuBr (0.13 g, 0.09 mmol) quickly added with the reaction mixture kept frozen under liquid nitrogen. After sealing the flask, it was further evacuated and purged with argon three times. The flask was then immersed in bath at 80 °C. After 4 h, the reaction was stopped by adding THF and exposing to air. The solvent was removed under reduced pressure to leave blue product that was purified by column chromatography on silica gel using dichloromethane. The solution was then concentrated and precipitated in hexane. For further purification, the product was dialysis against methanol with dialysis bag (2000 Da) for 12 h and the final product was dried in a vacuum oven for 24 h (0.31 g, yield: 77%). The corresponding 1 H NMR spectrum was shown in Fig. S1. IR (KBr): 3321 (s, C≡H), 2995, 1741 (vs, C=O), 1402, 1197, 617 cm−1. GPC (CHCl3): Mn,GPC = 1500 g/mol, Mw/Mn = 1.11. Preparation of O113An-Br. O113-Br macroinitiator (1.00 g, 0.2 mmol) was synthesized as described in supplementary material. DEAEMA (0.56 g, 3.0 mmol) and 2-propanol/water (v/v = 10:1, 5 ml) were added to a 25 ml Schlenk flask equipped with a magnetic stir bar and the mixture was degassed by three freeze-pump-thaw cycles. The flask was then filled with argon and PMDETA and CuBr were quickly added into the flask while the reaction mixture was kept frozen under liquid nitrogen. After sealing the flask, it was evacuated and purged with argon gas three times. The flask was then immersed in a water bath at 60 °C. After 4 h, the reaction was stopped by adding THF and exposing the reaction mixture to air. The solvent was removed under reduced pressure to give blue product that was further purified by column chromatography on silica gel using dichloromethane. The solution was concentrated and precipitated in hexane to yield O113A10-Br. For further purification, the product was dialysis against water with dialysis bag (7000 Da) for 12 h and the final product was dried in a vacuum oven for 24 h. The corresponding 1H NMR spectrum was shown in Fig. S2. IR(KBr): 2887, 1740 (vs, C=O), 1632, 1471, 1107, 961, 840 cm−1. GPC (CHCl3): Mn,GPC = 7068 g/mol, Mw/Mn = 1.20. Preparation of O113An-N3. A solution of O113An-Br (0.50 g, 7.134 mmol) and NaN3 (0.87 g, 8.56 mmol) in DMF (20 ml) was heated to 85 °C. After mixing for 48 h, the mixture was cooled to room temperature and 80 ml of dichloromethane was added. After the insoluble salt was filtered out, the solution was washed with deionized water three times, followed by drying with anhydrous MgSO4. The product was obtained after vacuum distillation and directly used in the next

2.2.2. Titration of amphiphilic fluorine copolymer OmAnFp In order to determine the pKa value of OmAnFp, a 5 mg/ml of copolymer solution (pH = 2) was titrated using 0.1 M NaOH solution. The pH value of the solution was monitored by a Corning Check-Mite pH sensor. 2.2.3. Preparation of OmAnFp@QDs OmAnFp was dissolved in 1 ml of CHCl3. The diluted oleylaminecapped CdSe (0.1 mg/ml, 1 ml) dispersed in CHCl3 were added and mixed intensively for 2h. After evaporation of CHCl3, 1 ml water was dropwise added to the mixture to trigger the self-assembly of the block copolymer and QDs. Then, the obtained mixture was centrifuged at 15000 rpm for 30 min. The precipitate was collected and re-dispersed in water. Finally, 5 ml of solution was obtained after precipitation and redispersion three times. 2.2.4. Test the colloidal stability of OmAnFp@QDs Stability tests. Each experiment was carried out in triplicate to determine the standard deviations of the measurements. Stability of OmAnFp@QDs solutions at different pH value. By mixing appropriate volumes of acids (0.4 M hydrochloric acid) and basic components (0.2 M sodium hydroxide), solutions with pH value from 4.0 to 12.0 were prepared. In order to test the stability of OmAnFp@QDs under different pH values, 1 ml of the resultant solution after the polymer coating was diluted to 5 ml solution at a corresponding pH and mixed for 10 min. The PL emission spectra, PL QY, zeta-potential, DLS and transmittance of the samples were measured 4 h later. Stability under different buffer media. Citrate buffer (pH = 5.0) and phosphate buffered saline (pH = 7.4) were used as received. Carbonate-bicarbonate buffer (pH = 9.5) was obtained by dissolving the commercial carbonate-bicarbonate buffer tablets in the corresponding volume of deionized water. The resultant solution (1 ml) after polymer coating was diluted in 5 ml buffer and mixed for 10 min. The PL emission spectra, PL QY and zeta-potential of the samples were recorded after 4 h incubation. Stability in NaCl solution. The resultant solution (1 ml) after polymer coating was diluted to 5 ml with NaCl solution at a concentration of 0, 200, 400, 600, 800 and 1000 mM, respectively. The PL emission spectra, PL QY and zeta potential of the samples in NaCl solution were recorded after 4 h incubation. The obtained OmAnFp@QDs (2 ml) were mixed with diluted FITCBSA (CBSA = 60 μg/ml, 2 ml) at 37 °C. After incubation for 1 h, OmAnFp@QDs were separated from the mixture by centrifuging at 3

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the GPC curves of F5-alkyne, O113A10-N3, and O113A10F5. As shown in Fig. 1a, the FT-IR band appears at 1197 cm−1 represents the characteristic vibrations of F5-alkyne (-CF-). Band at 1625 cm−1 is attributed to C-N stretching vibration for block A, and band at 1110 cm−1 belongs to the C-O-C stretching vibrations. The stretching vibration band of ≡CH band at 3321 cm−1 and vibration band of –N=N=N at 2137 cm−1 are missing, which indicates the successful coupling of F5alkyne and O113A10-N3. The structure of the amphiphilic fluorine copolymer is also confirmed by 1H NMR spectroscopy where the signals of the corresponding segments in every block were clearly shown in Fig. 1b. Comparing with the curves of F5-alkyne and O113A10-N3, the GPC curves of the resultant copolymer O113A10F5 shifts to shorter retention time (Fig. 1c). In addition, the molecular weight of O113A10F5 is approximately equal to the sum of F5-alkyne and O113A10-N3. This further confirms that F5-alkyne and O113A10-N3 are successfully bonded together. The upside down curve of F5-alkyne in Fig. 1c is the result of the lower refraction coefficient of F5-alkyne than the eluent chloroform. The amphiphilic fluorine copolymers with different blocks lengths (O113A10F5, O113A8F13 and O113A11F19) could be synthesized by changing the initial ratio of three reactants. The componential parameters of these copolymers are given in Table 1. According to the GPC results, the molecular weight characteristics and the degree of polymerization (DP) for each block are calculated and listed in Table S1. From the titration curves in Fig. 1d, pKa values of 7.40, 7.30 and 7.25 are obtained for the conjugate acid forms of the tertiary amine methacrylate residues in O113A10F5, O113A8F13, and O113A11F19, respectively.

15000 rpm for 5 min. After discarding the supernatant, the precipitate was re-dispersed into 4 ml deionized water. The fluorescence of the initial mixture and solution after re-dispersion was examined on a confocal laser scanning microscopy (CLSM) platform (Leica TCS SP8). In order to selectively measure the signal from QDs rather than FITCBSA, OmAnFp@QDs and FTIC-BSA were excited at 405 nm and 488 nm, respectively. The fluorescence was observed in the emission range from 510 to 550 nm for FITC-BSA (green channel) and in the range from 610 to 640 nm for OmAnFp@QDs (red channel). Image analysis was carried out using LAS AF software. The protein concentration in solutions before and after centrifugation was examined using Pierce BCA Protein Assay Kit. 2.2.6. Characterizations Nuclear magnetic resonance spectroscopy (NMR) and Fourier transform infrared spectroscopy (FT-IR) were used to characterize the structure of OmAnFp. Gel permeation chromatography (GPC) was conducted to determine the molecular weight and polydispersity index (PDI) of OmAnFp. Transmission electron microscopy (TEM) and dynamic light scattering (DLS) were used to characterize the size and zetapotential (ζ) of OmAnFp@QDs. Scanning transmission electron microscopy (STEM) and energy dispersive X-ray spectroscopy (EDS) with line-scan were performed on JEM-2100F. The optical properties of OmAnFp@QDs were characterized by ultraviolet–visible spectroscopy (UV–vis), photoluminescence spectroscopy (PL), time-resolved photoluminescence decay spectroscopy (Fig. S3) and Hamamatsu absolute PL quantum yields (QY) spectrometer. All these detail characterization methods were described in supplementary material.

3.2. Formation of OmAnFp@QDs assemblies in water

3. Results and discussion

When oleylamine-capped CdSe are dispersed in the selective nonsolvent (water), the van der Waals force directs them to the hydrophobic micelle core, inducing the formation of OmAnFp @ QDs. Fig. 2a, 2b and 2c are the TEM images, STEM images and their corresponding EDS of O113A10F5@QDs, O113A8F13@QDs and O113A11F19@QDs, respectively. The TEM and STEM images showed that QDs are

3.1. Characterization of amphiphilic fluorine copolymer O113A10F5 Fig. 1a shows the FT-IR spectra of F5-alkyne, O113A10-N3, and O113A10F5. Fig. 1b is the 1H NMR spectrum of O113A10F5 and Fig. 1c is

Fig. 1. (a) FT-IR (KBr) spectra of F5-alkyne, O113A10-N3 and O113A10F5. (b) 1H NMR spectrum of O113A10F5 in chloroform-d; (c) GPC curves of F5-alkyne, O113A10-N3 and O113A10F5. (d) Titration curves of OmAnFp copolymers. 4

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Fig. 2g shows the maximum absorption peak of oleylamine-capped CdSe at 619 nm and no difference is found at the maximum absorption peak locations between OmAnFp@QDs and oleylamine-capped CdSe in CHCl3. It could be seen from Table 2 that the absolute photoluminescence quantum yields (QY) were 14.9% for O113A10F5@QDs, 15.3% for O113A8F13@QDs and 16.8% for O113A11F19@QDs, respectively. In contrast, the QY of the original QDs in chloroform is 21.2%. The interdigitation between the native ligand of QDs and the hydrophobic block of O113A10F5 is stable enough to preserve most of the original QY in water (Fig. S6). By fitting decay profiles to bi-exponential function, we calculated the mean lifetime of these QDs as 11.7 ns, 11.9 ns and 12.5 ns for O113A10F5@QDs, O113A8F13@QDs and O113A11F19@QDs, respectively. The lifetime of OmAnFp@QDs is extended when having higher content of block F. With its amphiphobic nature, block F can easily get aggregate to form compact micellar core to be better protection QDs from their surrounding media. Therefore, O113A11F19@QDs shows the maximum QY and the longest lifetime in all three coated samples.

Table 1 Componential parameters of used OmAnFp copolymers. Sample

Mn,GPC (g/mol)

PDI

fphobic a

O113A10F5 O113A8F13 O113A11F19

8609 10800 12100

1.16 1.20 1.32

38% 45% 53%

a

fphobic represented the volume fraction of the hydrophobic portion in the ∑

fluorine copolymer, which can be calculated as fphobic =

Mphobic ρphobic ∑

Mi ρi

, where the Mi

is molar mass of each block in the copolymer and ρi is the corresponding density of each block.

encapsulated by a layer of OmAnFp to form a core-shell structure. We assume that those micelles consisted of the following three layers: a hydrophobic F core, an A interlayer and an O hydrophilic shell. Energydispersive X-ray spectroscopy line-scan profiles across nanoparticles shows bimodal distribution of F, Cd and Se localized toward the core, which also indicates the successful encapsulation. The exact number of QDs encapsulated in the core cannot be identified because they distributed in three-dimension. The strength of the van der Waals force directly affects the encapsulation of QDs. The volume fraction of hydrophobic blocks ( fphobic ) is a measure of the strength of the van der Waals force. For O113A10F5 with weak van der Waals force, it is easy to cause some QDs dispersed in an aqueous solution without encapsulation (Fig. 2a), while all QDs are successfully encapsulated in the micelles of O113A11F19 (high fphobic value) (Fig. 2c). The insets in Fig. 2a, 2b and 2c are the corresponding size histograms of QDs with three different O/A/F ratios determined by TEM. O113A11F19@QDs has the largest average diameter of 54 nm while O113A10F5@QDs has the smallest of 35 nm. Fig. 2d shows the hydrodynamic diameters (Dh,DLS) of these three kinds of OmAnFp@QDs. The average hydrodynamic diameters of O113A10F5@QDs, O113A8F13@QDs and O113A11F19@QDs are 40 nm, 48 nm, and 61 nm, respectively. The PDI of all three samples were lower than 0.13 as shown in Table 2, indicating particle aggregation is negligible. Note that the larger diameter found in DLS than what in TEM is caused by the hydration effects of the loose O shell. The sizes of OmAnFp@QDs are related to the volume fraction of hydrophobic blocks in OmAnFp, which increases with the value of fphobic going up. Theoretically, both A block and F block are hydrophobic in water (pH = 7.0) and their different block length are able to make the particle size change. Compared with the difference in polymerization degree of F block, the difference in polymerization degree of block A in O113A10F5, O113A8F13 and O113A11F19 is negligible. Thus, the different length of block F in our research plays a leading role in changing the particle size. Fig. 2e is the transmittance profile of OmAnFp@QDs solution. The decline trend of transmittance from O113A10F5@QDs to O113A11F19@QDs is attributed to the increase of the corresponding colloid diameters due to the surface coating, no aggregation in aqueous solution. The result form the benchmark of oleylamine-capped QDs suspended in CHCl3 is also presented as comparison purpose (Fig. 2d–g). As shown in Fig. 2f, the maximum emission peak of OmAnFp@QDs is at 631 nm. The maximum emission peak of oleylamine-capped CdSe is at 629 nm. A slight red shift (ca. 2 nm) for the maximum emission peak of the OmAnFp@QDs is observed, accompanied by a decreased PL intensity from 375 to 340 a.u.. We suspect this shift is attributed to the energy transfer among quantum dots due to the presence of multiple QDs in some individual polymeric micelles. When QDs are close enough in the same micelle, the excitation energy is preferentially transferred to the larger particle with a slightly small band gap, which gives rise to the red shift in the emission spectra [27–29]. Also, there is a broad feature in the emission spectra of OmAnFp@QDs, which is attributed to the different numbers of QDs packaged in polymeric micelles [30].

3.3. Colloidal stability of OmAnFp@QDs under different aqueous conditions The phase transfer of oleylamine-capped CdSe without the protection of OmAnFp is failed, because there is no absorption peak or emission peak in the UV–vis/PL spectrum and the PL is quenched (Fig. S5a). The average hydrodynamic diameter of oleylamine-capped CdSe in water is 2813 nm, and the PDI is 0.98, indicating CdSe particles are aggregated (Fig. S5b). Fig. 3a shows that O113A11F19@QDs has homogeneous fluorescence after 4 h of incubation in deionized water with pH value varying from 4.0 to 12.0 and no aggregates are formed. Fig. 3b shows that the PL intensity of O113A11F19@QDs decreases from 340 to 220 a.u. and the PL QY decreases from 15.3% to 8.3% when the pH value of the solution changes from 7.0 to 4.0. When adjusting the pH values from 7.0 to 4.0, zeta potential of the O113A11F19@QDs increased from +32.6 to +42.9 mV as shown in Fig. 3c, indicating the protonated block A endows the colloidal stability of O113A11F19@QDs. However, the shell of protonated block A is too loose to protect PL properties of QDs from the solvent effect, leading to the decrease of PL intensity and PL QY. The protonation of block A also leads to the decrease of Dh,DLS from 61 nm to 51 nm and the increase of the solution transmittance of O113A11F19@QDs solution from 78% to 92%. In the pH range from 8.0 to 12.0, the block A becomes hydrophobic and the dispersion of O113A11F19@QDs in deionized water no longer relies on the electrostatic stabilization mechanism from block A. QDs are not only covered by the block F, but also further protected by the hydrophobic block A, which contributes to the fairly consistent PL intensity (at 333 a.u.) and PL QY (15.3%) of O113A11F19@QDs. The Dh,DLS of the sample increases from 61 nm to 76 nm and the solution transmittance decreases from 75% to 68% when the solution pH changes from 8.0 to 12.0 (Fig. 3c). The influence of the NaCl concentration on the PL QY and PL intensity was also examined. As shown in Fig. 3d, the PL QY is almost unchanged and the PL intensity slight drops when the concentration of NaCl is below 400 mM. Further increasing the concentration, the PL QY and PL intensity decrease quickly and the fluorescence signal is almost quenched completely at the concentration of 1000 mM. The high ionic strength seems destroying the surface traps of QDs resulting in the loss of PL [23]. There is minor or no effect on the PL of the nanocomposite when they are dispersed in different common widely used buffers such as phosphate buffered saline (pH = 7.4), citrate buffer (pH = 5.0) and carbonate-bicarbonate buffer (pH = 9.5) (Fig. S7). It suggests that the encapsulation of OmAnFp is beneficial to maintain the fluorescence of QDs and endows superior colloidal stability of QDs in different aqueous solution.

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(caption on next page)

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Fig. 2. TEM images (top row, inset: size histograms of OmAnFp@QDs determined by TEM), STEM images (middle row, green arrows represents the trials that the linescan EDS performed) and corresponding EDS (bottom row, inset: line-scan EDS profile obtained along the green arrow) of (a) O113A10F5@QDs, (b) O113A8F13@QDs and (c) O113A11F19@QDs. (d) Hydrodynamic diameters of oleylamine-capped CdSe samples in CHCl3 and in deionized water wrapped by OmAnFp as determined by DLS. (e) Transmittance of different QD samples in CHCl3 and in deionized water. (f) Photoluminescence and (g) absorbance spectra of oleylamine-capped CdSe samples in CHCl3 and in deionized water wrapped by OmAnFp.

Table 2 Characters of oleylamine-capped CdSe samples in CHCl3 and OmAnFp@QDs in deionized water. Entry

Polydispersitya

λem [nm]b

QY [%]c

τ1 [ns]d

τ2 [ns]d

τ [ns]d

ζ [mV]e

QDs O113A10F5@QDs O113A8F13@QDs O113A11F19@QDs

0.08 0.13 0.12

629 631 631 631

21.2 14.9 15.3 16.8

20.6 7.8 8.4 7.4

57.7 21.7 23.2 18.4

27.3 11.7 11.9 12.5

+2.3± 0.8 +29.7± 1.2 +31.9± 0.7 +32.6± 1.0

a b c d e

Polydispersity determined by DLS. Maximum photoluminescence emission wavelength. Absolute photoluminescence quantum yields. Short (τ1), long (τ2) and average(τ) lifetime components. Zeta-potential.

Fig. 3. (a) Fluorescence images of OmAnFp@QDs in deionized water with pH values varying from 4.0 to 12.0 (Samples were excited using a hand-held UV lamp at 365 nm); (b) PL intensity and PL QY of OmAnFp@QDs in deionized water versus pH value; (c) Zeta-potential, Dh,DLS (inset) and transmittance(inset) evolution of the OmAnFp@QDs as a function of pH value from 4.0 to 12.0; (d) PL intensity, PL QY and the corresponding zeta-potential (inset) of OmAnFp@QDs as a function of NaCl concentration. All experiments were carried out in triplicate and error bars display the standard deviation ( ± SD) of the mean.

the initial mixture of O113A11F19@QDs and protein and the solution after re-dispersion were examined by using Pierce BCA Protein Assay Kit and laser scanning confocal microscopy, respectively. As shown in Fig. 4, there is minimal fluorescence associated with FITC-BSA and only 5 μg/ml is detected in the re-dispersion solution, indicating the negligible nonspecific protein bindings. This result is in good agreement with some previous reports that the presence of PEG and block F could minimize or eliminate the nonspecific binding [18,31–35].

3.4. Nonspecific Binding An important requirement for bio-imaging probes is that they should display minimal nonspecific binding to biological components such as protein, antibody, or DNA oligomers. To validate the biocompatibility of the OmAnFp@QDs, we mixed aliquots of FITC-BSA bovine serum albumin (60 μg/ml, 2 ml) with O113A11F19@QDs solutions (2 ml). After incubating for 1 h, O113A11F19@QDs was separated from the mixed solution by centrifuging at 15000 rpm for 5 min. The supernatant was discarded and the precipitate was re-dispersed in 4 ml deionized water. The protein concentration and fluorescent signal in 7

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Fig. 4. Nonspecific binding of O113A11F19@QDs to FITC-BSA. Mixing FITC-BSA with O113A11F19@QDs for 1h (top row), followed by centrifugation at 15000 rpm for 5 min and the precipitate re-dispersed into deionized water (bottom row). O113A11F19@ QDs channel (left column): QDs fluorescence at 613 nm with excitation at 405 nm; FITC-BSA channel (middle column): FITC-BSA fluorescence at 525 nm with excitation at 488 nm; O113A11F19@QDs and FITC-BSA merged channel (right column). Scale bars 0.5 μm.

4. Conclusions

References

A novel amphiphilic fluorine copolymer was successfully synthesized to transfer QDs from organic solvent to aqueous solution. The average hydrodynamic diameter of OmAnFp@QDs can be tuned between 40 nm and 61 nm when QDs is encapsulated by amphiphilic fluorine copolymers with different hydrophobic portions. The solution of obtained OmAnFp@QDs exhibits excellent colloidal stability with no sign of aggregation after stored for up to 4 h across a pH range of 4.0–12.0. In the pH range from 8.0 to 12.0, the hydrophobic block A has its amine groups deprotonated. The repulsion forces between the strong hydrated block O make OmAnFp@QDs nanoparticles stable in the aqueous solution. In the pH range from 7.0 to 4.0, the repulsion of block O and the cationic block A contributes to the colloidal stability of OmAnFp@QDs. The PL QY of obtained OmAnFp@QDs could reach to 16.8% when the pH value is 7.0 and its PL intensity changes only slightly in a NaCl solution of 400 mM. The compact shell from block F and block A protects the QDs from oxidation by molecules in the aqueous solutions. The compromise of PL properties of OmAnFp@QDs in the pH range from 7.0 to 4.0 is attributed to the loose protonated block A that fails to prevent the diffusing of oxide to the inner of OmAnFp@QDs. In the pH range from 12.0 to 8.0, the compact block F and block A help inhibit the diffusion of oxiders and maintain the PL QY of OmAnFp@QDs at 15.3%. The block O and block F are able to repel nonspecific binding proteins, such as BSA. These combined properties make OmAnFp@QDs an attractive candidate as ideal fluorescent probes in advanced biomedical research under in vitro and in vivo conditions.

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