Letter Cite This: ACS Appl. Mater. Interfaces XXXX, XXX, XXX-XXX
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Preparation of Monodisperse Hydrophilic Quantum Dots with Amphiphilic Polymers Zhi-Liang Chen, Yi Lin, Xiao-Juan Yu, Dong-Liang Zhu, San-Wei Guo, Jing-Jing Zhang, Jia-Jia Wang, Bao-Shan Wang, Zhi-Ling Zhang, and Dai-Wen Pang* Key Laboratory of Analytical Chemistry for Biology and Medicine (Ministry of Education), College of Chemistry and Molecular Sciences, State Key Laboratory of Virology, The Institute for Advanced Studies, and Wuhan Institute of Biotechnology, Wuhan University, Wuhan 430072, P. R. China S Supporting Information *
ABSTRACT: Monodisperse hydrophilic quantum dots (QDs) are promising labeling materials for biomedical applications. However, the controllable preparation of monodisperse hydrophilic QDs with amphiphilic polymers remains a challenge. Herein, the molecular structures of amphiphilic polymers assembled on different-sized QDs are investigated. Both the experimental results and the molecular dynamics (MD) calculation suggest that the grafting ratio of amphiphilic polymers assembled on QDs increases as the size of QDs increases. Thus, the controllable preparation of different-sized monodisperse hydrophilic QDs can be achieved by simply varying the grafting ratio of amphiphilic molecules and applied in the simultaneous labeling of three tumor biomarkers. KEYWORDS: amphiphilic polymers, hydrophobic encapsulation, selective assembly, monodisperse hydrophilic quantum dots, multicolor labeling, immunohistochemistry
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transfer of hydrophobic QDs into water (Figure 1c). After being purified with size exclusion chromatography (SEC) (Figure S3) and stained with 2% phosphotungstic acid,17,18 both individual QDs and OPA coating layers can be clearly visualized in TEM images (Figure 1a, lower, and Figure S4), indicating the assembly of OPA on QDs. Moreover, both the absorption and the photoluminescence (PL) spectra remain unchanged before and after OPA encapsulation (Figure 1d, e), indicating that the integrity of QDs is maintained during the OPA encapsulation and the purification. Molecular structures of OPA assembled on different-sized QDs were investigated with Fourier transform infrared (FTIR) spectra and X-ray photoelectron spectroscopy (XPS) spectra. In FTIR spectra, the peaks at 1650 and 1713 cm−1 can be attributed to the stretching vibrations of C = O (νC=O) in amide and carboxyl, respectively. The results (Figure 2a, Figure S5, and Table S1) show that on OPA encapsulated QDs (OPAQDs), the ratio of absorbance at 1650 cm−1 (νC=O in amide) to that at 1713 cm−1 (νC=O in carboxyl) increases significantly compared with those of the initially added OPA (Figure 2a, blue line), indicating that there are more carboxyls converted to amides in the OPA assembled on QDs than the initially added OPA. Moreover, the ratio increases gradually as the size of QDs increases (Figure 2a and Table S2), implying that the ratio of
onodisperse hydrophilic quantum dots (QDs) with unique properties, such as high fluorescence intensity, narrow size distribution, and outstanding colloidal stability, are ideal fluorescent labeling materials in biomedical fields including bioimaging and biosensing.1−4 Currently, the preparation of monodisperse hydrophilic QDs is generally achieved based on the ligand exchange strategy.5−7 Unfortunately, the exchange process influences inevitably the surface structures and the optical properties of QDs. Encapsulating QDs with amphiphilic molecules based on hydrophobic interactions, namely hydrophobic encapsulation strategy,8−10 is an alternative strategy to prepare monodisperse hydrophilic QDs while maintaining their optical properties. However, controllable preparation of monodisperse hydrophilic QDs with this strategy remains a challenge. Herein, different-sized hydrophobic CdSe/CdS QDs were synthesized by epitaxial-growth method.11−13 The uniformity is illustrated with transmission electron microscopy (TEM) images (Figure 1a, upper). The statistic diameters are 5.0 ± 1.1 nm (QD5), 8.0 ± 1.3 nm (QD8), and 11.0 ± 2.1 nm (QD11), respectively (Figure S1). Octylamine-grafted poly(acrylic acid) (OPA, Figure S2) was synthesized by grafting octylamine with the carboxyl in polyacrylic acids. Grafting ratio of OPA is defined as the ratio of carboxyl converted to amide. Based on hydrophobic interactions, the octyl chains of the resulting amphiphilic polymer OPA intercalate spontaneously to the surface ligands of hydrophobic QDs (Figure 1b).14−16 Thus, a coating layer of OPA forms on QDs and helps the © XXXX American Chemical Society
Received: July 3, 2017 Accepted: November 6, 2017
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DOI: 10.1021/acsami.7b09557 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX
Letter
ACS Applied Materials & Interfaces
Figure 1. (a) TEM images of different-sized CdSe/CdS QDs before (upper) and after (lower) encapsulation with OPA. Scale bar: 50 nm. (b) Scheme of OPA encapsulating QDs. (c) Photograph, (d) absorption spectra, and (e) corresponding photoluminescence (PL) spectra of differentsized QDs before (dashed curves) and after (solid curves) OPA encapsulation.
Figure 2. (a) FTIR spectra of QD5, OPA, and OPA-QDs of different sizes. (b) Grafting ratios of OPA assembled on different-sized QDs. (c) Model for selective assembly of OPA on different-sized QDs.
carboxyl converted to amide in the OPA assembled on QDs increases gradually as the size of QDs increases. Thus, the molecular structures of OPA assembled on QDs are different
compared with those of the initially added OPA in watersolubilization processes. Because no oxygen or nitrogen signal was detected on hydrophobic QDs (Figure S6a), the grafting B
DOI: 10.1021/acsami.7b09557 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX
Letter
ACS Applied Materials & Interfaces
Figure 3. (a−c) Hydrodynamic diameter distributions and (d−f) the corresponding zeta potential distributions of different-sized QDs coated with OPA of different grafting ratios (red: OPA41-QD5, OPA50-QD8, OPA56-QD11; green: OPA50-QD5, OPA56-QD8, OPA41-QD11). Agarose gel electrophoresis images of OPA41-QD5 (green), OPA50-QD8 (yellow), OPA56-QD11 (red); (g) and OPA50-QD5 (green), OPA56-QD8 (yellow), OPA41-QD11 (red) (h), respectively. The dashed lines indicate the location of the loading wells.
Briefly, OPA molecules with a higher grafting ratio assemble spontaneously on larger-sized QDs, whereas those of lower grafting ratio prefer to assemble on smaller-sized QDs. This model indicates that the surface nature of OPA-QDs, such as surface charge and functional groups, are likely to be dominated by the grafting ratio of OPA and the size of QDs. Similarly, FTIR spectra show a similar trend for OPA interacting with hydrophobic gold nanoparticles (Figures S9 and S10 and Table S5). Subsequently, OPA molecules with different grafting ratios were synthesized and utilized to encapsulate QDs with different sizes (Figure S11). Interestingly, when encapsulating QD5 with OPA of a grafting ratio of 41% (OPA41-QD5), QD8 with OPA of a grafting ratio of 50% (OPA50-QD8), and QD11 with OPA of a grafting ratio of 56% (OPA56-QD11), narrow size distributions of OPA-QDs are observed by dynamic light scattering (DLS) (Figure 3a-c, red line). In contrast, when encapsulating QD5 with OPA of a grafting ratio of 50% (OPA50-QD5), QD8 with OPA of a grafting ratio of 56% (OPA56-QD8), and QD11 with OPA of a grafting ratio of 41% (OPA41-QD11), broad size distributions are observed (Figure 3a-c, green line). Furthermore, the size of OPA itself dispersed in water was measured for comparison. As shown in Figures S12−S14 and Table S6, the size distributions of OPA-QDs are dominated by both the grafting ratio of OPA and the diameter of QDs. Thus, OPA-QDs with different sizes can be prepared by varying the diameter of QDs or the grafting ratio of OPA. Meanwhile, the corresponding zeta potential distributions show the same trend (Figure 3d−f), indicating that the size distributions and the zeta potential distributions of OPA-QDs are dominated by both the grafting ratio of OPA and the size of QDs. Gel electrophoresis results show that the fluorescent bands of the OPA41-QD5, OPA50-QD8 and OPA56-QD11 are narrow, and no obvious smearing observed (Figure 3g). However, the fluorescent bands of OPA50-QD5, OPA56-QD8 and OPA41-QD11 are rather broad (Figure 3h). This is in agreement with Smith’s results that the distributions of monodisperse QDs are narrow in DLS and gel electrophoresis
ratio of OPA assembled on different-sized QDs can be calculated according to the relative contents of nitrogen and oxygen numbers from the XPS spectra (Figure S6b) and eq S1.19 As shown in Figure 2b, the grafting ratios of OPA assembled on different-sized QDs are 42 ± 3% (OPA-QD5), 51 ± 4% (OPA-QD8), and 58 ± 4% (OPA-QD11), respectively. Since the same OPA was used in all QDs water-solubilization processes, it can be inferred that OPA molecules with a certain grafting ratio assemble preferentially on QDs with certain diameter. This is in agreement with Fabienne’s results that transferring ZnO nanocrystals into water solution can only be achieved with amphiphilic molecules of the certain molecular structures.20,21 Generally, the grafting ratio of OPA assembled on QDs increases as the diameter of QDs increases. Molecular dynamics (MD) calculation was used to investigate the assembly of OPA on different-sized QDs.22,23 Results show that as the grafting ratio of OPA increases, the gyration radius of OPA dispersed in water gradually increases (Figure S7 and Table S3). This is in accordance with previous reports that the size of micelle increases as the grafting ratio of amphiphilic molecules increases,24,25 indicating that it may be more suitable to accommodate larger QDs in the aggregates of OPA with higher grafting ratio. The interaction energy (ΔE) of OPA assembled on different-sized QDs was obtained in water.26 Results show that lower ΔE can be achieved when OPA with a higher grafting ratio assemble on the larger-sized QDs (Figure S8 and Table S4). Because much excess OPA was used in the water-solubilization processes, OPA with the “suitable” grafting ratio assembled spontaneously on QDs with a certain diameter, which minimized energy of the system. Therefore, the energy minimization may be the cause of the selective assembly of OPA molecules on the larger-sized QDs, which may be related to the density of ligand and the size of QDs. On the basis of the above results, a model of amphiphilic polymers selectively assembled on different-sized QDs is proposed. As shown in Figure 2c, the assembly of OPA on QDs depends on the grafting ratio of OPA and the size of QDs. C
DOI: 10.1021/acsami.7b09557 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX
Letter
ACS Applied Materials & Interfaces
Figure 4. (a) Simultaneous imaging of CR2, Ki-67 and PCK antigens coexpressed in tonsil tissues multilabeled with monodisperse OPA-QDs. (b) OPA-QDs emission spectra and tissue autofluorescence obtained from a. (c) Fluorescence images of CR2 positive, (d) Ki-67 positive, and (e) PCK positive tonsil tissues extracted from (a). (f−h) Corresponding DAB-based IHC of CR2, Ki-67, and PCK in tonsil tissues, respectively. Scale bar: 25 μm.
analysis.7 Thus, based on the selective assembly model of amphiphilic polymers on different-sized QDs, controllable preparation of monodisperse hydrophilic QDs is achieved by simply varying the grafting ratio of amphiphilic polymers. Furthermore, the fluorescence of OPA-QDs dispersed in water was compared with that of hydrophobic QDs dispersed in hexane (Figure S15). Retention rates of PL intensity from monodisperse OPA-QDs (OPA41-QD5, 85%; OPA50-QD8, 91%; OPA56-QD11, 92%) are much higher than those of the controls (OPA50-QD5, 71%; OPA56-QD8, 62%; OPA41QD11, 73%), indicating that fluorescence of monodisperse QDs is maintained to the largest extent. Compared with the ligand exchange strategy for the preparation of monodisperse hydrophilic QDs,5−7 three main advantages of our encapsulation strategy are to be highlighted. First, the surface structure change of QDs is not required during the water-solubilization processes, thus the optical properties are maintained. Second, the compact coating of amphiphilic molecules on QDs prevents the oxidation of surface structures and the leakage of heavy metal ions, which is beneficial to the improvement of biocompatibility.25 In addition, our method can be achieved at room temperature, making the entire process secure and energy saving. One unique property of QDs is their multicolor emission, offering the potential for the simultaneous labeling of multiple targets. Here, multiple monodisperse QDs were conjugated with antibodies and utilized to recognize three kinds of tumor markers in tonsil tissue. Simultaneous imaging and extraction of
spectra were achieved with multispectral microscopy (Figure 4a, b). As illustrated in Figure 4c−e, green fluorescence from human complement receptor type2 (CR2), yellow fluorescence from nuclear protein Ki-67 (Ki-67), and red fluorescence from pancytokeratin (PCK) can be clearly observed. The corresponding immunohistochemistry (IHC) images (Figure 4f−h) are consistent with the QDs-based IHC, indicating that simultaneous labeling of three tumor markers with triplecolored monodisperse QDs is reliable. Because CR2, Ki-67, and PCK are tumor markers of lymphoma, based on the simultaneous labeling with monodisperse QDs, multicolor imaging results can be associated with pathological diagnosis and the monitoring of the treatment process in lymphoma.27−29 Importantly, compared with conventional IHC in which only one kind of tumor biomarker can be stained at one time, the QD-based IHC can be used to label simultaneously multiple tumor biomarkers, facilitating the rapid clinical diagnosis.30 Our results here suggest that the asprepared monodisperse QDs are promising labeling materials in biomedical applications such as QDs-based IHC. In summary, based on a model of amphiphilic polymers selectively assembled on different-sized QDs, a method for the preparation of monodisperse hydrophilic QDs by matching the grafting ratio of OPA and the size of QDs is proposed. The asprepared monodisperse QDs with bright fluorescence are successfully applied in simultaneous labeling of three kinds of tumor biomarkers in tonsil tissues. This study provides a facile method to fabricate high-quality hydrophilic QDs for D
DOI: 10.1021/acsami.7b09557 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX
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ACS Applied Materials & Interfaces
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biomolecular detection and imaging, as well as offering new insights to understand the cooperative assembly of amphiphilic molecules on hydrophobic nanoparticles.
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.7b09557. Preparation, supplementary characterization, IHC procedures, and molecular dynamics calculations (PDF)
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. Tel: +86-27-68756759. Fax: +86-27-68754067. ORCID
Xiao-Juan Yu: 0000-0001-9412-6170 Bao-Shan Wang: 0000-0003-3417-9283 Zhi-Ling Zhang: 0000-0001-7807-2264 Dai-Wen Pang: 0000-0002-7017-5725 Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS Z.-L.C. is grateful to Yan-Hua Huang, Hui Yu, and Min Li (Wuhan Jiayuan Quantum Dots Co., Ltd., China) for their help in QD-based immunofluorescence imaging and IHC. This work was supported by the National Natural Science Foundation of China (21535005, 21275111), the 111 Project (111-2-10), the China Scholarship Council, and Collaborative Innovation Centre for Chemistry and Molecular Medicine.
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DOI: 10.1021/acsami.7b09557 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX
