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Green synthesis of biocompatible carboxylic curdlan-capped gold nanoparticles and its interaction with protein
Accepted Manuscript Title: Green synthesis of biocompatible carboxylic curdlan-capped gold nanoparticles and its interaction with protein Author: Jing-Kun Yan Jin-Lin Liu Yu-Jia Sun Shuang Tang Zheng-Ying Mo Yuan-Shuai Liu PII: DOI: Reference:
S0144-8617(14)01060-1 http://dx.doi.org/doi:10.1016/j.carbpol.2014.10.048 CARP 9401
To appear in: Received date: Revised date: Accepted date:
13-8-2014 27-9-2014 16-10-2014
Please cite this article as: Yan, J.-K., Liu, J.-L., Sun, Y.-J., Tang, S., Mo, Z.Y., and Liu, Y.-S.,Green synthesis of biocompatible carboxylic curdlan-capped gold nanoparticles and its interaction with protein, Carbohydrate Polymers (2014), http://dx.doi.org/10.1016/j.carbpol.2014.10.048 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Submitted to: Carbohydrate Polymers
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(Original Research MS)
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Green synthesis of biocompatible carboxylic curdlan-capped gold nanoparticles and its
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interaction with protein
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Jing-Kun Yan
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Liub,*
a,
, Jin-Lin Liu a, Yu-Jia Sun a, Shuang Tang a, Zheng-Ying Mo a, Yuan-Shuai
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an
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a
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b
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Science and Technology, Clear Water Bay, Kowloon, Hong Kong
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School of Food & Biological Engineering, Jiangsu University, Zhenjiang, 212013, China
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Department of Chemical and Biomolecular Engineering, The Hong Kong University of
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Corresponded authors: JK Yan, tel: +08615952819661, e-mail: [email protected]; YS Liu, tel: 852-23588409, e-mail: [email protected] 1 Page 1 of 29
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Abstract This study demonstrates a facile, green strategy for the preparation of gold nanoparticles
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(AuNPs) from chloroauric acid (HAuCl4) using carboxylic curdlan (Cc) as both reducing and
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stabilizing agent. The as-prepared AuNPs are characterized by UV-Vis spectroscopy, high
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resolution transmission electron microscopy, X-ray diffraction, energy dispersive X-ray
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spectrometry and Fourier transform infrared spectroscopy. The results indicated that the
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particle size of the AuNPs changes with variations in the reaction time and concentrations of
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Cc and HAuCl4. The spherical AuNPs are well dispersed, exhibiting high stability even after
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six months storage. The carboxylic groups (COO-) in the Cc molecules tend to adsorb and
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stabilize the surface of the AuNPs. The interaction between BSA and the Cc-capped AuNPs
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was investigated using fluorescence and circular dichroism spectroscopies. The results
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indicated that the BSA molecules adsorb on the surface of the AuNPs, without significant
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change in its helical structure even after conjugation with the AuNPs.
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Carboxylic
curdlan;
Keywords:
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Protein-nanoparticle interaction
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Gold
nanoparticles;
Green
synthesis;
Structure;
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1. Introduction Metal nanoparticles, especially gold nanoparticles (AuNPs), have recently gained
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significant attentions because of their unique properties and potential applications in catalysis,
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optoelectronics, sensors, biotechnology, and biomedicine (Daniel & Astruc, 2004; Dykman &
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Khlebtsov, 2012; Li, Zhao & Astruc, 2014; Sau, Rogach, Jackel, Klar, & Feldmann, 2010;
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Yeh, Creran & Rotello, 2012). In order to explore the biological processes that are critical for
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diagnostics and modulation of cell functions, a lot of work has been done on unraveling the
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surface interaction of biomolecules with metal nanoparticles (NPs) (Lacerda et al., 2010).
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Obviously, AuNPs are the natural starting points for understanding the protein-NP
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interactions due to their promise for diverse biomedical applications, such as molecular
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diagnostics, bioengineering, biomolecular imaging, and drug delivery for cancer therapeutics
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(Andres et al., 1996; Galletto, Brevet, Girault, Antoine, & Broyer, 1999; Ghosh, Han, De,
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Kim, & Rotello, 2008; Mirkin, Letsinger, Mucic, & Storhoff, 1999; Burt, Gutierrez-Wing,
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Miki-Yoshida, & Jose-Yacaman, 2004).
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To date, AuNPs are being synthesized with a variety of bottom-up methods and
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technologies, including chemical reduction (Chen, Wang, Ballato, Foulger, & Carroll, 2003),
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seed-growth (Busbee, Obare, & Murphy, 2003), photochemistry (Kim, Song, & Yang, 2002),
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electrochemistry (Ye, Yan, Ye, & Zhou, 2010), sonochemistry (Qiu, Zhu, & Chen, 2003), and
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template-assisted supramolecular chemistry (Lehn, 1995). Among these techniques, the
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chemical reduction method, which involves the use of various reducing agents, such as
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hydrazine, sodium borohydride (NaBH4), dimethyl formamide (DMF), and other organic
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compounds, is considered as the most simple and conventional method for the preparation of
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AuNPs. However, these reducing agents are often associated with potential environmental
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toxicity or biological hazards. Therefore, more and more interests are being put on the
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development of “green” synthesis strategies for the preparation of AuNPs using non-toxic,
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environmentally friendly and renewable materials.
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Polysaccharides derived from renewable sources, including plants, animals, and
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microorganisms, are being extensively studied in a wide variety of biomedical applications
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and functional foods due to their notable and excellent bioactivities, such as
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immunostimulatory, antitumor, and antioxidant activities (Giavasis, 2014; Stachowiak &
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Reguła, 2012). In particular, polysaccharides possessing good biocompatibility and
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biodegradability play an important role in nanoscience and drug delivery owing to the
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presence of a large number of functional groups, including hydroxyl, carboxyl, and amino
63
groups, in their structures. Raveendran et al. (2003) firstly reported a facile “green” method
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for the synthesis of metal nanoparticles using glucose as the reducing agent and starch as the
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protecting agent. Then many other polysaccharides and their derivatives have been employed
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for the “green” synthesis of AuNPs, such as chitosan (Huang et al., 2007), dextran (Wang,
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Zhan, & Huang, 2010), pullulan (Choudhury, Malhotra, Bhattacharjee, & Prasad, 2014),
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glucan (Jia, Xu & Zhang, 2013; Sen, Maity, & Islam, 2013), cellulose (Tan et al., 2010), and
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guar gum (Pandey, Goswami, & Nanda, 2013). Our group has also successfully developed a
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facile, simple, and eco-friendly method to synthesize silver nanoparticles (AgNPs) using
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aqueous
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4-acetamido-2,2,6,6-tetramethypiperidine-1-oxyl radical-mediated oxidation curdlan, as both
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reducing and stabilizing agents (Yan et al., 2013). However, no studies have been reported on
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solution
of
carboxylic
curdlan
(Cc),
which
was
prepared
by
the
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the green synthesis of AuNPs using Cc solution. Herein, for the first time, we developed a
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green strategy for the synthesis of AuNPs from chloroauric acid (HAuCl4) using Cc as the
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reducing as well as stabilizing agent. The characteristics and mechanism of the as-prepared
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Cc-AuNPs have been systematically elucidated using UV-Vis spectroscopy, high-resolution
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transmission electron microscopy (HR-TEM), X-ray diffraction (XRD), energy-dispersive
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X-ray (EDX) analysis, and Fourier-transform infrared (FT-IR) spectroscopy. In addition, the
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interaction between the prepared Cc-AuNPs and bovine serum albumin (BSA) has been
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investigated using fluorescence spectroscopy and circular dichroism (CD) spectroscopy.
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2. Materials and methods
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2.1. Materials and chemicals
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Carboxylic curdlan (Cc) bearing the β-1,3-polyglucuronic acid structure was prepared
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from 4-acetamido-TEMPO-mediated oxidation, details being shown in our previous work
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(Yan et al., 2014). The carboxylate content and molecular weight (MW) of Cc are 4.87
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mmol/g and 1.2×105 Da, respectively. Hydrogen tetrachloroaurate (III) tetrahydrate
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(HAuCl4·3H2O, 99%), and bovine serum albumin (BSA, MW 6.6×104 kDa) were obtained
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from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). All other chemicals and solvents
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were of laboratory grade, without further purification. Ultrapure Milli-Q water (18.2 MΩ cm
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at 25 ºC) was used in all experiments.
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2.2. Green synthesis of Cc-capped gold nanoparticles (Cc-AuNPs) Stock solutions of HAuCl4 with concentrations ranging from 0.5 to 2.0 mM were 5 Page 5 of 29
prepared by adding gold (III) chloride hydrate in deionized water. For the synthesis of AuNPs,
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2 mL of the HAuCl4 stock solution was added to an equal volume of Cc (0.2 %, w/v). The
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resulting mixture was maintained at 100 ºC under continuous stirring in a water bath for
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different reaction time (15–120 min) to obtain AuNPs.
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2.3. Characterization
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The UV-Visible absorption spectra of the as-prepared Cc-AuNPs solutions were
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obtained from a Perkin-Elmer Lambda 35 spectrophotometer in the wavelength range of
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400–700 nm with the interval of 1.0 nm. The change in the color was directly recorded by a
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digital camera. The particle size and morphology of the Cc-AuNPs was observed using High
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Resolution Transmission Electron Microscopy (HRTEM, Tecnai 12, Philips, 120 kV). For
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TEM analysis, the sample was prepared by placing a drop of the Cc-AuNPs solution on a
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carbon coated copper grid (300 meshes), followed by drying at room temperature (20 ºC) for
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30 min. The elemental composition of the nanoparticles was determined by an EDX
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spectroscope (Inca Energy-350, Oxford Co., UK) attached to the SEM (JSM-7001F, JEOL
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Ltd., Japan). The critical structure of the Cc-AuNPs was determined by a wide-angle XRD
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(D8-Advance, Bruker, Co., Germany). The XRD patterns were recorded with Cu Kα
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radiation (λ = 0.1541 nm) at 40 kV and 40 mA in the 2θ ranging from 30° to 80° with a step
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speed of 4°/min. Fourier-transform infrared (FT-IR) spectra of Cc and freeze-dried
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Cc-AuNPs were obtained from a Nexus 670 FT-IR spectrometer (Thermo Nicolet Co., USA)
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in the wavenumber range of 500–4000 cm−1 with KBr pellets and referenced against air.
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2.4. Preparation and characterization of Cc-AuNPs and BSA interactant The interactant was prepared by adding 50 μL of the as-prepared Cc-AuNPs solution
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(with different concentrations ranging from 10-12 to 10-6 M) to 1 mL of BSA solution (0.01
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mg/mL). Then the mixture was incubated at 25 ºC in a water bath for 3 h under continuous
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stirring. After incubation, the mixture was maintained at 4 ºC before analysis.
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The fluorescence spectra of BSA and BSA/Cc-AuNPs interactants were recorded on a
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Perkin Elmer LS35 spectrofluorimeter in the wavelength range of 300–450 nm using the
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excitation wavelength of 290 nm. The width of both the excitation and emission slit was 5.0
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nm. Furthermore, the CD spectra were recorded on a circular dichroism spectrometer
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(JASCO J-815, Japan) at room temperature using a 0.1 cm path length quartz cell in the
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wavelength range of 190–290 nm.
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3. Results and discussion
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3.1. Influence of reaction time on the synthesis of Cc-AuNPs
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Owing to the presence of a large number of hydroxyl and carboxyl (COO-) groups, the
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Cc molecules prepared via the 4-acetamido-TEMPO-mediated oxidation of curdlan exhibited
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a strong ability to adsorb metallic cations. The Cc molecules thus played the role of both
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reducing and stabilizing agents for the one-pot, green synthesis of AuNPs in an aqueous
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medium. Moreover, the functional groups in the Cc molecules cover and passivate the surface
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of the AuNPs, thereby preventing any further aggregation (Liu, Raveendran, Qin & Ikushima,
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2005).
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Fig. 1 shows the UV-Vis absorption spectra of the AuNPs prepared with 0.2% (w/v) Cc 7 Page 7 of 29
and 2.0 mM HAuCl4 solution at 100 ºC for different time intervals. After 15 min of
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incubation, the UV-Vis spectra show a broad absorption band at around 530 nm. When the
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incubation time increases from 15 to 90 min, the bands become stronger. The appearance of
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this typical surface plasmon resonance (SPR) band of AuNPs further indicates the formation
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of Cc-AuNPs (Mulvaney, 1996). The inset image of Fig. 1 shows that the HAuCl4 aqueous
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solution is colorless in the beginning, and with the ongoing of reaction, the color becomes
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dark-blue and then wine red, without the formation of precipitates. This indicates that Au3+ in
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the solution is reduced to Au0 by the Cc molecules (Zhou et al., 2009). In general, metal
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nanoparticles with various shapes and sizes show different colors in solutions, therefore the
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shape of the nanoparticles can be visually evaluated. For instance, metallic gold is yellow,
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spherical AuNPs are wine red or purple, while gold nanorods are blue or black in solution
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(Burda, Chen, Narayanan & El-Sayed, 2005; Thakor, Jokerst, Zavaleta, Massoud & Gambhir,
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2011). These results also reveal that the Cc molecules in the solution could act as both
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reducing and stabilizing agents for the synthesis of well-dispersed spherical AuNPs, without
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the need of any additional reducing agents or stabilizers. In addition, no significant increase
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in absorption intensity could be observed after 90 min of reaction time, suggesting that the
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reaction is completed within 90 min.
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3.2. Influence of Cc concentration on the synthesis of Cc-AuNPs
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According to our previous work (Yan et al., 2013), the concentration of Cc has a
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significant influence on the formation of AgNPs. Therefore, in the present study, we analyzed
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the effects of Cc concentration on the synthesis of the as-prepared AuNPs. Fig. 2 shows the
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UV-Vis absorption spectra of the AuNPs prepared with different concentrations of Cc (0.05% 8 Page 8 of 29
to 0.2%, w/v) and 2.0 mM HAuCl4 at 100 ºC for 90 min. When the Cc concentration is below
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0.1%, Au3+ was not sufficiently reduced and stabilized for the preparation of AuNPs. It was
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found that there is a gradual increase in the absorption intensity, by increasing the Cc
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concentration up to 0.2% (w/v). Moreover, the width of absorption peak gradually becomes
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narrower with the increase in the concentration of Cc from 0.1% to 0.2% (w/v) suggesting
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that the size distribution of the as-prepared AuNPs becomes narrower (Babapour et al., 2006).
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In addition, the SPR band of the as-synthesized AuNPs blue-shifted from 540 nm to 530 nm
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with increasing Cc concentration from 0.1% to 0.2% (w/v) indicating that the particle size
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was decreased with increasing Cc concentration. In addition, the effect of Cc concentration
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on the formation of AuNPs was also evaluated by visually observing the color changes in the
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solution. As can be seen from the inset image shown in Fig. 2, the color of the solution
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changes from colorless to light blue or blue color and subsequently to wine red color with
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increase in the concentration of Cc from 0.05% to 0.2%. This indicates that the shape of the
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as-prepared particles can be tuned by controlling the concentration of Cc. In the present study,
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the concentration of Cc suitable for the preparation of AuNPs was found to be 0.2% (w/v).
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Neither AuNP aggregation nor polymer phase separation was observed under this condition.
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3.3. Influence of HAuCl4 concentration on the synthesis and morphology of the Cc-AuNPs
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Fig. 3A shows the UV-Vis absorption spectra of the AuNPs prepared by reducing
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different concentrations of HAuCl4 solution with 0.2% (w/v) Cc at 100 ºC for 90 min. The
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absorption peaks appear at about 530 nm, which is a characteristic SPR band of AuNPs and
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confirms the formation of AuNPs under different concentrations of HAuCl4 solution. With an
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increase in the concentration of HAuCl4 from 0.5 to 2.0 mM, the intensity of the absorption
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peak increased gradually, suggesting the formation of more nanoparticles at higher 9 Page 9 of 29
concentration of HAuCl4. Meanwhile, the effects of HAuCl4 concentration on the formation
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of AuNPs can also be visually observed through the color change of the solution. As shown in
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the inset images of Fig. 3A, the AuNPs formed under lower concentration of HAuCl4 (0.5
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mM) exhibits a light purple color in solution, and the color becomes deeper to wine red or
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purple at higher concentration (2.0 mM).
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The particle size and morphology of the AuNPs, formed by reducing 0.5, 1.0, and 2.0
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mM HAuCl4 solution with 0.2% (w/v) Cc, respectively, was recorded by HR-TEM (Figs.
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3B-D). Fig. 3B shows the formation of well-dispersed spherical particles with an average size
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of ~17 nm, at the HAuCl4 concentration of 0.5 mM. When HAuCl4 concentration increases to
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2.0 mM, the particle size also increased to 24 nm (Fig. 3D), correspondingly, higher
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concentration of HAuCl4 leading to the formation of larger sized AuNPs. This result agrees
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well with the UV-Vis analysis and visual observation of color formation. It is reasonable
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because higher concentration of Au3+ ions in the HAuCl4 solution provides more nuclei,
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which favors further growth of the nanoparticles (Wu, Zhang & Zhang, 2012). More
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specifically, an increase in the concentration of HAuCl4 may result in the aggregation of
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particles to some extent, perhaps due to the role of polysaccharides as stabilizers or
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nucleating agents (Sen, Maity & Islam, 2013). The amount of 0.2% (w/v) Cc used in this
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work is sufficient enough to reduce the Au3+ ions in the high HAuCl4 concentration of 2.0
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mM, but could not completely prevent the aggregation of the as-formed AuNPs. However, at
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lower concentrations, the same concentration of Cc is completely sufficient to stabilize the
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AuNPs and prevent further aggregation, forming smaller size of AuNPs.
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3.4. Characterization of Cc-AuNPs through XRD and EDX Fig. 4A shows the XRD pattern of the AuNPs prepared with 0.2% (w/v) Cc and 2.0 mM
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HAuCl4 solution at 100 ºC for 90 min. The four diffraction peaks at 2θ corresponding to
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38.2°, 44.4°, 64.6°, and 77.7° can be attributed to the (111), (200), (220), and (311) planes,
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respectively, of the face-centered cubic (fcc) gold. The Bragg reflections (200), (220), and
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(311) are relatively weaker and broader compared to the intense (111) reflection, implying
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that the gold nanocrystals are preferentially oriented along the (111) plane. The EDX
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spectrum of the AuNPs (Fig. 4B) reveals a strong and typical optical absorption peak at
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approximately 200 eV, which could be attributed to the SPR of the metallic Au nanocrystals,
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and confirms the formation of AuNPs in the reaction medium. In addition, the presence of Au,
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C, and O elements in the EDX spectrum indicates that the nanocomposite consists of both Cc
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and AuNPs.
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3.5. FT-IR analysis and mechanism underlying the formation of Cc-AuNPs
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Fig. 5 shows the FT-IR spectra of Cc and Cc-AuNPs prepared from 0.2% (w/v) Cc and
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2.0 mM HAuCl4 at 100 ºC for 90 min, aiming to investigate the possible interaction between
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the Cc molecules and the Cc-capped AuNPs in the solution. The FT-IR spectrum of Cc (Fig.
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5A) shows two characteristic absorptions of the polysaccharides. The strong and wide
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stretching peak at approximately 3470 cm−1 can be ascribed to the O–H stretching vibration,
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while the weak absorption peak at approximately 2935 cm−1 can be associated with the C–H
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stretching vibration. The absorption band at 1636 cm-1 can be attributed to the asymmetrical
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COO- stretching vibration, and that at 1426 cm-1 to the symmetrical COO- stretching
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vibration (Delattre et al., 2009). The absorption peaks at 1087 and 1050 cm-1 are ascribed to
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the stretching vibration of C-OH. Additional peaks at 3450, 2932, 1632, 1426, 1087, and
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1050 cm-1 (Fig. 5B) correspond to O-H, C-H, COO- and C-OH, respectively, coincident with
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the characteristic peaks of the polysaccharides. This suggests that the skeleton structure of Cc
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remains unaltered after the formation of AuNPs. Interestingly, a new peak appeared at around
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1730 cm-1 can be ascribed to the stretching vibration of free -COOH group, indicating the
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reduction of Au3+ to Au0 by the polysaccharide in the solution (Shi & Sun, 1988). Sun et al.,
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(2008) has reported a number of glycosidic linkages in the polysaccharide chain are broken
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into shorter segments in an acidic HAuCl4 solution. Besides, some -CHO groups, present in
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the opened structure, may be oxidized to -COOH groups along with the reduction of Au3+ to
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Au0 in the presence of Au3+. Moreover, the peak at 1636 cm-1 (COO- stretching vibration) is
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slightly shifted towards 1632 cm-1 in the FT-IR spectrum of Cc-AuNPs, meaning that the
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combination between Au nanoparticles and Cc molecules is through affinity or electrostatic
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interaction with carboxylate (COO-) groups, and leads to no traces of blank AuNPs in the
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suspension. Therefore, the functional groups present in the Cc molecules play a dual role of
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both reducing agent and stabilizing agent in synthesizing as well as protecting the as-formed
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AuNPs.
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Our
previous
work
have
reported
that
the
Cc
molecules
bearing
the
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β-1,3-polyglucuronic acid structure existed as a semi-flexible chain in the aqueous solution
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and had the potential for producing gene carriers, bio-nanomaterials, and other chiral
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nanowires, through hydrogen bonding interactions, hydrophobic interactions, and
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electrostatic attraction (Yan et al., 2014; Lien et al., 2011). Based on this study, a possible 12 Page 12 of 29
mechanism underlying the green synthesis of AuNPs is proposed in Fig. 6, Cc serving both as
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a reducing agent as well as a capping agent for the synthesis and stabilization of AuNPs.
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Firstly, the extensive carboxylic groups (COO-) on the C-6 position of the Cc chains play an
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active role in the adsorption and stabilization of the Au3+ ions through electrostatic attraction
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or affinity interaction. Secondly, the hydroxyl groups in the Cc chain reduce the adsorbed
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Au3+ ions to Au0 and metallic Au, which further nucleate and grow to form AuNPs. The
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AuNPs are subsequently capped and stabilized by Cc because of its oxygen-rich structure
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(Huang et al., 2007). Meanwhile, the hydroxyl groups in the polymeric chains and networks
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will also promote the stabilization of the AuNPs. The AuNPs formed by this method are
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highly stable and well dispersed, and there is no any color change or precipitates formation
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after six months of storage at room temperature from direct visual observation.
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3.6. Studies on the interaction of Cc with BSA
Generally, the adsorption of proteins on the surface of the NPs alters the surface
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functionality and thus may strongly influence the translocation behavior in the biological
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systems (Treuel, Malissek, Gebauer, & Zellner, 2010). In order to understand the fate of the
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NPs in the biological systems, we analyzed the interactions between BSA and the AuNPs
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prepared from 0.2% (w/v) Cc and 2mM HAuCl4 at 100 ºC for 90 min using fluorescence
271
spectroscopy and CD measurements. Fig. 7A show that the fluorescence intensity of BSA at
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around 365 nm (the presence of tyrosine, tryptophan, and phenylalanine residues in BSA
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molecule), tends to decrease when the concentration of Cc-AuNPs increases from 10-12 M to
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10-6 M, suggesting that the addition of Cc-AuNPs quenches BSA fluorescence emission,
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without any peak shift or the formation of new peak. The observed quenching of the
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fluorescence intensity of BSA may be attributed to the formation of conjugate between
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Cc-AuNPs and BSA. CD is powerful in studying the interactions of proteins with other molecules and
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investigating the protein conformation in solution or adsorbed onto other molecules. In
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general, the presence of high percentage of α-helical (67%) structure in the BSA molecules
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exhibits a characteristic CD signal in the far UV region. Typically, changes in the ellipticity at
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208 and 222 nm are considered useful probes for visualizing the variations in α-helical
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content. Fig. 7B shows the CD spectra of BSA in the absence and presence of different
284
concentrations of AuNPs. The ellipticity at 208 and 222 nm of BSA gradually decrease with
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an increase in the concentration of Cc-AuNPs from 10-12 M to 10-6 M, implying that the
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AuNPs capped with the carboxylic groups of Cc influence the conformation of BSA to a
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certain extent. Nonetheless, the change in α-helical content of BSA is not significant even
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after conjugation with 10-6 M AuNPs. It is well known that the changes in the original
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structure of the protein result in loss of biological activity or the activation of immune
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response (Brandes, Welzel, Werner, & Kroh, 2006). Remarkably, the BSA molecule could
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maintain most of its α-helical structure after conjugation in this study, which offers great
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potentials as a novel carrier of proteins for biomedical applications.
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4. Conclusions
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In summary, we developed a facile and green method for the preparation of
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well-dispersed spherical AuNPs using Cc as both reducing agent and stabilizing agent in 14 Page 14 of 29
aqueous medium. The particle size, shape, morphology, structure and elemental composition
298
of the as-prepared AuNPs were thoroughly characterized using UV-Vis spectroscopy,
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HRTEM, XRD, and EDX analyses, and the interactions between Cc and AuNPs were
300
examined using FT-IR spectroscopy. Based on the FT-IR analysis, we proposed the possible
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mechanism underlying the synthesis of Au nanoparticles using Cc. In addition, the interaction
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between BSA and the Cc-capped AuNPs was also investigated using fluorescence
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spectroscopy and CD spectroscopy. The results suggest the potential use of non-toxic, benign,
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biodegradable, and biocompatible Cc as a reducing agent as well as stabilizer for the
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synthesis of metal nanoparticles, and a broad spectrum of their potential applications in
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biomedicine and bioanalytical fields.
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Acknowledgements
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This work was supported financially by the Natural Science Foundation of Jiangsu Province
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(2013M531292), the Priority Academic Program Development (PAPD) of Jiangsu Higher
312
Education Institutions, and Jiangsu university students' scientific research project (13A097).
China
Postdoctoral
Science
Foundation
funded
project
Ac ce
313
(BK20140542),
pt
310
314
References
315
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Langmuir, 26(3), 2093-2098.
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Wang, Y., Zhan, L., & Huang, C.Z. (2010). One-pot preparation of dextran-capped gold
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of
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using
4-acetamido-TEMPO-oxidized
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Yan, J.K., Ma, H.L., Cai, P.F., Zhang, Q., Hu, N.Z., Feng, X.B., & Wu, J.Y. (2014) Structural
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characteristics and antioxidant activities of different families of 4-acetamido-TEMPO-
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-oxidized curdlan. Food Chemistry, 143, 530-535.
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Ye, W., Yan, J., Ye, Q., & Zhou, F. (2010). Template-free and direct electrochemical
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and applications in bionanotechnology. Nanoscale, 4, 1871-1880. Zhou, M., Wang, B., Rozynek, Z., Xie, Z., Fossum, J. O., Yu, X., & Raaen, S. (2009). Minute synthesis of extremely stable gold nanoparticles. Nanotechnology. 20, 505606.
pt
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Yeh, Y.C., Creran, B., & Rotello, V.M. (2012). Gold nanoparticles: preparation, properties,
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an
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:
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Fig. 1. UV-Vis spectra absorption of Cc-AuNPs prepared with 0.2% (w/v) Cc and 2.0 mM
425
HAuCl4 at 100 ºC for different reaction times (inset images: solution color in different
426
time interval).
ip t
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Fig. 2. UV-Vis spectra absorption of Cc-AuNPs prepared with different concentrations of Cc
428
and 2.0 mM HAuCl4 at 100 ºC for 90 min (inset images: solution color with different
429
concentrations of Cc)
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Fig. 3. (A) UV-Vis spectra absorption of Cc-AuNPs prepared with 0.2% (w/v) Cc and
431
different concentrations of HAuCl4 at 100 ºC for 90 min (inset images: solution color
432
with different concentrations of HAuCl4); HRTEM images of Cc-AuNPs prepared with
433
0.2% (w/v) Cc and 0.5 mM HAuCl4 (B), 1.0 mM HAuCl4 (C), and 2.0 mM HAuCl4 (D).
434
Fig. 4. (A) XRD pattern of Cc-AuNPs; (B) EDX of Cc-AuNPs. Cc-AuNPs prepared with
437
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pt
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0.2% (w/v) Cc and 2 mM HAuCl4 at 100 ºC for 90 min. Fig. 5. FT-IR spectra of (A) Cc and (B) Cc-AuNPs synthesized in 0.2% (w/v) Cc and 2 mM
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an
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HAuCl4 at 100 ºC for 90 min.
438
Fig. 6. Proposed scheme for the preparation of AuNPs by Cc.
439
Fig. 7. (A) Fluorescence spectra of BSA in the presence of Cc-AuNPs. The concentrations of
440
Cc-AuNPs are: a. 0 M, b. 10-12 M, c. 10-10 M, d. 10-8 M, and e. 10-6 M; (B) CD spectra of
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native BSA (curve a) and BSA conjugated with different concentrations of Cc-AuPNs: b.
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10-12 M, c. 10-10 M, d. 10-8 M, and e. 10-6 M.
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0.8 0
0 min 15 min 30 min 60 min 90 min 120 min
15 30 60 90 120
0.5 0.4 0.3
cr
Absorbance
0.6
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0.1 400
450
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Wavelength (nm)
650
an
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Fig. 1 Yan et al.
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0.8 0.7 0 0.05 0.10 0.15 0.20
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cr
Absorbance
0.6
0% 0.05% 0.10% 0.15% 0.20%
0.2
400
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650
700
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Fig. 2 Yan et al.
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0.8
A 0
0.5 1.0 1.5 2.0
0.5
B
0.4 0.3
cr
Absorbance
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0 mM 0.5 mM 1.0 mM 1.5 mM 2.0 mM
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0.7
0.2
400
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50 nm
an
Wavelength (nm)
449 450
450
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0.1
C
D
50 nm
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Fig. 3 Yan et al.
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1000 A (111)
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600 (200)
400
cr
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800
(220)
(311)
40
50
60
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1000
cps/eV
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ed
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400
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Fig. 4 Yan et al.
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A
1310 2935
Transmittance
941
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1426 1087 1051
cr
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3500
3000
2500
2000
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1000
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pt
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3500
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-1
Wavenumber (cm )
Fig. 5 Yan et al.
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COO -
- OOC
Au3+
COO -
-OOC
Reducing
-
Stabilizing
COOCOO -
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COOCOO-
R
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458
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Fig. 6 Yan et al.
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20
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Fig. 7 Yan et al.
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Highlights
467 468
◆ A facile, green strategy was developed for the preparation of AuNPs.
470
◆ Use of carboxylic curdlan (Cc) both as reducing and stabilizing agent.
471
◆ The Cc-capped AuNPs with high stability are spherical and well dispersed in solution.
472
◆ Carboxylic groups in Cc molecules tend to absorb and stabilize the surface of AuNPs.
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◆ The AuNPs can be developed as a potential candidate in biomedicine and biotechnology.
an
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S0144-8617(14)01060-1 http://dx.doi.org/doi:10.1016/j.carbpol.2014.10.048 CARP 9401
To appear in: Received date: Revised date: Accepted date:
13-8-2014 27-9-2014 16-10-2014
Please cite this article as: Yan, J.-K., Liu, J.-L., Sun, Y.-J., Tang, S., Mo, Z.Y., and Liu, Y.-S.,Green synthesis of biocompatible carboxylic curdlan-capped gold nanoparticles and its interaction with protein, Carbohydrate Polymers (2014), http://dx.doi.org/10.1016/j.carbpol.2014.10.048 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
1
Submitted to: Carbohydrate Polymers
2
(Original Research MS)
3
Green synthesis of biocompatible carboxylic curdlan-capped gold nanoparticles and its
5
interaction with protein
ip t
4
Jing-Kun Yan
8
Liub,*
a,
, Jin-Lin Liu a, Yu-Jia Sun a, Shuang Tang a, Zheng-Ying Mo a, Yuan-Shuai
us
7
cr
6
an
9 10
a
11
b
12
Science and Technology, Clear Water Bay, Kowloon, Hong Kong
M
School of Food & Biological Engineering, Jiangsu University, Zhenjiang, 212013, China
ed
Department of Chemical and Biomolecular Engineering, The Hong Kong University of
Ac ce
pt
13
Corresponded authors: JK Yan, tel: +08615952819661, e-mail: [email protected]; YS Liu, tel: 852-23588409, e-mail: [email protected] 1 Page 1 of 29
14
Abstract This study demonstrates a facile, green strategy for the preparation of gold nanoparticles
16
(AuNPs) from chloroauric acid (HAuCl4) using carboxylic curdlan (Cc) as both reducing and
17
stabilizing agent. The as-prepared AuNPs are characterized by UV-Vis spectroscopy, high
18
resolution transmission electron microscopy, X-ray diffraction, energy dispersive X-ray
19
spectrometry and Fourier transform infrared spectroscopy. The results indicated that the
20
particle size of the AuNPs changes with variations in the reaction time and concentrations of
21
Cc and HAuCl4. The spherical AuNPs are well dispersed, exhibiting high stability even after
22
six months storage. The carboxylic groups (COO-) in the Cc molecules tend to adsorb and
23
stabilize the surface of the AuNPs. The interaction between BSA and the Cc-capped AuNPs
24
was investigated using fluorescence and circular dichroism spectroscopies. The results
25
indicated that the BSA molecules adsorb on the surface of the AuNPs, without significant
26
change in its helical structure even after conjugation with the AuNPs.
27
pt
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Carboxylic
curdlan;
Keywords:
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Protein-nanoparticle interaction
30
Ac ce
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Gold
nanoparticles;
Green
synthesis;
Structure;
2 Page 2 of 29
30
1. Introduction Metal nanoparticles, especially gold nanoparticles (AuNPs), have recently gained
32
significant attentions because of their unique properties and potential applications in catalysis,
33
optoelectronics, sensors, biotechnology, and biomedicine (Daniel & Astruc, 2004; Dykman &
34
Khlebtsov, 2012; Li, Zhao & Astruc, 2014; Sau, Rogach, Jackel, Klar, & Feldmann, 2010;
35
Yeh, Creran & Rotello, 2012). In order to explore the biological processes that are critical for
36
diagnostics and modulation of cell functions, a lot of work has been done on unraveling the
37
surface interaction of biomolecules with metal nanoparticles (NPs) (Lacerda et al., 2010).
38
Obviously, AuNPs are the natural starting points for understanding the protein-NP
39
interactions due to their promise for diverse biomedical applications, such as molecular
40
diagnostics, bioengineering, biomolecular imaging, and drug delivery for cancer therapeutics
41
(Andres et al., 1996; Galletto, Brevet, Girault, Antoine, & Broyer, 1999; Ghosh, Han, De,
42
Kim, & Rotello, 2008; Mirkin, Letsinger, Mucic, & Storhoff, 1999; Burt, Gutierrez-Wing,
43
Miki-Yoshida, & Jose-Yacaman, 2004).
pt
ed
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an
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To date, AuNPs are being synthesized with a variety of bottom-up methods and
45
technologies, including chemical reduction (Chen, Wang, Ballato, Foulger, & Carroll, 2003),
46
seed-growth (Busbee, Obare, & Murphy, 2003), photochemistry (Kim, Song, & Yang, 2002),
47
electrochemistry (Ye, Yan, Ye, & Zhou, 2010), sonochemistry (Qiu, Zhu, & Chen, 2003), and
48
template-assisted supramolecular chemistry (Lehn, 1995). Among these techniques, the
49
chemical reduction method, which involves the use of various reducing agents, such as
50
hydrazine, sodium borohydride (NaBH4), dimethyl formamide (DMF), and other organic
51
compounds, is considered as the most simple and conventional method for the preparation of
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3 Page 3 of 29
AuNPs. However, these reducing agents are often associated with potential environmental
53
toxicity or biological hazards. Therefore, more and more interests are being put on the
54
development of “green” synthesis strategies for the preparation of AuNPs using non-toxic,
55
environmentally friendly and renewable materials.
ip t
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Polysaccharides derived from renewable sources, including plants, animals, and
57
microorganisms, are being extensively studied in a wide variety of biomedical applications
58
and functional foods due to their notable and excellent bioactivities, such as
59
immunostimulatory, antitumor, and antioxidant activities (Giavasis, 2014; Stachowiak &
60
Reguła, 2012). In particular, polysaccharides possessing good biocompatibility and
61
biodegradability play an important role in nanoscience and drug delivery owing to the
62
presence of a large number of functional groups, including hydroxyl, carboxyl, and amino
63
groups, in their structures. Raveendran et al. (2003) firstly reported a facile “green” method
64
for the synthesis of metal nanoparticles using glucose as the reducing agent and starch as the
65
protecting agent. Then many other polysaccharides and their derivatives have been employed
66
for the “green” synthesis of AuNPs, such as chitosan (Huang et al., 2007), dextran (Wang,
67
Zhan, & Huang, 2010), pullulan (Choudhury, Malhotra, Bhattacharjee, & Prasad, 2014),
68
glucan (Jia, Xu & Zhang, 2013; Sen, Maity, & Islam, 2013), cellulose (Tan et al., 2010), and
69
guar gum (Pandey, Goswami, & Nanda, 2013). Our group has also successfully developed a
70
facile, simple, and eco-friendly method to synthesize silver nanoparticles (AgNPs) using
71
aqueous
72
4-acetamido-2,2,6,6-tetramethypiperidine-1-oxyl radical-mediated oxidation curdlan, as both
73
reducing and stabilizing agents (Yan et al., 2013). However, no studies have been reported on
Ac ce
pt
ed
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an
us
cr
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solution
of
carboxylic
curdlan
(Cc),
which
was
prepared
by
the
4 Page 4 of 29
the green synthesis of AuNPs using Cc solution. Herein, for the first time, we developed a
75
green strategy for the synthesis of AuNPs from chloroauric acid (HAuCl4) using Cc as the
76
reducing as well as stabilizing agent. The characteristics and mechanism of the as-prepared
77
Cc-AuNPs have been systematically elucidated using UV-Vis spectroscopy, high-resolution
78
transmission electron microscopy (HR-TEM), X-ray diffraction (XRD), energy-dispersive
79
X-ray (EDX) analysis, and Fourier-transform infrared (FT-IR) spectroscopy. In addition, the
80
interaction between the prepared Cc-AuNPs and bovine serum albumin (BSA) has been
81
investigated using fluorescence spectroscopy and circular dichroism (CD) spectroscopy.
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2. Materials and methods
84
2.1. Materials and chemicals
M
83
an
82
Carboxylic curdlan (Cc) bearing the β-1,3-polyglucuronic acid structure was prepared
86
from 4-acetamido-TEMPO-mediated oxidation, details being shown in our previous work
87
(Yan et al., 2014). The carboxylate content and molecular weight (MW) of Cc are 4.87
88
mmol/g and 1.2×105 Da, respectively. Hydrogen tetrachloroaurate (III) tetrahydrate
89
(HAuCl4·3H2O, 99%), and bovine serum albumin (BSA, MW 6.6×104 kDa) were obtained
90
from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). All other chemicals and solvents
91
were of laboratory grade, without further purification. Ultrapure Milli-Q water (18.2 MΩ cm
92
at 25 ºC) was used in all experiments.
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93 94 95
2.2. Green synthesis of Cc-capped gold nanoparticles (Cc-AuNPs) Stock solutions of HAuCl4 with concentrations ranging from 0.5 to 2.0 mM were 5 Page 5 of 29
prepared by adding gold (III) chloride hydrate in deionized water. For the synthesis of AuNPs,
97
2 mL of the HAuCl4 stock solution was added to an equal volume of Cc (0.2 %, w/v). The
98
resulting mixture was maintained at 100 ºC under continuous stirring in a water bath for
99
different reaction time (15–120 min) to obtain AuNPs.
ip t
96
100
2.3. Characterization
cr
101
The UV-Visible absorption spectra of the as-prepared Cc-AuNPs solutions were
103
obtained from a Perkin-Elmer Lambda 35 spectrophotometer in the wavelength range of
104
400–700 nm with the interval of 1.0 nm. The change in the color was directly recorded by a
105
digital camera. The particle size and morphology of the Cc-AuNPs was observed using High
106
Resolution Transmission Electron Microscopy (HRTEM, Tecnai 12, Philips, 120 kV). For
107
TEM analysis, the sample was prepared by placing a drop of the Cc-AuNPs solution on a
108
carbon coated copper grid (300 meshes), followed by drying at room temperature (20 ºC) for
109
30 min. The elemental composition of the nanoparticles was determined by an EDX
110
spectroscope (Inca Energy-350, Oxford Co., UK) attached to the SEM (JSM-7001F, JEOL
111
Ltd., Japan). The critical structure of the Cc-AuNPs was determined by a wide-angle XRD
112
(D8-Advance, Bruker, Co., Germany). The XRD patterns were recorded with Cu Kα
113
radiation (λ = 0.1541 nm) at 40 kV and 40 mA in the 2θ ranging from 30° to 80° with a step
114
speed of 4°/min. Fourier-transform infrared (FT-IR) spectra of Cc and freeze-dried
115
Cc-AuNPs were obtained from a Nexus 670 FT-IR spectrometer (Thermo Nicolet Co., USA)
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in the wavenumber range of 500–4000 cm−1 with KBr pellets and referenced against air.
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2.4. Preparation and characterization of Cc-AuNPs and BSA interactant The interactant was prepared by adding 50 μL of the as-prepared Cc-AuNPs solution
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(with different concentrations ranging from 10-12 to 10-6 M) to 1 mL of BSA solution (0.01
121
mg/mL). Then the mixture was incubated at 25 ºC in a water bath for 3 h under continuous
122
stirring. After incubation, the mixture was maintained at 4 ºC before analysis.
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The fluorescence spectra of BSA and BSA/Cc-AuNPs interactants were recorded on a
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Perkin Elmer LS35 spectrofluorimeter in the wavelength range of 300–450 nm using the
125
excitation wavelength of 290 nm. The width of both the excitation and emission slit was 5.0
126
nm. Furthermore, the CD spectra were recorded on a circular dichroism spectrometer
127
(JASCO J-815, Japan) at room temperature using a 0.1 cm path length quartz cell in the
128
wavelength range of 190–290 nm.
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3. Results and discussion
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3.1. Influence of reaction time on the synthesis of Cc-AuNPs
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Owing to the presence of a large number of hydroxyl and carboxyl (COO-) groups, the
133
Cc molecules prepared via the 4-acetamido-TEMPO-mediated oxidation of curdlan exhibited
134
a strong ability to adsorb metallic cations. The Cc molecules thus played the role of both
135
reducing and stabilizing agents for the one-pot, green synthesis of AuNPs in an aqueous
136
medium. Moreover, the functional groups in the Cc molecules cover and passivate the surface
137
of the AuNPs, thereby preventing any further aggregation (Liu, Raveendran, Qin & Ikushima,
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2005).
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Fig. 1 shows the UV-Vis absorption spectra of the AuNPs prepared with 0.2% (w/v) Cc 7 Page 7 of 29
and 2.0 mM HAuCl4 solution at 100 ºC for different time intervals. After 15 min of
141
incubation, the UV-Vis spectra show a broad absorption band at around 530 nm. When the
142
incubation time increases from 15 to 90 min, the bands become stronger. The appearance of
143
this typical surface plasmon resonance (SPR) band of AuNPs further indicates the formation
144
of Cc-AuNPs (Mulvaney, 1996). The inset image of Fig. 1 shows that the HAuCl4 aqueous
145
solution is colorless in the beginning, and with the ongoing of reaction, the color becomes
146
dark-blue and then wine red, without the formation of precipitates. This indicates that Au3+ in
147
the solution is reduced to Au0 by the Cc molecules (Zhou et al., 2009). In general, metal
148
nanoparticles with various shapes and sizes show different colors in solutions, therefore the
149
shape of the nanoparticles can be visually evaluated. For instance, metallic gold is yellow,
150
spherical AuNPs are wine red or purple, while gold nanorods are blue or black in solution
151
(Burda, Chen, Narayanan & El-Sayed, 2005; Thakor, Jokerst, Zavaleta, Massoud & Gambhir,
152
2011). These results also reveal that the Cc molecules in the solution could act as both
153
reducing and stabilizing agents for the synthesis of well-dispersed spherical AuNPs, without
154
the need of any additional reducing agents or stabilizers. In addition, no significant increase
155
in absorption intensity could be observed after 90 min of reaction time, suggesting that the
156
reaction is completed within 90 min.
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3.2. Influence of Cc concentration on the synthesis of Cc-AuNPs
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According to our previous work (Yan et al., 2013), the concentration of Cc has a
160
significant influence on the formation of AgNPs. Therefore, in the present study, we analyzed
161
the effects of Cc concentration on the synthesis of the as-prepared AuNPs. Fig. 2 shows the
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UV-Vis absorption spectra of the AuNPs prepared with different concentrations of Cc (0.05% 8 Page 8 of 29
to 0.2%, w/v) and 2.0 mM HAuCl4 at 100 ºC for 90 min. When the Cc concentration is below
164
0.1%, Au3+ was not sufficiently reduced and stabilized for the preparation of AuNPs. It was
165
found that there is a gradual increase in the absorption intensity, by increasing the Cc
166
concentration up to 0.2% (w/v). Moreover, the width of absorption peak gradually becomes
167
narrower with the increase in the concentration of Cc from 0.1% to 0.2% (w/v) suggesting
168
that the size distribution of the as-prepared AuNPs becomes narrower (Babapour et al., 2006).
169
In addition, the SPR band of the as-synthesized AuNPs blue-shifted from 540 nm to 530 nm
170
with increasing Cc concentration from 0.1% to 0.2% (w/v) indicating that the particle size
171
was decreased with increasing Cc concentration. In addition, the effect of Cc concentration
172
on the formation of AuNPs was also evaluated by visually observing the color changes in the
173
solution. As can be seen from the inset image shown in Fig. 2, the color of the solution
174
changes from colorless to light blue or blue color and subsequently to wine red color with
175
increase in the concentration of Cc from 0.05% to 0.2%. This indicates that the shape of the
176
as-prepared particles can be tuned by controlling the concentration of Cc. In the present study,
177
the concentration of Cc suitable for the preparation of AuNPs was found to be 0.2% (w/v).
178
Neither AuNP aggregation nor polymer phase separation was observed under this condition.
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3.3. Influence of HAuCl4 concentration on the synthesis and morphology of the Cc-AuNPs
181
Fig. 3A shows the UV-Vis absorption spectra of the AuNPs prepared by reducing
182
different concentrations of HAuCl4 solution with 0.2% (w/v) Cc at 100 ºC for 90 min. The
183
absorption peaks appear at about 530 nm, which is a characteristic SPR band of AuNPs and
184
confirms the formation of AuNPs under different concentrations of HAuCl4 solution. With an
185
increase in the concentration of HAuCl4 from 0.5 to 2.0 mM, the intensity of the absorption
186
peak increased gradually, suggesting the formation of more nanoparticles at higher 9 Page 9 of 29
concentration of HAuCl4. Meanwhile, the effects of HAuCl4 concentration on the formation
188
of AuNPs can also be visually observed through the color change of the solution. As shown in
189
the inset images of Fig. 3A, the AuNPs formed under lower concentration of HAuCl4 (0.5
190
mM) exhibits a light purple color in solution, and the color becomes deeper to wine red or
191
purple at higher concentration (2.0 mM).
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The particle size and morphology of the AuNPs, formed by reducing 0.5, 1.0, and 2.0
193
mM HAuCl4 solution with 0.2% (w/v) Cc, respectively, was recorded by HR-TEM (Figs.
194
3B-D). Fig. 3B shows the formation of well-dispersed spherical particles with an average size
195
of ~17 nm, at the HAuCl4 concentration of 0.5 mM. When HAuCl4 concentration increases to
196
2.0 mM, the particle size also increased to 24 nm (Fig. 3D), correspondingly, higher
197
concentration of HAuCl4 leading to the formation of larger sized AuNPs. This result agrees
198
well with the UV-Vis analysis and visual observation of color formation. It is reasonable
199
because higher concentration of Au3+ ions in the HAuCl4 solution provides more nuclei,
200
which favors further growth of the nanoparticles (Wu, Zhang & Zhang, 2012). More
201
specifically, an increase in the concentration of HAuCl4 may result in the aggregation of
202
particles to some extent, perhaps due to the role of polysaccharides as stabilizers or
203
nucleating agents (Sen, Maity & Islam, 2013). The amount of 0.2% (w/v) Cc used in this
204
work is sufficient enough to reduce the Au3+ ions in the high HAuCl4 concentration of 2.0
205
mM, but could not completely prevent the aggregation of the as-formed AuNPs. However, at
206
lower concentrations, the same concentration of Cc is completely sufficient to stabilize the
207
AuNPs and prevent further aggregation, forming smaller size of AuNPs.
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3.4. Characterization of Cc-AuNPs through XRD and EDX Fig. 4A shows the XRD pattern of the AuNPs prepared with 0.2% (w/v) Cc and 2.0 mM
211
HAuCl4 solution at 100 ºC for 90 min. The four diffraction peaks at 2θ corresponding to
212
38.2°, 44.4°, 64.6°, and 77.7° can be attributed to the (111), (200), (220), and (311) planes,
213
respectively, of the face-centered cubic (fcc) gold. The Bragg reflections (200), (220), and
214
(311) are relatively weaker and broader compared to the intense (111) reflection, implying
215
that the gold nanocrystals are preferentially oriented along the (111) plane. The EDX
216
spectrum of the AuNPs (Fig. 4B) reveals a strong and typical optical absorption peak at
217
approximately 200 eV, which could be attributed to the SPR of the metallic Au nanocrystals,
218
and confirms the formation of AuNPs in the reaction medium. In addition, the presence of Au,
219
C, and O elements in the EDX spectrum indicates that the nanocomposite consists of both Cc
220
and AuNPs.
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3.5. FT-IR analysis and mechanism underlying the formation of Cc-AuNPs
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Fig. 5 shows the FT-IR spectra of Cc and Cc-AuNPs prepared from 0.2% (w/v) Cc and
224
2.0 mM HAuCl4 at 100 ºC for 90 min, aiming to investigate the possible interaction between
225
the Cc molecules and the Cc-capped AuNPs in the solution. The FT-IR spectrum of Cc (Fig.
226
5A) shows two characteristic absorptions of the polysaccharides. The strong and wide
227
stretching peak at approximately 3470 cm−1 can be ascribed to the O–H stretching vibration,
228
while the weak absorption peak at approximately 2935 cm−1 can be associated with the C–H
229
stretching vibration. The absorption band at 1636 cm-1 can be attributed to the asymmetrical
230
COO- stretching vibration, and that at 1426 cm-1 to the symmetrical COO- stretching
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11 Page 11 of 29
vibration (Delattre et al., 2009). The absorption peaks at 1087 and 1050 cm-1 are ascribed to
232
the stretching vibration of C-OH. Additional peaks at 3450, 2932, 1632, 1426, 1087, and
233
1050 cm-1 (Fig. 5B) correspond to O-H, C-H, COO- and C-OH, respectively, coincident with
234
the characteristic peaks of the polysaccharides. This suggests that the skeleton structure of Cc
235
remains unaltered after the formation of AuNPs. Interestingly, a new peak appeared at around
236
1730 cm-1 can be ascribed to the stretching vibration of free -COOH group, indicating the
237
reduction of Au3+ to Au0 by the polysaccharide in the solution (Shi & Sun, 1988). Sun et al.,
238
(2008) has reported a number of glycosidic linkages in the polysaccharide chain are broken
239
into shorter segments in an acidic HAuCl4 solution. Besides, some -CHO groups, present in
240
the opened structure, may be oxidized to -COOH groups along with the reduction of Au3+ to
241
Au0 in the presence of Au3+. Moreover, the peak at 1636 cm-1 (COO- stretching vibration) is
242
slightly shifted towards 1632 cm-1 in the FT-IR spectrum of Cc-AuNPs, meaning that the
243
combination between Au nanoparticles and Cc molecules is through affinity or electrostatic
244
interaction with carboxylate (COO-) groups, and leads to no traces of blank AuNPs in the
245
suspension. Therefore, the functional groups present in the Cc molecules play a dual role of
246
both reducing agent and stabilizing agent in synthesizing as well as protecting the as-formed
247
AuNPs.
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Our
previous
work
have
reported
that
the
Cc
molecules
bearing
the
249
β-1,3-polyglucuronic acid structure existed as a semi-flexible chain in the aqueous solution
250
and had the potential for producing gene carriers, bio-nanomaterials, and other chiral
251
nanowires, through hydrogen bonding interactions, hydrophobic interactions, and
252
electrostatic attraction (Yan et al., 2014; Lien et al., 2011). Based on this study, a possible 12 Page 12 of 29
mechanism underlying the green synthesis of AuNPs is proposed in Fig. 6, Cc serving both as
254
a reducing agent as well as a capping agent for the synthesis and stabilization of AuNPs.
255
Firstly, the extensive carboxylic groups (COO-) on the C-6 position of the Cc chains play an
256
active role in the adsorption and stabilization of the Au3+ ions through electrostatic attraction
257
or affinity interaction. Secondly, the hydroxyl groups in the Cc chain reduce the adsorbed
258
Au3+ ions to Au0 and metallic Au, which further nucleate and grow to form AuNPs. The
259
AuNPs are subsequently capped and stabilized by Cc because of its oxygen-rich structure
260
(Huang et al., 2007). Meanwhile, the hydroxyl groups in the polymeric chains and networks
261
will also promote the stabilization of the AuNPs. The AuNPs formed by this method are
262
highly stable and well dispersed, and there is no any color change or precipitates formation
263
after six months of storage at room temperature from direct visual observation.
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3.6. Studies on the interaction of Cc with BSA
Generally, the adsorption of proteins on the surface of the NPs alters the surface
267
functionality and thus may strongly influence the translocation behavior in the biological
268
systems (Treuel, Malissek, Gebauer, & Zellner, 2010). In order to understand the fate of the
269
NPs in the biological systems, we analyzed the interactions between BSA and the AuNPs
270
prepared from 0.2% (w/v) Cc and 2mM HAuCl4 at 100 ºC for 90 min using fluorescence
271
spectroscopy and CD measurements. Fig. 7A show that the fluorescence intensity of BSA at
272
around 365 nm (the presence of tyrosine, tryptophan, and phenylalanine residues in BSA
273
molecule), tends to decrease when the concentration of Cc-AuNPs increases from 10-12 M to
274
10-6 M, suggesting that the addition of Cc-AuNPs quenches BSA fluorescence emission,
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13 Page 13 of 29
275
without any peak shift or the formation of new peak. The observed quenching of the
276
fluorescence intensity of BSA may be attributed to the formation of conjugate between
277
Cc-AuNPs and BSA. CD is powerful in studying the interactions of proteins with other molecules and
279
investigating the protein conformation in solution or adsorbed onto other molecules. In
280
general, the presence of high percentage of α-helical (67%) structure in the BSA molecules
281
exhibits a characteristic CD signal in the far UV region. Typically, changes in the ellipticity at
282
208 and 222 nm are considered useful probes for visualizing the variations in α-helical
283
content. Fig. 7B shows the CD spectra of BSA in the absence and presence of different
284
concentrations of AuNPs. The ellipticity at 208 and 222 nm of BSA gradually decrease with
285
an increase in the concentration of Cc-AuNPs from 10-12 M to 10-6 M, implying that the
286
AuNPs capped with the carboxylic groups of Cc influence the conformation of BSA to a
287
certain extent. Nonetheless, the change in α-helical content of BSA is not significant even
288
after conjugation with 10-6 M AuNPs. It is well known that the changes in the original
289
structure of the protein result in loss of biological activity or the activation of immune
290
response (Brandes, Welzel, Werner, & Kroh, 2006). Remarkably, the BSA molecule could
291
maintain most of its α-helical structure after conjugation in this study, which offers great
292
potentials as a novel carrier of proteins for biomedical applications.
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4. Conclusions
295
In summary, we developed a facile and green method for the preparation of
296
well-dispersed spherical AuNPs using Cc as both reducing agent and stabilizing agent in 14 Page 14 of 29
aqueous medium. The particle size, shape, morphology, structure and elemental composition
298
of the as-prepared AuNPs were thoroughly characterized using UV-Vis spectroscopy,
299
HRTEM, XRD, and EDX analyses, and the interactions between Cc and AuNPs were
300
examined using FT-IR spectroscopy. Based on the FT-IR analysis, we proposed the possible
301
mechanism underlying the synthesis of Au nanoparticles using Cc. In addition, the interaction
302
between BSA and the Cc-capped AuNPs was also investigated using fluorescence
303
spectroscopy and CD spectroscopy. The results suggest the potential use of non-toxic, benign,
304
biodegradable, and biocompatible Cc as a reducing agent as well as stabilizer for the
305
synthesis of metal nanoparticles, and a broad spectrum of their potential applications in
306
biomedicine and bioanalytical fields.
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Acknowledgements
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This work was supported financially by the Natural Science Foundation of Jiangsu Province
311
(2013M531292), the Priority Academic Program Development (PAPD) of Jiangsu Higher
312
Education Institutions, and Jiangsu university students' scientific research project (13A097).
China
Postdoctoral
Science
Foundation
funded
project
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(BK20140542),
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and applications in bionanotechnology. Nanoscale, 4, 1871-1880. Zhou, M., Wang, B., Rozynek, Z., Xie, Z., Fossum, J. O., Yu, X., & Raaen, S. (2009). Minute synthesis of extremely stable gold nanoparticles. Nanotechnology. 20, 505606.
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Yeh, Y.C., Creran, B., & Rotello, V.M. (2012). Gold nanoparticles: preparation, properties,
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Fig. 1. UV-Vis spectra absorption of Cc-AuNPs prepared with 0.2% (w/v) Cc and 2.0 mM
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HAuCl4 at 100 ºC for different reaction times (inset images: solution color in different
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time interval).
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Fig. 2. UV-Vis spectra absorption of Cc-AuNPs prepared with different concentrations of Cc
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and 2.0 mM HAuCl4 at 100 ºC for 90 min (inset images: solution color with different
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concentrations of Cc)
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Fig. 3. (A) UV-Vis spectra absorption of Cc-AuNPs prepared with 0.2% (w/v) Cc and
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different concentrations of HAuCl4 at 100 ºC for 90 min (inset images: solution color
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with different concentrations of HAuCl4); HRTEM images of Cc-AuNPs prepared with
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0.2% (w/v) Cc and 0.5 mM HAuCl4 (B), 1.0 mM HAuCl4 (C), and 2.0 mM HAuCl4 (D).
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Fig. 4. (A) XRD pattern of Cc-AuNPs; (B) EDX of Cc-AuNPs. Cc-AuNPs prepared with
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M
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0.2% (w/v) Cc and 2 mM HAuCl4 at 100 ºC for 90 min. Fig. 5. FT-IR spectra of (A) Cc and (B) Cc-AuNPs synthesized in 0.2% (w/v) Cc and 2 mM
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HAuCl4 at 100 ºC for 90 min.
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Fig. 6. Proposed scheme for the preparation of AuNPs by Cc.
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Fig. 7. (A) Fluorescence spectra of BSA in the presence of Cc-AuNPs. The concentrations of
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Cc-AuNPs are: a. 0 M, b. 10-12 M, c. 10-10 M, d. 10-8 M, and e. 10-6 M; (B) CD spectra of
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native BSA (curve a) and BSA conjugated with different concentrations of Cc-AuPNs: b.
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10-12 M, c. 10-10 M, d. 10-8 M, and e. 10-6 M.
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0.8 0
0 min 15 min 30 min 60 min 90 min 120 min
15 30 60 90 120
0.5 0.4 0.3
cr
Absorbance
0.6
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0.1 400
450
500
550
600
Wavelength (nm)
650
an
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0.2
Fig. 1 Yan et al.
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0.8 0.7 0 0.05 0.10 0.15 0.20
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cr
Absorbance
0.6
0% 0.05% 0.10% 0.15% 0.20%
0.2
400
450
500
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Wavelength (nm)
650
700
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Fig. 2 Yan et al.
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0.8
A 0
0.5 1.0 1.5 2.0
0.5
B
0.4 0.3
cr
Absorbance
0.6
0 mM 0.5 mM 1.0 mM 1.5 mM 2.0 mM
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0.2
400
500
550
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650
700
50 nm
an
Wavelength (nm)
449 450
450
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0.1
C
D
50 nm
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50 nm
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Fig. 3 Yan et al.
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1000 A (111)
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600 (200)
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cr
Intensity (a.u.)
800
(220)
(311)
40
50
60
2Theta(Degree)
1000
cps/eV
C
ed
O
600
80
M
B 800
70
an
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Na
400
Cl
pt
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Au
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Fig. 4 Yan et al.
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A
1310 2935
Transmittance
941
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1426 1087 1051
cr
1636
3500
3000
2500
2000
us
3470 1500 -1
Wavenumber (cm )
1306
M
B
942
1730
2932
ed
Transmittance
500
an
455
1000
1426 1160 1087 1051
pt
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Ac ce
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3500
456 457
3000
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1500
1000
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-1
Wavenumber (cm )
Fig. 5 Yan et al.
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COO -
- OOC
Au3+
COO -
-OOC
Reducing
-
Stabilizing
COOCOO -
OOC -OOC
Cc:
COOCOO-
R
HO O
O
n OH R = COONa or CH2OH
gold yellow
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Au0
wine red
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Cc:
COO -
-OOC
cr
COO -
an
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Fig. 6 Yan et al.
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a
200
e
cr
Flurescence Intensity (a.u.)
300
300
320
340
360
Wavelength (nm)
20
440
ed
0
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CD (mdeg)
10
464
420
M
B
-20
400
an
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463
380
us
100
-10
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A
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210
e a 220
230
240
250
260
Wavelength (nm)
Fig. 7 Yan et al.
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Highlights
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◆ A facile, green strategy was developed for the preparation of AuNPs.
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◆ Use of carboxylic curdlan (Cc) both as reducing and stabilizing agent.
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◆ The Cc-capped AuNPs with high stability are spherical and well dispersed in solution.
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◆ Carboxylic groups in Cc molecules tend to absorb and stabilize the surface of AuNPs.
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◆ The AuNPs can be developed as a potential candidate in biomedicine and biotechnology.
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