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Topographic characterization of the self-assembled nanostructures of chitosan on mica surface by atomic force microscopy
Accepted Manuscript Title: Topographic Characterization of the Self-Assembled Nanostructures of Chitosan on Mica Surface by Atomic Force Microscopy Author: Li Wang Jiafeng Wu Yan Guo Coucong Gong Yonghai Song PII: DOI: Reference:
S0169-4332(15)01541-X http://dx.doi.org/doi:10.1016/j.apsusc.2015.06.193 APSUSC 30714
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Received date: Revised date: Accepted date:
9-4-2015 28-6-2015 29-6-2015
Please cite this article as:http://dx.doi.org/10.1016/j.apsusc.2015.06.193 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.
*Highlights (for review)
Highlights
Nanocomposites of chitosan film were prepared by simple self-assembly from solvent media. Chitosan molecules assembled on mica surface in the morphology of nanoparticles, fibril and
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membrane with varied chitosan concentration.
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Chitosan molecules assembled with different nanostructure under varied pH.
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The optimum drying temperature for forming chitosan membrane is about 65 °C.
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Graphical Abstract (for review)
Topographic Characterization of the Self-Assembled Nanostructures of Chitosan on Mica Surface by Atomic Force Microscopy
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Li Wang, Jiafeng Wu, Yan Guo, Coucong Gong and Yonghai Song
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Key Laboratory of Functional Small Organic Molecule, Ministry of Education, Key Laboratory of Chemical Biology, Jiangxi Province, College of Chemistry and Chemical Engineering, Jiangxi
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Normal University, Nanchang, 330022, China.
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Graphical abstract
Corresponding author: Tel/Fax: +86-791-88120861. E-mail: [email protected] (Y. Song).
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*Manuscript Click here to view linked References
Topographic Characterization of the Self-Assembled Nanostructures of Chitosan on Mica Surface by Atomic Force
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Microscopy
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Li Wang, Jiafeng Wu, Yan Guo, Coucong Gong and Yonghai Song∗
Key Laboratory of Functional Small Organic Molecule, Ministry of Education, Key Laboratory of
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Chemical Biology, Jiangxi Province, College of Chemistry and Chemical Engineering, Jiangxi
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Normal University, Nanchang, 330022, China.
Li Wang: [email protected] Jiafeng Wu: [email protected] Yan Guo: [email protected]
Coucong Gong: [email protected]
∗Corresponding author: Tel/Fax: +86-791-88120861. E-mail: [email protected] (Y. Song).
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ABSTRACT In this work, the self-assembled nanostructures of chitosan on mica surface formed from various
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solvents were investigated by using atomic force microscopy. The effects of various factors on the self-assembled nanostructures of chitosan on mica surface, including solvents, the concentration of
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chitosan, the pH of solution and the drying temperature, were explored in detail. Our experimental
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data resulted in the conclusion that chitosan molecules could self-assemble on mica surface to form various nanostructures such as nanoparticles, fibril and film. Nanopartilces were always formed on
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mica surface from CCl4, C6H6, CH2Cl2 solution, fibril preferred to form on mica surface from CH3CH2OH and CH3OH solution and the optimal solvent to form film was found to be CH3CN.
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Low concentration, pH and temperature were helpful for the formation of nanoparticles, medium
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concentration, pH and temperature resulted in fibril and high concentration, pH and temperature
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was often beneficial to forming chitosan films. The study of self-assembled nanostructures of chitosan on mica surface would provide new insight into the development of chitosan-based
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load-bearing materials.
Keywords: Self-assembly; Chitosan; Nanostructures; Mica; Atomic force microscopy
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1. Introduction Chitosan (CS), a copolymer made of N- acetyl- D-glucosamine and D-glucosamine units, joined
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together by β-1,4-glycosidic bonds, is a white, translucent, amorphous solid with pearly luster [1-5], showing attractive properties such as biocompatibility, antibacterial property, biodegradability, low
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toxicity and metal binding ability [6-10]. CS has received a great deal of attention for its broad
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range of applications in biosensors, water treatment, textiles, separation membrane, food package, tissue engineering and drug delivery [11-16], owing to its ability to bind to a variety of natural or
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synthetic molecules such as nucleic acid, protein, polymers, metal ions, halogen and nanoparticles. Accordingly, it is necessary to develop approaches to prepare self-assembled nanostructures of CS
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including nanowires, nanotubes, nanaorods, films, hydrogels and emulsions which could be utilized in numerous fields [17-19].
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CS can form various self-assembled nanostructures based on hydrogen-bond networks in aqueous solutions by controlling local environments such as pH, solvent, temperature, the degree of
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acetylation of CS, types of salt and types of acids [11,12,17-21]. As a hydrophilic biopolymer, CS is a compound with both strong inter- and intramolecular hydrogen bond due to the presence of a large number of free –NH2 and –OH in each unit [13,20-22]. When CS is introduced to acids, amine groups of CS can be protonated, which results in periodic positive charges along the polymer backbone to exhibit noticeable repulsion between CS molecules [16,17]. At basic medium, most of the amine groups are not protonated and the solubility of CS decreases drastically, which leads to noticeable hydrophobic attraction. CS is also a stimulus-responsive polymer with a solubility that can be reversibly alerted by pH changes [17,23]. CS micro- or nanofibers have been widely accepted as biomedical scaffolding materials to restore, maintain, or improve the functions of
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various tissues [24,25]. Recently, Gong et al reported an nonaqueous electrochemical approach to synthesize various one-dimensional CS nanostructures (nanowires, nanotubes, and nanorods) via
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adjusting deposition time, current, and electrochemical potential [18]. Three-dimensional CS nanostructures were also prepared to serve as nanotemplates for subsequent biomineralization of
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calcium carbonate [19]. Lee et al investigated the effects of pH and time on nanostructures of CS by
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using a surface forces apparatus [22]. Yang et al prepared CS/graphene oxide (GO) films by simple self-assembly of CS and GO in aqueous media [20]. Caridade et al produced a thick membrane by
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layer-by-layer assembly of CS and alginate to study the permeation of model drugs and the adhesion of muscle cells [26]. Although CS nanostructures with controllable morphology are highly
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desirable for various application [27,28], very little attention have been focused on the self-assembled nanostructures of chitosan on mica surface, especially the effects of local
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environments on the self-assembled nanostructures of chitosan on mica surface which might be very important to control self-assembled nanostructures..
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In this work, unlike the previous kinetic study and optical microscopic study, CS nanostructures on a mica surface in a controllable fashion were investigated by atomic force microscopy (AFM). A lot of information on the self-assembled nanostructures of chitosan on mica surface was revealed by AFM studies [29-35]. It was found that solvents, concentration, pH and temperature were all key factors for the formation of self-assembled nanostructures of chitosan on mica surface. This approach does not require template, catalysis, or surfactant, and therefore it is ideal for exploring large-scale industrial production of CS nanostructures.
2. Materials and methods
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2.1 Materials. CS with a ≥80% degree of deacetylation (Fig. 1), H2SO4, NaOH, CCl4, C6H6, CH2Cl2,
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CH3CH2OH, CH3OH, and CH3CN were all purchased from Sinopharm Group Chemical Reagent Co. Ltd (Shanghai, China). All reagents (AG) were used as received without any further
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purification. The muscovite mica substrate [KAl2(AlSi3)O10(OH)2 ] were purchased from Linhe
“Here in Fig. 1”
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2.2 Sample preparation.
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Street Commodity Marketplace (Changchun, China).
In a typical experiment, 0.8 mg of CS was dispersed into 4 mL of CH3CN or other solvents
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solution under mild ultrasonication (70 W) for 2 h. The solutions were kept at a pH of ∼4.0 by adding dropwise an appropriate amount of aqueous H2SO4. Then 20 µL of CS solution was applied
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to a freshly cleaved muscovite mica surface (about 1.2×1.2 cm2 square) and incubated at room temperature for about 10 min in a moisture chamber to evaporation. After that, the sample was
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heated at 65 °C in an air oven overnight for complete drying. Finally the dried mica sheet was affixed on a metal disk for AFM imaging. To study the effect of the pH on the chitosan assembly, the assembling solutions were kept at a pH of 2.0, 4.0, 5.0, 6.0, 7.0 and 8.0 by adding dropwise an appropriate amount of aqueous H2SO4 and NaOH. In the temperature controlling experiment, the heating temperature was controlled at room temperature, 50 °C, 65 °C and 80 °C, respectively. 2.3 AFM imaging. The AFM instrument (NanoscopeⅤ, MultiMode®8, BRUKER) was employed in ScanAsyst mode at room temperature under ambient conditions. Standard silicon nitride cantilevers with Al reflective coating (BRUKER Probes, SCANASYST-AIR, spring constant of
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0.4N/m, radius of curvature less than 2 nm) were used. All the AFM data were obtained close to their resonance frequencies of the cantilever (typically, 60-150 kHz). The signal-to-noise ratio was
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maintained higher than 10. The scan rate was 1 Hz for small-area scans (<2 µm) and decreased when larger areas were scanned to ensure image quality. All images were typically obtained from at
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least five macroscopically separated regions on each sample. Changing scan direction did not affect
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the patterns, and there was no other evidence of tip-induced perturbations in the samples. Image analysis and processing were performed with the NanoScope Software 9.0. The AFM images
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presented in this work were raw data, except for minimal post-processing (a simple flatten for eliminating the background effect). Flattening modifies the image in a line-by-line way to remove
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out of the vertical offset between scan lines in the fast scan direction (X at 0° scan angle) by
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3. Results and discussion
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fit from the original scan line.
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calculating a 1st order least-squares fit polynomial for each scan line and subtracting the polynomial
3.1 Effect of solvent on self-assembled nanostructures of CS on mica surface
At first, we investigated the effect of solvent on self-assembled nanostructures of CS on mica surface. After 0.8 mg CS were dissolved in 4 mL of six kinds of different solvents, 20 µL CS solution was dropped onto a freshly cleaved mica surface (dried at 65 °C in air) and used for AFM imaging. And the results were shown in Fig. 2. When CS was dissolved in CCl4, the CS/CCl4 solution self-assembled on mica surface and CS formed some big spherical particles with diameter of about 387.95 nm (Fig. 2a). When CS was dissolved in C6H6, some spherical CS particles with diameter of about 166.28 nm were also observed on mica surface (Fig. 2b), smaller than those 6
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formed from CS/CCl4. As commented above, CS was a polar hydrophilic biopolymer with both strong inter- and intramolecular hydrogen bond, while CCl4 and C6H6 were both nonpolar
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hydrophobic solvents. Thus it was very hard for CS to disperse well in CCl4 and C6H6 solutions. After CCl4 or C6H6 was completely evaporated during drying process, the CS molecules aggregated
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into big CS sphere on mica surface owing to the intermolecular hydrogen bond. Although CCl4 and
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C6H6 were both nonpolar solvents, the polarity of C6H6 was a little stronger than that of CCl4. Thus CS particles obtained from CS/C6H6 were smaller than that obtained from CS/CCl4. In Fig. 2c, the
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morphology of self-assembled CS nanoparticles also appeared on mica surfaces when CS dissolved in CH2Cl2. It was noticeable that the diameter of these CS nanoparticles was about 3.4 nm. The
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polarity of CH2Cl2 was stronger than that of C6H6, thus CS could partially disperse in CH2Cl2 and
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formed smaller CS particle.
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Next, we also investigated three polar solvents including CH3OH, CH3CH2OH and CH3CN. As
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could see form Fig. 2d-f, the self-assembled CS nanostructures was very different due to their different polarity. Fig. 2d showed the morphology of self-assembled CS nanostructures obtained in CH3CH2OH. There was a great deal of CS fragments on mica surface. Some CS fragments were gathered together to form small films. When CS was dissolved in CH3OH, CS fibril networks with about 1.4 nm in height were formed on mica surface (Fig. 2e). When CS was dissolved in CH3CN, flat CS films were self-assembled on mica surfaces (Fig. 2f). The height of these CS films was estimated to be about 1.7 nm. Since CS molecules were well dispersed in all these polar solvent, some fragments, fibrils and films were formed on mica surface. And CH3CN was chosen as the optimum solvent for the following study.
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“Here in Fig. 2”
3.2 Effect of temperature on self-assembled nanostructures of CS on mica surface
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The evaporated rate of solvent and the movement rate of CS mica surface might affect
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self-assembled nanostructures of CS on mica surface and they strongly depended on the temperature. Thus, the effect of temperature on self-assembled nanostructures of CS on mica
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surface was also investigated in this work. Fig. 3a showed the representative AFM images of CS
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self-assembled nanostructures on mica dried at room temperature (0.2 mg/mL, pH=4.0). Some uniform CS nanoparticles were observed to be distributed over the substrate. Section analysis
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indicated that the heights of CS nanoparticles were about 1.7 nm for those big one and 0.6 nm for those small one, respectively. At low temperature, molecule movement was very slow. Most of CS
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molecules had not been thoroughly spread all the surface of mica, which led to the aggregation of
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CS into many small nanoparticles. Fig. 3b displayed self-assembled CS nanostructures obtained at
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50 °C, and uniform CS nanoparticles were observed on mica surface. Section analysis indicated that the heights of CS nanoparticles were about 1.5 nm. As the drying temperature of CS/CH3CN solution was further increased, the CS molecule movement was more and more quick. CS molecules could well spread all the surface of mica. Consequently, many smaller spherical particles in the same scale were self-assembled on mica surface. When the CS/CH3CN solution was dried at 65 °C, some incomplete CS films were formed. A higher movement rate of CS on mica surface was induced by an increased drying temperature, which prompted the self-assembly rate of CS directly and resulted in the film architecture (Fig. 3c). Upon a increase of the drying temperature to 80 °C, many small CS films were formed in Fig. 3d. Evidently, the higher temperature also resulted in
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large evaporated rate of solvent, and accordingly CS molecules could not well spread all the surface of mica. As a result, some little CS films formed. Thus 65 °C was chosen as the optimum drying
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temperature for the following study.
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“Here in Fig. 3”
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3.3 Effect of CS concentration on self-assembled CS nanostructures on mica surface
To investigate the effect of CS concentration on self-assembled CS nanostructures on mica
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surface, the CS/CH3CN solutions with different concentrations were firstly dropped onto the surface
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of freshly cleaved mica and dried at 65 °C in air. Then the samples were characterized by AFM and the results were shown in Fig. 4. At 0.02 mg/mL (Fig. 4a), only a few CS nanoparticles appeared on
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the mica surface. At 0.05 mg/mL (Fig. 4b), the mica surface was almost covered with CS
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nanoparticles. With the increase of CS concentration, the morphology of the assembled CS
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nanostructures changed gradually. Some small thin CS films appeared, and finally large thin CS films were observed. At 0.1 mg/mL (Fig. 4c), no CS nanoparticle was observed, but many small CS films with defects were formed on mica surface. The small CS films with different shapes and sizes were consisted of CS fibrils whose average thickness was estimated to be about 1.65 nm. Continuing to increase the CS/CH3CN concentration to 0.2 mg/mL (Fig. 4d), the formed CS films became larger and more compacted and only few holes were observed in these CS films [18,20,22]. In Fig. 4e, the self-assembled CS films on mica surface were connected with one another to form large CS films at 0.3 mg/mL. The complete CS films covered the whole mica surfaces when the concentration of CS/CH3CN was further increased to 0.4 mg/mL. There were many holes in the CS films. Section analysis (insets in Fig. 4d-f) indicated that the height of the films in Fig. 4d-f was 9
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about 1.6 nm, 1.9 nm and 2.1 nm, respectively (also shown in Fig. 5a). The plot of root mean square (Rms) roughness of CS films assembled on mica surface at pH 4 and 65 °C versus CS concentration
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was shown in Fig. 5b. The result clearly indicated that the Rms increased gradually as the CS concentration increased from 0.02 mg/mL to 0.4 mg/mL.
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At pH=4, the amino groups of CS were completely protonated and accordingly the inter- and
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intramolecular hydrogen bonds were very weak, which resulted in good dispersion of CS molecules in CH3CN solution. At low concentration of CS, the rapid removal of CH3CN originated from the
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high drying temperature of 65 °C led to the formation of CS nanoparticles on mica surface under
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the aid of the strong electrostatic attraction interaction between positively charged CS and negatively charged mica surface and the weak intermolecular hydrogen bonds. As the concentration
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of CS increased, the intermolecular hydrogen bonds were enhanced gradually. Under the help of the
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intermolecular hydrogen bonds, the CS molecules connected together gradually to form CS films
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when they were assembled on mica surface. Finally, a complete CS formed at high concentration. However, owing to the electrostatic repulsion between positively charged CS molecules, a large number of pores also formed in the CS film.
“Here in Fig. 4” “Here in Fig. 5”
3.4 Effect of pH on CS nanostructures on mica surface
CS was known as a stimulus-responsive polymer which could be reversibly alerted by changing pH. In order to further investigate the pH effect on CS/CH3CN assembly on mica surface, six 10
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different pHs from 2.0 to 8.0 of CS/CH3CN solutions were employed in the following experiments at the same other conditions (concentration of CS and temperature) and the results were shown in
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Fig. 6. In these experiments, the concentration of CS/CH3CN solutions was 0.2 mg/mL and drying was carried out at 65 °C in air. At pH 2.0 (Fig. 6a), many CS fibrils formed on mica surface and
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they were connected together to form small CS films with many big pores. At pH 4.0, the CS films
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became larger and the holes became smaller as shown in Fig. 6b. With the increase of pH of CS/CH3CN solution, the self-assembled CS nanostructures were changed greatly. At pH 5.0 (Fig.
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6c), the fragments of CS films connected together to form large CS film with many holes. At pH 6.0 and 7.0 (Fig. 6d and e), the CS films became more compacted and complete, and the holes
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obviously decreased. At pH 8.0 (Fig. 6f), a complete CS film covered the whole mica but a great many of round holes formed. Section analysis (inset in Fig. 6b-f) indicated that the heights of CS
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films in Fig. 6b-f were about 1.6 nm, 1.3 nm, 1.2 nm, 1.3 nm and 1.3 nm, respectively (also shown in Fig. 7a). The plot of Rms of CS films assembled on mica surface at 65 °C versus pH was shown
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in Fig. 7b. The result showed that the Rms decreased gradually as the pH increased and then increased at pH 8.0 suddenly.
Generally, the protonation/deprotonation and inter- and intramolecular hydrogen bond of CS depended on pH of solution due to a large number of free –NH2 and –OH in each unit [13,17,22]. The pH-responsive behaviors of self-assembled CS nanostructures on mica surface could be understood by using pH-stimulated alterations of conformation and charging state of CS. At low pH (pH<4), the amino groups of CS were protonated to gave a large number of positive charges and accordingly the inter- and intramolecular hydrogen bonds were very weak. The positively charged CS molecules were highly extended and dispersed under electrostatic repulsion in a solution, so CS 11
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molecules formed fibrils and small thin CS films on mica surface based on the strong electrostatic attraction interaction between positively charged CS and negatively charged mica surface and the
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weak inter- and intramolecular hydrogen bonds. At middle pH (4
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partial inter- and intramolecular hydrogen bonds formed. The small number of positive charges in
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CS molecules resulted in the weak interaction between CS and negatively charged mica surface and accordingly the CS molecules could rapidly remove on mica surface when they were assembled on
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mica surface. Under the help of the strong inter- and intramolecular hydrogen bonds, a large and compacted film of CS formed on mica surface. As the pH further increased, the CS molecules
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become completely deprotonated and the interaction between CS and negatively charged mica surface was minimized. At the same time, intermolecular association (hydrogen bonding) of CS
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molecules became dominant, and accordingly the films of CS mica became larger and larger (pH>8). Besides, the solubility of CS decreased when the pH exceeded 8 because the pKa value of CS was
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around 6.5-7.0 [36,37]. Thus only a little CS molecules on mica could be observed.
“Here in Fig. 6” “Here in Fig. 7”
4. CONCLUSION
In conclusion, we investigated the self-assembled CS nanostructures on mica surface using AFM. Our data demonstrated that the solvents, CS concentration, temperature and pH of the CS solution were all key factors for the formation of self-assembled CS nanostructures on mica surface. The polarity of solvents played a vital role in the formation of self-assembled CS nanostructures on mica
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surface and the strong polar CH3CN could lead to formation of flat CS films and weak polarity resulted in the fibrils and nanoparticles. The optimum drying temperature to form CS films was 65
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°C. To obtain a complete and compacted CS films, 0.2 mg/ml CS concentration was needed. CS was a stimulus-responsive polymer that could form different nanostructures by changing pH
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changes and the suitable pH was 7.0 for the CS films. This approach need not require template,
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catalysis or surfactant, therefore it is ideal for exploring large-scale industrial production of thin CS
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film and this study might shed some new light on the formation of CS thin film.
ACKNOWLEDGEMENT
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This work was financially supported by National Natural Science Foundation of China (21165010, 21465014 and 21465015), Natural Science Foundation of Jiangxi Province
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(20142BAB203101), The Ministry of Education by the Specialized Research Fund for the Doctoral Program of Higher Education (20133604110002) and the Ground Plan of Science and Technology
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Projects of Jiangxi Educational Committee (KJLD14023) and the Open Project Program of Key Laboratory of Functional Small Organic Molecule, Ministry of Education, Jiangxi Normal University (No. KLFS-KF-201410; KLFS-KF-201416).
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membrane by solution spreading: scanning probe microscopy study, Chemistry and Physics of Lipids, 123 (2003) 177.
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[30] Y. Song, Z. Li, Z. Liu, G. Wei, L. Wang, L. Sun, C. Guo, Y. Sun, T. Yang,A Novel Strategy to Construct a Flat-Lying DNA Monolayer on a Mica Surface, The Journal of Physical Chemistry B,
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110 (2006) 10792.
[31] K. Bierbaum, M. Grunze, A.A. Baski, L.F. Chi, W. Schrepp, H. Fuchs,Growth of
Langmuir, 11 (1995) 2143.
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Self-Assembled n-Alkyltrichlorosilane Films on Si(100) Investigated by Atomic Force Microscopy,
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[32] L. Wang, J. Jiang, Y. Song, B. Zhang, E. Wang,Self-Assembled Monolayer Growth of Octanol on Mica:Ⅴ Atomic Force Microscopy and Fourier-Transform Infrared Spectroscopy Studies, Langmuir, 19 (2003) 4953.
[33] J. Tang, A. Ebner, N. Ilk, H. Lichtblau, C. Huber, R. Zhu, D. Pum, M. Leitner, V. Pastushenko, H.J. Gruber, U.B. Sleytr, P. Hinterdorfer,High-Affinity Tags Fused to S-Layer Proteins Probed by Atomic Force Microscopy†, Langmuir, 24 (2008) 1324. [34] H. Wang, X. Hao, Y. Shan, J. Jiang, M. Cai, X. Shang,Preparation of cell membranes for high resolution imaging by AFM, Ultramicroscopy, 110 (2010) 305. [35] J. Preiner, J. Tang, V. Pastushenko, P. Hinterdorfer,Higher Harmonic Atomic Force Microscopy:
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Imaging of Biological Membranes in Liquid, Physical Review Letters, 99 (2007) 046102. [36] M.W. Anthonsen, O. Smidsrød,Hydrogen ion titration of chitosans with varying degrees of
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N-acetylation by monitoring induced 1H-NMR chemical shifts, Carbohydrate Polymers, 26 (1995) 303.
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[37] S. Danielsen, K.M. Vårum, B.T. Stokke,Structural Analysis of Chitosan Mediated DNA
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Condensation by AFM:Ⅴ Influence of Chitosan Molecular Parameters, Biomacromolecules, 5
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d
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(2004) 928.
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Figure captions Fig. 1. The CS structure of CS in acid and alkali solution.
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Fig. 2. AFM images of self-assembled CS nanostructures on mica surface obtained from 0.2 mg/mL CS different solution of pH 4 at 65 °C: (a) CCl4, (b) C6H6, (c) CH2Cl2, (d) CH3CH2OH, (e) CH3OH
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and (f) CH3CN. Scale bare =1250 nm. Vertical scale: (a) 800 nm, (b) 350 nm, (c) 2.0 nm, (d) 14.0
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nm, (e) 5.0 nm and (f) 5.0 nm.
Fig. 3 AFM images of self-assembled CS nanostructures on mica surface obtained from 0.2 mg/mL
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CS/CH3CN of pH 4.0 at different temperature: (a) room temperature, (b) 50 °C, (c) 65 °C and (d) 80 °C. Scale bare =1250 nm. Vertical scale: 4.0 nm for (a, b, d) and 5.0 nm for (c).
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Fig. 4. AFM images of self-assembled CS nanostructures on mica surface obtained from CS/CH3CN with different concentration at pH 4 and 65 °C: (a) 0.02 mg/mL, (b) 0.05 mg/mL, (c)
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0.1 mg/mL, (d) 0.2 mg/mL, (e) 0.3 mg/mL and (f) 0.4 mg/mL. Insets in Fig. 5d-f were their corresponding section analysis. Scale bare =1250 nm. Vertical scale: 2.0 nm for (a), 3.0 nm for (b),
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4.0 nm for (c, e, f) and 5.0 nm for (d).
Fig. 5. (a) The histogram of height and (b) the plot of Rms of self-assembled CS nanostructures on mica surface obtained from CS/CH3CN at pH 4 and 65 °C versus CS concentration. Fig. 6. AFM images of self-assembled CS nanostructures on mica surface obtained from 0.2 mg/mL CS/CH3CN with different pH 4 at 65 °C: (a) pH=2, (b) pH=4, (c) pH=5, (d) pH=6, (e) pH=7 and (f) pH=8. Scale bare =1250 nm. Vertical scale: 4.0 nm for (a, c, d, e, f) and 5.0 nm for (b). Fig. 7. The histogram of height and (b) the plot of Rms of self-assembled CS nanostructures on mica surface obtained from 0.2 mg/mL CS/CH3CN at 65 °C versus pH.
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S0169-4332(15)01541-X http://dx.doi.org/doi:10.1016/j.apsusc.2015.06.193 APSUSC 30714
To appear in:
APSUSC
Received date: Revised date: Accepted date:
9-4-2015 28-6-2015 29-6-2015
Please cite this article as:
*Highlights (for review)
Highlights
Nanocomposites of chitosan film were prepared by simple self-assembly from solvent media. Chitosan molecules assembled on mica surface in the morphology of nanoparticles, fibril and
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membrane with varied chitosan concentration.
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Chitosan molecules assembled with different nanostructure under varied pH.
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ed
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The optimum drying temperature for forming chitosan membrane is about 65 °C.
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Graphical Abstract (for review)
Topographic Characterization of the Self-Assembled Nanostructures of Chitosan on Mica Surface by Atomic Force Microscopy
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Li Wang, Jiafeng Wu, Yan Guo, Coucong Gong and Yonghai Song
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Key Laboratory of Functional Small Organic Molecule, Ministry of Education, Key Laboratory of Chemical Biology, Jiangxi Province, College of Chemistry and Chemical Engineering, Jiangxi
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Normal University, Nanchang, 330022, China.
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Graphical abstract
Corresponding author: Tel/Fax: +86-791-88120861. E-mail: [email protected] (Y. Song).
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*Manuscript Click here to view linked References
Topographic Characterization of the Self-Assembled Nanostructures of Chitosan on Mica Surface by Atomic Force
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Microscopy
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Li Wang, Jiafeng Wu, Yan Guo, Coucong Gong and Yonghai Song∗
Key Laboratory of Functional Small Organic Molecule, Ministry of Education, Key Laboratory of
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Chemical Biology, Jiangxi Province, College of Chemistry and Chemical Engineering, Jiangxi
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Normal University, Nanchang, 330022, China.
Li Wang: [email protected] Jiafeng Wu: [email protected] Yan Guo: [email protected]
Coucong Gong: [email protected]
∗Corresponding author: Tel/Fax: +86-791-88120861. E-mail: [email protected] (Y. Song).
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ABSTRACT In this work, the self-assembled nanostructures of chitosan on mica surface formed from various
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solvents were investigated by using atomic force microscopy. The effects of various factors on the self-assembled nanostructures of chitosan on mica surface, including solvents, the concentration of
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chitosan, the pH of solution and the drying temperature, were explored in detail. Our experimental
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data resulted in the conclusion that chitosan molecules could self-assemble on mica surface to form various nanostructures such as nanoparticles, fibril and film. Nanopartilces were always formed on
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mica surface from CCl4, C6H6, CH2Cl2 solution, fibril preferred to form on mica surface from CH3CH2OH and CH3OH solution and the optimal solvent to form film was found to be CH3CN.
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Low concentration, pH and temperature were helpful for the formation of nanoparticles, medium
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concentration, pH and temperature resulted in fibril and high concentration, pH and temperature
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was often beneficial to forming chitosan films. The study of self-assembled nanostructures of chitosan on mica surface would provide new insight into the development of chitosan-based
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load-bearing materials.
Keywords: Self-assembly; Chitosan; Nanostructures; Mica; Atomic force microscopy
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1. Introduction Chitosan (CS), a copolymer made of N- acetyl- D-glucosamine and D-glucosamine units, joined
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together by β-1,4-glycosidic bonds, is a white, translucent, amorphous solid with pearly luster [1-5], showing attractive properties such as biocompatibility, antibacterial property, biodegradability, low
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toxicity and metal binding ability [6-10]. CS has received a great deal of attention for its broad
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range of applications in biosensors, water treatment, textiles, separation membrane, food package, tissue engineering and drug delivery [11-16], owing to its ability to bind to a variety of natural or
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synthetic molecules such as nucleic acid, protein, polymers, metal ions, halogen and nanoparticles. Accordingly, it is necessary to develop approaches to prepare self-assembled nanostructures of CS
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including nanowires, nanotubes, nanaorods, films, hydrogels and emulsions which could be utilized in numerous fields [17-19].
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CS can form various self-assembled nanostructures based on hydrogen-bond networks in aqueous solutions by controlling local environments such as pH, solvent, temperature, the degree of
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acetylation of CS, types of salt and types of acids [11,12,17-21]. As a hydrophilic biopolymer, CS is a compound with both strong inter- and intramolecular hydrogen bond due to the presence of a large number of free –NH2 and –OH in each unit [13,20-22]. When CS is introduced to acids, amine groups of CS can be protonated, which results in periodic positive charges along the polymer backbone to exhibit noticeable repulsion between CS molecules [16,17]. At basic medium, most of the amine groups are not protonated and the solubility of CS decreases drastically, which leads to noticeable hydrophobic attraction. CS is also a stimulus-responsive polymer with a solubility that can be reversibly alerted by pH changes [17,23]. CS micro- or nanofibers have been widely accepted as biomedical scaffolding materials to restore, maintain, or improve the functions of
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various tissues [24,25]. Recently, Gong et al reported an nonaqueous electrochemical approach to synthesize various one-dimensional CS nanostructures (nanowires, nanotubes, and nanorods) via
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adjusting deposition time, current, and electrochemical potential [18]. Three-dimensional CS nanostructures were also prepared to serve as nanotemplates for subsequent biomineralization of
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calcium carbonate [19]. Lee et al investigated the effects of pH and time on nanostructures of CS by
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using a surface forces apparatus [22]. Yang et al prepared CS/graphene oxide (GO) films by simple self-assembly of CS and GO in aqueous media [20]. Caridade et al produced a thick membrane by
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layer-by-layer assembly of CS and alginate to study the permeation of model drugs and the adhesion of muscle cells [26]. Although CS nanostructures with controllable morphology are highly
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desirable for various application [27,28], very little attention have been focused on the self-assembled nanostructures of chitosan on mica surface, especially the effects of local
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environments on the self-assembled nanostructures of chitosan on mica surface which might be very important to control self-assembled nanostructures..
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In this work, unlike the previous kinetic study and optical microscopic study, CS nanostructures on a mica surface in a controllable fashion were investigated by atomic force microscopy (AFM). A lot of information on the self-assembled nanostructures of chitosan on mica surface was revealed by AFM studies [29-35]. It was found that solvents, concentration, pH and temperature were all key factors for the formation of self-assembled nanostructures of chitosan on mica surface. This approach does not require template, catalysis, or surfactant, and therefore it is ideal for exploring large-scale industrial production of CS nanostructures.
2. Materials and methods
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2.1 Materials. CS with a ≥80% degree of deacetylation (Fig. 1), H2SO4, NaOH, CCl4, C6H6, CH2Cl2,
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CH3CH2OH, CH3OH, and CH3CN were all purchased from Sinopharm Group Chemical Reagent Co. Ltd (Shanghai, China). All reagents (AG) were used as received without any further
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purification. The muscovite mica substrate [KAl2(AlSi3)O10(OH)2 ] were purchased from Linhe
“Here in Fig. 1”
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2.2 Sample preparation.
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Street Commodity Marketplace (Changchun, China).
In a typical experiment, 0.8 mg of CS was dispersed into 4 mL of CH3CN or other solvents
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solution under mild ultrasonication (70 W) for 2 h. The solutions were kept at a pH of ∼4.0 by adding dropwise an appropriate amount of aqueous H2SO4. Then 20 µL of CS solution was applied
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to a freshly cleaved muscovite mica surface (about 1.2×1.2 cm2 square) and incubated at room temperature for about 10 min in a moisture chamber to evaporation. After that, the sample was
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heated at 65 °C in an air oven overnight for complete drying. Finally the dried mica sheet was affixed on a metal disk for AFM imaging. To study the effect of the pH on the chitosan assembly, the assembling solutions were kept at a pH of 2.0, 4.0, 5.0, 6.0, 7.0 and 8.0 by adding dropwise an appropriate amount of aqueous H2SO4 and NaOH. In the temperature controlling experiment, the heating temperature was controlled at room temperature, 50 °C, 65 °C and 80 °C, respectively. 2.3 AFM imaging. The AFM instrument (NanoscopeⅤ, MultiMode®8, BRUKER) was employed in ScanAsyst mode at room temperature under ambient conditions. Standard silicon nitride cantilevers with Al reflective coating (BRUKER Probes, SCANASYST-AIR, spring constant of
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0.4N/m, radius of curvature less than 2 nm) were used. All the AFM data were obtained close to their resonance frequencies of the cantilever (typically, 60-150 kHz). The signal-to-noise ratio was
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maintained higher than 10. The scan rate was 1 Hz for small-area scans (<2 µm) and decreased when larger areas were scanned to ensure image quality. All images were typically obtained from at
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least five macroscopically separated regions on each sample. Changing scan direction did not affect
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the patterns, and there was no other evidence of tip-induced perturbations in the samples. Image analysis and processing were performed with the NanoScope Software 9.0. The AFM images
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presented in this work were raw data, except for minimal post-processing (a simple flatten for eliminating the background effect). Flattening modifies the image in a line-by-line way to remove
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out of the vertical offset between scan lines in the fast scan direction (X at 0° scan angle) by
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3. Results and discussion
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fit from the original scan line.
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calculating a 1st order least-squares fit polynomial for each scan line and subtracting the polynomial
3.1 Effect of solvent on self-assembled nanostructures of CS on mica surface
At first, we investigated the effect of solvent on self-assembled nanostructures of CS on mica surface. After 0.8 mg CS were dissolved in 4 mL of six kinds of different solvents, 20 µL CS solution was dropped onto a freshly cleaved mica surface (dried at 65 °C in air) and used for AFM imaging. And the results were shown in Fig. 2. When CS was dissolved in CCl4, the CS/CCl4 solution self-assembled on mica surface and CS formed some big spherical particles with diameter of about 387.95 nm (Fig. 2a). When CS was dissolved in C6H6, some spherical CS particles with diameter of about 166.28 nm were also observed on mica surface (Fig. 2b), smaller than those 6
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formed from CS/CCl4. As commented above, CS was a polar hydrophilic biopolymer with both strong inter- and intramolecular hydrogen bond, while CCl4 and C6H6 were both nonpolar
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hydrophobic solvents. Thus it was very hard for CS to disperse well in CCl4 and C6H6 solutions. After CCl4 or C6H6 was completely evaporated during drying process, the CS molecules aggregated
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into big CS sphere on mica surface owing to the intermolecular hydrogen bond. Although CCl4 and
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C6H6 were both nonpolar solvents, the polarity of C6H6 was a little stronger than that of CCl4. Thus CS particles obtained from CS/C6H6 were smaller than that obtained from CS/CCl4. In Fig. 2c, the
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morphology of self-assembled CS nanoparticles also appeared on mica surfaces when CS dissolved in CH2Cl2. It was noticeable that the diameter of these CS nanoparticles was about 3.4 nm. The
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polarity of CH2Cl2 was stronger than that of C6H6, thus CS could partially disperse in CH2Cl2 and
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formed smaller CS particle.
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Next, we also investigated three polar solvents including CH3OH, CH3CH2OH and CH3CN. As
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could see form Fig. 2d-f, the self-assembled CS nanostructures was very different due to their different polarity. Fig. 2d showed the morphology of self-assembled CS nanostructures obtained in CH3CH2OH. There was a great deal of CS fragments on mica surface. Some CS fragments were gathered together to form small films. When CS was dissolved in CH3OH, CS fibril networks with about 1.4 nm in height were formed on mica surface (Fig. 2e). When CS was dissolved in CH3CN, flat CS films were self-assembled on mica surfaces (Fig. 2f). The height of these CS films was estimated to be about 1.7 nm. Since CS molecules were well dispersed in all these polar solvent, some fragments, fibrils and films were formed on mica surface. And CH3CN was chosen as the optimum solvent for the following study.
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“Here in Fig. 2”
3.2 Effect of temperature on self-assembled nanostructures of CS on mica surface
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The evaporated rate of solvent and the movement rate of CS mica surface might affect
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self-assembled nanostructures of CS on mica surface and they strongly depended on the temperature. Thus, the effect of temperature on self-assembled nanostructures of CS on mica
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surface was also investigated in this work. Fig. 3a showed the representative AFM images of CS
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self-assembled nanostructures on mica dried at room temperature (0.2 mg/mL, pH=4.0). Some uniform CS nanoparticles were observed to be distributed over the substrate. Section analysis
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indicated that the heights of CS nanoparticles were about 1.7 nm for those big one and 0.6 nm for those small one, respectively. At low temperature, molecule movement was very slow. Most of CS
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molecules had not been thoroughly spread all the surface of mica, which led to the aggregation of
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CS into many small nanoparticles. Fig. 3b displayed self-assembled CS nanostructures obtained at
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50 °C, and uniform CS nanoparticles were observed on mica surface. Section analysis indicated that the heights of CS nanoparticles were about 1.5 nm. As the drying temperature of CS/CH3CN solution was further increased, the CS molecule movement was more and more quick. CS molecules could well spread all the surface of mica. Consequently, many smaller spherical particles in the same scale were self-assembled on mica surface. When the CS/CH3CN solution was dried at 65 °C, some incomplete CS films were formed. A higher movement rate of CS on mica surface was induced by an increased drying temperature, which prompted the self-assembly rate of CS directly and resulted in the film architecture (Fig. 3c). Upon a increase of the drying temperature to 80 °C, many small CS films were formed in Fig. 3d. Evidently, the higher temperature also resulted in
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large evaporated rate of solvent, and accordingly CS molecules could not well spread all the surface of mica. As a result, some little CS films formed. Thus 65 °C was chosen as the optimum drying
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temperature for the following study.
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“Here in Fig. 3”
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3.3 Effect of CS concentration on self-assembled CS nanostructures on mica surface
To investigate the effect of CS concentration on self-assembled CS nanostructures on mica
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surface, the CS/CH3CN solutions with different concentrations were firstly dropped onto the surface
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of freshly cleaved mica and dried at 65 °C in air. Then the samples were characterized by AFM and the results were shown in Fig. 4. At 0.02 mg/mL (Fig. 4a), only a few CS nanoparticles appeared on
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the mica surface. At 0.05 mg/mL (Fig. 4b), the mica surface was almost covered with CS
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nanoparticles. With the increase of CS concentration, the morphology of the assembled CS
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nanostructures changed gradually. Some small thin CS films appeared, and finally large thin CS films were observed. At 0.1 mg/mL (Fig. 4c), no CS nanoparticle was observed, but many small CS films with defects were formed on mica surface. The small CS films with different shapes and sizes were consisted of CS fibrils whose average thickness was estimated to be about 1.65 nm. Continuing to increase the CS/CH3CN concentration to 0.2 mg/mL (Fig. 4d), the formed CS films became larger and more compacted and only few holes were observed in these CS films [18,20,22]. In Fig. 4e, the self-assembled CS films on mica surface were connected with one another to form large CS films at 0.3 mg/mL. The complete CS films covered the whole mica surfaces when the concentration of CS/CH3CN was further increased to 0.4 mg/mL. There were many holes in the CS films. Section analysis (insets in Fig. 4d-f) indicated that the height of the films in Fig. 4d-f was 9
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about 1.6 nm, 1.9 nm and 2.1 nm, respectively (also shown in Fig. 5a). The plot of root mean square (Rms) roughness of CS films assembled on mica surface at pH 4 and 65 °C versus CS concentration
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was shown in Fig. 5b. The result clearly indicated that the Rms increased gradually as the CS concentration increased from 0.02 mg/mL to 0.4 mg/mL.
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At pH=4, the amino groups of CS were completely protonated and accordingly the inter- and
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intramolecular hydrogen bonds were very weak, which resulted in good dispersion of CS molecules in CH3CN solution. At low concentration of CS, the rapid removal of CH3CN originated from the
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high drying temperature of 65 °C led to the formation of CS nanoparticles on mica surface under
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the aid of the strong electrostatic attraction interaction between positively charged CS and negatively charged mica surface and the weak intermolecular hydrogen bonds. As the concentration
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of CS increased, the intermolecular hydrogen bonds were enhanced gradually. Under the help of the
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intermolecular hydrogen bonds, the CS molecules connected together gradually to form CS films
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when they were assembled on mica surface. Finally, a complete CS formed at high concentration. However, owing to the electrostatic repulsion between positively charged CS molecules, a large number of pores also formed in the CS film.
“Here in Fig. 4” “Here in Fig. 5”
3.4 Effect of pH on CS nanostructures on mica surface
CS was known as a stimulus-responsive polymer which could be reversibly alerted by changing pH. In order to further investigate the pH effect on CS/CH3CN assembly on mica surface, six 10
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different pHs from 2.0 to 8.0 of CS/CH3CN solutions were employed in the following experiments at the same other conditions (concentration of CS and temperature) and the results were shown in
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Fig. 6. In these experiments, the concentration of CS/CH3CN solutions was 0.2 mg/mL and drying was carried out at 65 °C in air. At pH 2.0 (Fig. 6a), many CS fibrils formed on mica surface and
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they were connected together to form small CS films with many big pores. At pH 4.0, the CS films
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became larger and the holes became smaller as shown in Fig. 6b. With the increase of pH of CS/CH3CN solution, the self-assembled CS nanostructures were changed greatly. At pH 5.0 (Fig.
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6c), the fragments of CS films connected together to form large CS film with many holes. At pH 6.0 and 7.0 (Fig. 6d and e), the CS films became more compacted and complete, and the holes
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obviously decreased. At pH 8.0 (Fig. 6f), a complete CS film covered the whole mica but a great many of round holes formed. Section analysis (inset in Fig. 6b-f) indicated that the heights of CS
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films in Fig. 6b-f were about 1.6 nm, 1.3 nm, 1.2 nm, 1.3 nm and 1.3 nm, respectively (also shown in Fig. 7a). The plot of Rms of CS films assembled on mica surface at 65 °C versus pH was shown
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in Fig. 7b. The result showed that the Rms decreased gradually as the pH increased and then increased at pH 8.0 suddenly.
Generally, the protonation/deprotonation and inter- and intramolecular hydrogen bond of CS depended on pH of solution due to a large number of free –NH2 and –OH in each unit [13,17,22]. The pH-responsive behaviors of self-assembled CS nanostructures on mica surface could be understood by using pH-stimulated alterations of conformation and charging state of CS. At low pH (pH<4), the amino groups of CS were protonated to gave a large number of positive charges and accordingly the inter- and intramolecular hydrogen bonds were very weak. The positively charged CS molecules were highly extended and dispersed under electrostatic repulsion in a solution, so CS 11
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molecules formed fibrils and small thin CS films on mica surface based on the strong electrostatic attraction interaction between positively charged CS and negatively charged mica surface and the
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weak inter- and intramolecular hydrogen bonds. At middle pH (4
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partial inter- and intramolecular hydrogen bonds formed. The small number of positive charges in
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CS molecules resulted in the weak interaction between CS and negatively charged mica surface and accordingly the CS molecules could rapidly remove on mica surface when they were assembled on
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mica surface. Under the help of the strong inter- and intramolecular hydrogen bonds, a large and compacted film of CS formed on mica surface. As the pH further increased, the CS molecules
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become completely deprotonated and the interaction between CS and negatively charged mica surface was minimized. At the same time, intermolecular association (hydrogen bonding) of CS
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molecules became dominant, and accordingly the films of CS mica became larger and larger (pH>8). Besides, the solubility of CS decreased when the pH exceeded 8 because the pKa value of CS was
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around 6.5-7.0 [36,37]. Thus only a little CS molecules on mica could be observed.
“Here in Fig. 6” “Here in Fig. 7”
4. CONCLUSION
In conclusion, we investigated the self-assembled CS nanostructures on mica surface using AFM. Our data demonstrated that the solvents, CS concentration, temperature and pH of the CS solution were all key factors for the formation of self-assembled CS nanostructures on mica surface. The polarity of solvents played a vital role in the formation of self-assembled CS nanostructures on mica
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surface and the strong polar CH3CN could lead to formation of flat CS films and weak polarity resulted in the fibrils and nanoparticles. The optimum drying temperature to form CS films was 65
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°C. To obtain a complete and compacted CS films, 0.2 mg/ml CS concentration was needed. CS was a stimulus-responsive polymer that could form different nanostructures by changing pH
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changes and the suitable pH was 7.0 for the CS films. This approach need not require template,
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catalysis or surfactant, therefore it is ideal for exploring large-scale industrial production of thin CS
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film and this study might shed some new light on the formation of CS thin film.
ACKNOWLEDGEMENT
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This work was financially supported by National Natural Science Foundation of China (21165010, 21465014 and 21465015), Natural Science Foundation of Jiangxi Province
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(20142BAB203101), The Ministry of Education by the Specialized Research Fund for the Doctoral Program of Higher Education (20133604110002) and the Ground Plan of Science and Technology
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Projects of Jiangxi Educational Committee (KJLD14023) and the Open Project Program of Key Laboratory of Functional Small Organic Molecule, Ministry of Education, Jiangxi Normal University (No. KLFS-KF-201410; KLFS-KF-201416).
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Figure captions Fig. 1. The CS structure of CS in acid and alkali solution.
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Fig. 2. AFM images of self-assembled CS nanostructures on mica surface obtained from 0.2 mg/mL CS different solution of pH 4 at 65 °C: (a) CCl4, (b) C6H6, (c) CH2Cl2, (d) CH3CH2OH, (e) CH3OH
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and (f) CH3CN. Scale bare =1250 nm. Vertical scale: (a) 800 nm, (b) 350 nm, (c) 2.0 nm, (d) 14.0
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nm, (e) 5.0 nm and (f) 5.0 nm.
Fig. 3 AFM images of self-assembled CS nanostructures on mica surface obtained from 0.2 mg/mL
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CS/CH3CN of pH 4.0 at different temperature: (a) room temperature, (b) 50 °C, (c) 65 °C and (d) 80 °C. Scale bare =1250 nm. Vertical scale: 4.0 nm for (a, b, d) and 5.0 nm for (c).
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Fig. 4. AFM images of self-assembled CS nanostructures on mica surface obtained from CS/CH3CN with different concentration at pH 4 and 65 °C: (a) 0.02 mg/mL, (b) 0.05 mg/mL, (c)
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0.1 mg/mL, (d) 0.2 mg/mL, (e) 0.3 mg/mL and (f) 0.4 mg/mL. Insets in Fig. 5d-f were their corresponding section analysis. Scale bare =1250 nm. Vertical scale: 2.0 nm for (a), 3.0 nm for (b),
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4.0 nm for (c, e, f) and 5.0 nm for (d).
Fig. 5. (a) The histogram of height and (b) the plot of Rms of self-assembled CS nanostructures on mica surface obtained from CS/CH3CN at pH 4 and 65 °C versus CS concentration. Fig. 6. AFM images of self-assembled CS nanostructures on mica surface obtained from 0.2 mg/mL CS/CH3CN with different pH 4 at 65 °C: (a) pH=2, (b) pH=4, (c) pH=5, (d) pH=6, (e) pH=7 and (f) pH=8. Scale bare =1250 nm. Vertical scale: 4.0 nm for (a, c, d, e, f) and 5.0 nm for (b). Fig. 7. The histogram of height and (b) the plot of Rms of self-assembled CS nanostructures on mica surface obtained from 0.2 mg/mL CS/CH3CN at 65 °C versus pH.
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