Synthesis and Stabilization of Gold Nanoparticles Induced by

May 9, 2013 - Metal nanoparticles, especially gold nanoparticles (AuNPs), have attracted great attention over the past decades due in part to specific...
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Synthesis and Stabilization of Gold Nanoparticles Induced by Denaturation and Renaturation of Triple Helical β‑Glucan in Water Xuewei Jia, Xiaojuan Xu,* and Lina Zhang Department of Chemistry, Wuhan University, Wuhan 430072, China ABSTRACT: We report on a green procedure for the synthesis and stabilization of gold nanoparticles (AuNPs) from chlorauric acid (HAuCl4) with the use of a β-glucan known as Lentinan (LNT) without external reducing or stabilizing agents in aqueous medium. LNT adopted triple helical conformation in water, which was first denatured into single chains (s-LNT) at a high temperature of 140 °C before mixing with HAuCl4. Results from UV−vis absorption spectroscopy, transmission electron microscopy (TEM), and energy dispersive X-ray (EDX) spectra suggested that AuCl4− was rapidly reduced to AuNPs by s-LNT. Moreover, the as-prepared AuNPs could be converted into nanobelt, spherical nanoparticles, and nanowire morphology simply by controlling the s-LNT concentration, reaction time, and temperature. In particular, the AuNPs nanowire was confirmed as the most stable shape in water, which was predominately ascribed to the hydrophobic cavity in the helical center of the renatured triple helical LNT (r-LNT) from s-LNT. Namely, AuNPs were entrapped in the hydrophobic cavity of r-LNT to form nanowire with an outer layer of water-soluble r-LNT, leading to stable dispersion of AuNPs. All the data demonstrated that the β-glucan of s-LNT can be used as a reducing and stabilizing agent to synthesize and disperse AuNPs in water. The whole process of reduction and stabilization was free of organic solvent and thus very safe, which is important for the potential application of AuNPs in biotechnology and biomedicine.

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INTRODUCTION Metal nanoparticles, especially gold nanoparticles (AuNPs), have attracted great attention over the past decades due in part to specific chemical and physical properties such as optics, photonics, catalytic activity, nanostructure fabrication, chemical/biochemical sensing, special stability, and bioaffinity, leading to the wide range of applicability in catalysis, chemical/biochemical sensing, photonics, and biotechnology.1−10 AuNPs have genernally been synthesized by various methods such as wet chemical reduction,11 thermolysis,12 femtosecond laser ablation,13 UV irradiation,14 ultrasonolysis,15 rediolysis,16 and two-phase reaction method.17 Of these, wet chemical reduction (e.g., the synthesis of AuNPs by the citrate reduction of Au salt in aqueous media under boiling conditions or seed-mediated growth) is the most widely used.18−21 As summarized in the literature,22 the majority of the methods reported to date on the synthesis of metal nanoparticles use reducing agents such as hydrazine, sodium borohydride (NaBH4), and dimethyl formamide (DMF), which are highly reactive chemicals and pose potential environmental and biological risks. Much effort has thus been devoted to developing “green” methods for the feasible synthesis of metal nanoparticles, including gold nanoparticles. For instance, Raveendran et al. reported on a simple and green method through which metal nanoparticles, including Au, Ag, and Au− Ag alloy nanoparticles, were synthesized by glucose as a reducing agent.22,23 In addition, Huang et al. used a polysaccharide of chitosan to successfully prepare gold nanoparticles.24 More recently, a procedure for the formation © XXXX American Chemical Society

of colloidal gold nanoparticles derived from the supramolecular self-assembled structure of a cyclodextrin (CD)/Au salt complex was presented.25,26 It is well-known that metal nanoparticles have high surface energy that ensures extreme reactivity and easy aggregation without protection or passivation of their surfaces. Various approaches have thus been developed to prevent the nanoparticles from aggregation including protection by selfassembled monolayers,27 encapsulation by adding capping agents,28 and dispersion in polymeric matrixes.29 Biomacromolecule-stabilized nanoparticles are especially interesting due to their potential biological activities and excellent biocompatibilities. Very recently, we successfully used a water-soluble hyperbranched polysaccharide as a capping agent to disperse Se nanoparticles.30 Three points which were used to evaluate the environmental friendliness of the synthetic method, including the solvent system used for synthesis, the benign reducing agent, and the nontoxic material used for stabilization of the nanoparticles.22 In this instance, β-D-glucose was used as the reducing agent and starch as the stabilizer to synthesize and stabilize the gold nanoparticles.23 However, Chung et al. have demonstrated a synthetic method for CD-stabilized gold nanoparticles in an aqueous medium derived from the supramolecular self-assembled structure of a CD/Au salt complex using thermal treatment without external reducing Received: February 4, 2013 Revised: May 9, 2013

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Figure 1. (a) UV−vis absorption spectra of the aqueous mixture of HAuCl4 (0.1 mg/mL) and t-LNT or s-LNT (3.0 mg/mL). The inset images are the photos of the corresponding mixture. “0” stands for only HAuCl4 in the vial, “t-LNT” stands for HAuCl4, which was reduced by t-LNT at 100 °C for 30 min, and “s-LNT” signifies the reduction of HAuCl4 by s-LNT at 100 °C for 30 min. (b, c) TEM images of AuNPs reduced by t-LNT and sLNT at 100 °C for 30 min, respectively.

agents.25,26 To be precise, CD was proved suitable as both a reducing agent and a capping agent. β-Glucans have also been used as protecting agents in the preparation of metal nanoparticles such as chitosan and schizophyllan,31,32 among others. It is well-known that monosaccharides possess a free aldehyde or ketone group with reducibility, whereas polysaccharide has a free aldehyde or ketone group at the end of the long chain showing little reducibility. Generally speaking, polysaccharide without free aldehyde or ketone groups tends to have nonreducibility qualities. It has been reported that ethylene glycol can reduce PtCl2 or AgNO3 to Pt or Ag nanoparticles at ∼160 °C,33 suggesting that hydroxyl (−OH) groups have clear reducibility. Although polysaccharide has many −OH groups, reports of its reducibility are scarce. There is a clearly a great need for further investigation into the potential of polysaccharides for use as a mild reducing agent and a stabilizer that can reduce gold ion to generate and stabilize AuNPs in aqueous medium. Lentinan (LNT) is a neutral β-(1,3)-D-glucan from the fruiting bodies of Lentinus edodes, which possesses two β-(1,6)glucoside branches for every five β-(1,3)-glucoside linear linkages.34 It adopts a triple helical conformation (coded as tLNT) in aqueous solution and single chains (coded as s-LNT) in dimethyl sulfoxide (DMSO) or at a temperature higher than 130 °C.35−38 In addition, s-LNT also has the capacity to be renatured into a new triple helix (coded as r-LNT).39,40 It has been shown that t-LNT can be used as a protecting agent to stabilize Ag nanoparticles in water through the interaction between hydroxyl and Ag nanoparticles.41 In the present study, LNT was not just highlighted as a stabilizer but also as a mild reducing agent for the synthesis of gold nanoparticles. Moreover, the whole reaction was effectively performed in an aqueous medium with gentle heating. This work will therefore prove valuable in providing an alternative, green, and simple method for the synthesis and stabilization of AuNPs, while avoiding a need for additional reducing or capping agents.

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Preparation of a Single Chain of t-LNT. The sample t-LNT was dissolved in the deionized water and then heated at 140 °C for half an hour to break the intra- and intermolecular hydrogen bonds supporting the triple helical structure. A single chain of t-LNT (coded as s-LNT) was thus obtained in accordance with our previous work;37 this was immediately used in the subsequent experiments. Preparation of the Reduced Au from HAuCl by s-LNT. The aforementioned freshly prepared s-LNT aqueous solution was stirred continuously in a temperature-controlled oil bath with concentrations ranging from 1.0 to 10.0 mg/mL. This was maintained until the solution was stabilized at the desired reaction temperatures (100 °C or room temperature). Meanwhile, a t-LNT/water solution with a concentration of 6.0 mg/mL was also prepared by directly dissolving tLNT in water at room temperature. An equal volume of HAuCl4 (0.2 mg/mL) was rapidly added into the s-LNT or t-LNT aqueous solutions. The mixed solutions were kept at the reaction temperature with stirring for the desired time until the reduced Au was delivered for subsequent use in the following characterization. Characterization. The reduction of Au3+ was examined by UV− vis absorption spectrometry (UV-6100PCS) in the range of 200−700 nm with 1 nm resolution. Quartz cuvettes with an optical length of 1 cm were used. The morphologies of the reduced Au were observed by transmission electron microscopy (TEM, JEM-2010HT) and highresolution transmission electron microscopy (HRTEM, JEM2010FEF) operating at 200 kV. Samples for TEM observation were obtained by dropping the reaction solutions of 2 μL onto carboncoated copper grids without any purification or inspissation. A filter paper was used for rapid removal of the liquid. The reaction solution was dispersed onto a V3 TEM grid with a holey carbon support film to obtain the energy dispersive X-ray (EDX) spectroscopy.

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RESULTS AND DISCUSSION Reduction of Au3+ to Au by β-Glucan. It is well-known that size, shape, and surface morphology play a key role in controlling the physical, chemical, optical, and electronic properties of metal nanoparticles. Nanoparticles with different shapes appear in different colors that can be used as a visualized evidence to judge the shape of the nanoparticles formed. For example, metallic gold is golden yellow, while spherical AuNPs have a visible red wine color and gold nanorods are blue (aspect ratio 2−3) or black (aspect ratio 3) in solution.41−43 Initial assumptions about the incidence of a reaction could therefore be made through the color changes in the solutions. As shown in the inset images of Figure 1a, the HAuCl4 aqueous solution was colorless, while the solutions containing s-LNT or t-LNT (3.0 mg/mL) without any precipitates displayed red wine and purple coloring, respectively, indicating that Au3+ was reduced to Au by s-LNT and t-LNT. As suggested by the coloring changes in the solution, strong and broad surface plasmon resonance (SPR) bands, which resulted from the collective

EXPERIMENTAL SECTION

Materials. Lentinan (LNT), one branched β-(1→3)-D-glucan,34 was isolated from the fruiting bodies of Lentinus edodes by extraction with 1.25 M NaOH/0.05% NaBH4. The detailed procedures have previously been reported.35 As described in the Introduction, LNT occurs as a triple helical chain in water and so it was herein coded as tLNT. The weight-average molecular weight (Mw) of t-LNT was determined as 8.0 × 105 by laser light scattering. Chlorauric acid (HAuCl4) was purchased from Shanghai Chemical Reagent and used without any additional purification. Deionized water (Millipore) was utilized in all of the following experiments. B

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the AuNPs. These results verified the reduction and stabilization of Au3+ ions by s-LNT, which was achieved without external reducing reagent and stabilizer even at the low concentration of 0.5 mg/mL. As expected based on the color changes of the solutions with different s-LNT concentrations, the different shapes and sizes of the reduced Au were confirmed by TEM observation as shown in Figure 3. Interestingly, a belt-like shape with the

oscillations of the conduction band electrons, were observed within the 500−600 nm range, indicating good dispersion of Au nanoparticles (AuNPs) (Figure 1a). These results verified the formation of AuNPs and subsequent stabilization by β-glucans, including s-LNT and t-LNT. This was achieved without external reducing reagent or stabilizer, indicating that s-LNT or t-LNT can be used as a reducing agent and a stabilizer of metal nanoparticles as predicted. There were huge variations between the colors of the reduced AuNPs by s-LNT and t-LNT solutions, which signified AuNPs with different size and shape. Figure 1b and c illustrates the TEM images of AuNPs with sLNT and t-LNT as reducing agents, respectively. AuNPs were seen to adopt spherical morphology in both cases. Compared with the fluctuating size of the Au reduced by t-LNT, AuNPs from the s-LNT solution showed a more uniform size. In particular, the wine red coloring indicated the formation of well-dispersed spherical AuNPs.42,43 Moreover, the color change of the AuNPs in s-LNT was observed within 30 s, which was much faster than the change seen in t-LNT, indicating that s-LNT has a higher reaction rate than t-LNT. Based on these results, it was concluded that s-LNT is the most favorable reducing and stabilizing agent, and thus s-LNT was used in the following experiments to investigate the effects of reaction conditions such as s-LNT concentration, reaction temperature, and reaction time during the reduction of Au3+ and Au morphology. Effect of s-LNT Concentration on the Reduction of Au3+ and Morphology of the Reduced Au. As shown in Figure 2, after mixing with HAuCl4 and reacting for 15 min at

Figure 3. TEM images of AuNPs obtained at 100 °C for 15 min at different s-LNT concentrations of (a) 0.5, (b) 2.5, and (c) 5.0 mg/mL. (d) Size distribution of AuNPs in (c).

width of 10−15 nm (which was termed nanobelt) occurred at the low s-LNT concentration of 0.5 mg/mL (Figure 3a). With an increase in s-LNT concentration, the Au nanobelt was partially broken, resulting in the appearance of inerratic spherical or polygonal nanoparticles (Figure 3b). At the high s-LNT concentration of 5.0 mg/mL, spherical particles with an average size of ∼18 nm were observed (Figure 3c,d), while the belt-like shape completely disappeared. Only ∼5% of the spherical particles showed an unusally large size of ∼40 nm, suggesting that this simple method prepared relatively uniform AuNPs. The shape changes closely corresponded to the reported color transitions of the reaction solutions described above. More specifically, the very light blue at the low s-LNT concentration corresponded to the Au nanobelt, while the wine red at the high concentration indicated the occurrence of spherical AuNPs. This essentially demonstrated that the size and shape of the reduced Au can be controlled through changes in the concentration of s-LNT. Moreover, a higher s-LNT concentration was shown to lead to more AuNPs with spherical shape and uniform size form. Effect of Time on Au Synthesis and Morphology. As shown in Figure 3a, an Au nanobelt formed within 15 min at the low concentration of 0.5 mg/mL, implying that the reduction of Au3+ by s-LNT occurred very rapidly. To further investigate this assumption, the next component of our research focused on the influence of reaction time on the formation and morphology of Au. As shown in the inset images of Figure 4a, the aqueous solution with a s-LNT concentration of 0.5 mg/ mL was seen to change color from firt achromaticity to light purple and then to red purple all within the time scale of 5 to 60 min, indicating a continuous evolution of AuNPs in aqueous media.18,20,21,44 The UV−vis absorption spectra also verified the reduction of Au3+ and growth of the AuNPs, as shown in

Figure 2. Concentration-variant photo images and UV−vis absorption spectra of the aqueous mixture of s-LNT (four concentrations as indicated in the figure) and HAuCl4 (0.1 mg/mL). After stirring the mixture for 15 min at 100 °C, the aqueous solution was immediately used for photo-taking and UV−vis measurements.

100 °C, the solutions without precipitates displayed different colors that correlated with increasing s-LNT concentration. When cs‑LNT was 0.5 mg/mL, light blue was observed after 10 min. When cs‑LNT was 1.0 mg/mL, very light purple appeared after 5 min. When cs‑LNT was increased to 2.5 mg/mL, a lavender color appeared after 2−3 min, and finally, red purple occurred. Moreover, the solution color immediately changed to wine red following the addition of HAuCl4 solution at cs‑LNT of 5.0 mg/mL. Evidently, increasing s-LNT concentration led to more rapid color changes, which was indicative of a more rapid reduction. Additionally, strong and broad SPR bands within a 500−600 nm range were also observed as shown in Figure 2. These were seen to shift to lower wavelengths with increasing sLNT concentration, which was indicative of the size changes in C

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Figure 4. UV−vis absorption spectra (inset images: solution color at different reaction times; the arrow indicates the direction of increasing time) (a) and TEM images of AuNPs obtained at 100 °C with cs‑LNT of 0.5 mg/mL for reaction times of (b) 5, (c) 10, (d) 15, (e) 30, and (f) 60 min. Image (g) stands for the AuNPs formed in (d), which were placed at room temperature for 30 days and then used for TEM observation.

Figure 5. TEM images (inset image: solution color of AuNPs at different reaction times) of AuNPs prepared at 100 °C for 30 s (a) and 10 min (b) with cs‑LNT of 5.0 mg/mL, and UV−vis absorption spectra of AuNPs at cs‑LNT of 5.0 mg/mL for different reaction times (c). The TEM samples were obtained by dropping the reaction mixture immediately without any further treatment after reaction for the desired time.

were not observed. More interestingly, when the mixture of sLNT and HAuCl4 was reacted at 100 °C for 15 min and then kept at room temperature for 30 days, the original Au nanobelt, as seen in Figure 4d, evolved to spherical AuNPs, as shown in Figure 4g. More specifically, increased storage time caused further dispersion in the Au nanobelt into spherical AuNPs. It can thus be concluded that the shape of Au reduced by s-LNT may also be modifiable through changes in the reaction time. The influence of time on Au synthesis at the high s-LNT concentration of 5.0 mg/mL was also investigated. As shown in Figure 5a, a 30 s reaction produced a color change in the solution to red purple and the formation of a small amount of spherical AuNPs with a diameter of ∼20 nm together with some aggregates (∼30 nm). With increasing reaction time, the

Figure 4a. The strong SPR band was seen to gradually develop and shift to lower wavelengths with time after mixing s-LNT with HAuCl4 at 100 °C. Moreover, the 220 nm band assigned to the charge transfer of the ion of AuCl4− gradually decreased, while the 280 nm band gradually increased. This was attributed to the long pair interaction between the hydroxyl groups of βglucan and the gold surface.26,45−48 The gradual size and shape evolution of the reduced Au is clearly demonstrated in Figure 4b−f. During the period of 5− 30 min, an Au nanobelt with a diameter of 10−20 nm was observed together with a small amount of substantially sized aggregates, further confirming the occurrence of rapid reduction. After a 60 min reduction, spherical AuNPs with a diameter of 10−20 nm were predominant, while nanobelts D

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Figure 6. UV−vis absorption spectra of AuNPs prepared at room temperature for different reaction times (a), TEM image (b), and EDX spectra (c) of AuNPs obtained at room temperature after a 24 h reaction. The final s-LNT concentrations and HAuCl4 were 5.0 and 0.1 mg/mL, respectively, in (a), (b), and (c).

spectra of AuNPs obtained at room temperature after a 24 h reaction where an oxygen peak occurred. This suggests that Au stabilized through the formation of a composite with LNT. To verify this assumption, the reaction solution with cs‑LNT of 5.0 mg/mL that reacted for 10 min at 100 °C was kept at room temperature for 7 days and then observed with TEM. As shown in Figure 7a, a uniform linear arrangement of AuNPs was

solution color changed to red wine and further AuNPs formed (Figure 5b). The size of the AuNPs also became more uniform. Similarly, the SPR band appeared at 538 nm in less than 30 s. With an increase in reaction time, the intensity of the absorption peak increased and the maximum absorption of the SPR band showed a small blue-shift (Figure 5c), indicating that the number of AuNPs had increased and the morphology had become more smooth and regular. It was thus concluded that a higher concentration of s-LNT will lead to reduced time for the formation of AuNPs with spherical shape and uniform size. Effect of Temperature on Au Synthesis. It has previously been reported that the cyclodextrin (CD)/Au salt complex requires heating to produce Au seed for the growth of AuNPs. It has been shown that AuNPs can not be formed without heating, and black precipitates will form in their place.25 To investigate the necessity of thermal treatment in the present case, s-LNT (10.0 mg/mL) was mixed with equal volumes of HAuCl4 (0.2 mg/mL) at room temperature for the desired time, as indicated in Figure 6. This was then used for UV−vis and TEM observations. After a 24 h incubation, amaranth was observed in the mixture solution, revealing the formation of AuNPs as shown in the inset images of Figure 6a. As described above, a purple red color appeared after 30 s at 100 °C, suggesting that the thermal treatment accelerated the formation of spherical AuNPs but not the necessary condition. The UV−vis spectra (Figure 6a) confirmed the formation of AuNPs as a result of the SPR bands between 500 and 600 nm after a 24 h reduction. Figure 6b illustrates the prevalence of spherical AuNPs sequentially arranged in one direction to form a wire-like shape (which we have termed nanowire), which was similar to the shape of t-LNT reported in our previous work.37 It was also greatly different from the Au nanobelts shown in Figures 3 and 4. This distinction would be discussed further in the next section. In contrast to the method of Chung,25 thermal treatment proved unnecessary for the production of Au seed and the synthesis of Au. The thermal treatment and room temperature conditions only differentiated slightly, in that thermal treatment could accelerate the formation of spherical AuNPs. Stabilization of AuNPs in Aqueous Medium. Stable dispersion of AuNPs in aqueous medium is very important for applications in biotechnology. As illustrated in Figures 1, 2, and 4−6, the reaction solutions without any further treatments were very transparent, which was indicative of the good dispersion of AuNPs in aqueous medium. It was thus acknowledged that the nanobelts, nanowires, and the individual spherical nanoparticles were all dispersed in water without any visible precipitates. It has been reported that AuNPs can be stabilized by CD through capping on the surface of AuNPs.25,26 Figure 6c shows the EDX

Figure 7. (a, b) TEM images of AuNPs; (c) the typical EDX spectrum from HRTEM (b); and (d) TEM images of AuNPs in DMSO. AuNPs were obtained under conditions of the final cs‑LNT of 5.0 mg/mL and the reaction time of 10 min, which was kept at room temperature for 7 days. They were then used to prepare TEM samples for (a); (b) is the enlarged image of (a), and the inset image in (b) is the HRTEM images of (b). DMSO was added into an AuNPs/water solution, and the supernatant after centrifugation was used to prepare a sample for TEM observation in (d).

displayed (herein also termed nanowire) similar to that shown in Figure 6b. The HRTEM image of an Au nanowire in Figure 7b clearly shows the sequential regular linear arrangement of AuNPs with a relatively uniform size of less than ∼15 nm. The EDX photographs (Figure 7c) for the microarea of the Au nanowire shown in Figure 7b also displayed the characteristic absorption peaks of the three elements C, Au, and O, indicating that the Au nanowire was composed of Au and β-glucan. Namely, a composite consisting of AuNPs and LNT formed in the aqueous solution. In contrast to the aforementioned results, Figure 5b suggests that most of the AuNPs were randomly distributed. However, Au nanowire formed following storage for 7 days (Figure 7a). The rationale behind both the linear arrangement of AuNPs and the formation of nanowire after storage was deliberated. It E

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as-prepared AuNPs/r-LNT, AuNPs/s-LNT, and AuNPs/tLNT solutions, as shown in the left pictures of Figure 8a1,

has been reported and widely accepted that the 2-OH side of the glucose unit is hydrophobic, whereas the 6-OH side of branch is hydrophilic.49 The triple helical structure of β-(1, 3)D-glucans such as schizophyllan, curdlan, and LNT may thus be stabilized by the hydrogen-bonding network among the hydrophobic 2-OH groups formed inside the helical center.49−51 Therefore, one may presume that the inside hollow center is considerably hydrophobic. Additionally, the single chains of schizophyllan could be reassociated into the triplex with a controllable hydrophobic cavity that fits with the size of the added guest molecules. For example, the singlewalled carbon nanotubes (SWNTs) could be entrapped in the hydrophobic cavity of the reformed triplex of schizophyllan resulting in good dispersion of SWNTs in water.52 In our previous work,34,40 it was reported that s-LNT can also be rapidly renatured into triple helix (called r-LNT) in water at room temperature with a concentration of higher than 0.4 mg/ mL. In this work, the s-LNT concentration (5.0 mg/mL) utilized was far higher than 0.4 mg/mL. It was thus proposed that the s-LNT also renatured into triple helical r-LNT with hydrophobic cavity, even in the presence of hydrophobic guest molecules of AuNPs. AuNPs would have been entrapped in the cavity with the aid of the hydrophobic force to provide a nanocomposite of AuNPs/r-LNT with a unique one-dimensional structure as observed in Figure 6b. To confirm this assumption, we added ample DMSO to an AuNPs/r-LNT solution with the aim of breaking the triple helix. This was followed by the appearance of black Au precipitates at the bottom of the vial as shown in the inset image of Figure 7d. The solution color was also seen to change from wine red (Figure 7a) to achromaticity (Figure 7d). In the supernatant, only a few clusters composed of spherical AuNPs were observed as shown in the TEM images of Figure 7d. It is widely accepted that DMSO has strong polarity that has the capacity to break hydrogen bonds in polysaccharides and interact with the hydroxyl groups.53 It can therefore be assumed that DMSO added in the AuNPs/r-LNT solution cleaved the hydrogen bonds in the reformed triple helical structure of rLNT, causing the hydrophobic cavity to disappear. Moreover, DMSO interacted with the hydroxyl groups of r-LNT, leading to complete exposure and final sedimentation of AuNPs in aqueous medium. We thus deduced that AuNPs were mainly stabilized via the hydrophobic interaction in the reformed hydrophobic cavity of r-LNT. As in our previous work,41 Ag nanoparticles were stabilized in t-LNT aqueous solution through interactions between Ag nanoparticles and the hydroxyl groups outside the helical center of t-LNT. It was questioned if AuNPs were indeed stabilized by r-LNT through the interaction between AuNPs and the hydroxyl group of r-LNT. Studies have shown that the ability of different ligands to bind to AuNPs widely varied as RSH ≈ RNH2 ≈ R3P ≈ RSiH3 > RI > ROH ≈ RBr.54 To verify our assumption, dodecanethiol was thus used to elucidate the interaction between AuNPs and r-LNT. Three AuNPs/LNT aqueous solutions were first prepared as follows: s-LNT solution was obtained as described in the Experimental Section and then reacted with HAuCl4 at 100 °C for 30 min before being maintained at room temperature for 3 days to obtain AuNPs/r-LNT (cs‑LNT = 3.0 mg/mL) and AuNPs/s-LNT (cs‑LNT = 0.5 mg/mL) solutions. The AuNPs/t-LNT solution was obtained by directly mixing t-LNT and HAuCl4 at 100 °C (ct‑LNT = 3.0 mg/mL) for 30 min followed by sustained room temperature for 3 days. Dodecanethiol was then added to the

Figure 8. Color changes in the AuNPs/r-LNT (a1−3), AuNPs/t-LNT (b1−3) and AuNPs/s-LNT(c1−3) solutions mixed with dodecanethiol. AuNPs/LNT aqueous solutions were prepared as follows: sLNT solution was obtained as described in the Experimental Section and then reacted with HAuCl4 at 100 °C for 30 min. It was then stood at room temperature for 3 days to obtain AuNPs/r-LNT (cs‑LNT = 3.0 mg/mL) and AuNPs/s-LNT (cs‑LNT = 0.5 mg/mL) solutions. Whereas, the AuNPs/t-LNT solution was obtained by directly mixing t-LNT and HAuCl4 at 100 °C (ct‑LNT = 3.0 mg/mL) for 30 min and then standing at room temperature for 3 days; 1 indicates the addition of dodecanethiol into the AuNPs/LNT solutions without stirring, 2 indicates mixture 1 after stirring for 1 h, and 3 stands for mixture 2 after centrifugation.

b1, and c1. The colorless dodecanethiol with lower density than water was distributed on the upper layer. Noticeably, AuNPs/rLNT, AuNPs/s-LNT, and AuNPs/t-LNT aqueous solutions exhibited completely different colors, indicating that the reduced Au was presented in different morphologies or size. After stirring for 1 h, the AuNPs/r-LNT aqueous solution turned slightly turbid as a result of an air bubble that arose from stirring. Nevertheless, the wine red color was not changed (Figure 8a2). In contrast, the purple of AuNPs/t-LNT and AuNPs/s-LNT was seen to disappear in the water layer and moved to the upper dodecanethiol layer (Figure 8b2,b3). Even after centrifugation, the wine red color of AuNPs/r-LNT aqueous still endured (Figure 8a3), while the purple in the dodecanethiol layer of AuNPs/s-LNT and AuNPs/t-LNT completely disappeared, leaving some black Au precipitates at the bottom of the vial, as shown in Figure 8b3,c3. These results implied that almost all of the AuNPs reduced by s-LNT at high concentration were entrapped in the reformed hydrophobic cavity of r-LNT, leading to noncontact of dodecanethiol with AuNPs and thus stable dispersion of AuNPs in water. In the case of the t-LNT with a chain diameter of ∼1.2 nm,37,55 it was supposed that the original helical core was so small that the AuNPs with an average size of ∼18 nm could not be entrapped in the hydrophobic cavity. Its dispersion was thus ascribed to the interaction between AuNPs and the hydroxyl group outside the helical core of t-LNT, similar to the dispersion of Ag by tLNT.41 At the low s-LNT concentration of 0.5 mg/mL, s-LNT could not reform into triple helix, and thus, the dispersion of AuNPs by s-LNT resulted from the interaction between AuNPs and the hydroxyl group of s-LNT. Once dodecanethiol with stronger polarity was mixed with AuNPs/t-LNT and AuNPs/sLNT, AuNPs were seen to preferentially interact with dodecanethiol and then move to the upper layer, as shown in Figure 8b2,c2. The scarcity of the observed AuNPs nanowires was also considered (Figure 5). It was supposed that few t-LNT could reform in such a short time as 10 min. On the other hand, most of the AuNPs were randomly distributed but were dispersed in water. As discussed in Figure 4a, the 280 nm band was ascribed to the long pair interaction between β-glucan hydroxyl groups F

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Figure 9. Scheme of synthesis and dispersion of AuNPs by s-LNT at different concentrations.

and the gold surface.26,45−48 It was thus determined that AuNPs interacted with the hydroxyl groups of LNT except the hydrophobic interaction, leading to good dispersion in water. After a sufficient timespan, t-LNT reformed to a greater extent and AuNPs were mainly displayed as nanowire structures as shown in Figure 7a,b. At high concentrations, s-LNT rapidly reduced Au3+ into AuNPs at the initial stage. They were subsequently stabilized through the interaction between the surface of AuNPs and hydroxyl groups and then entrapped in the hydrophobic cavity of the gradually renatured t-LNT to form more stable Au nanowire. Schematic Model of Au Synthesis and Dispersion in Water. Based on the aforementioned results, a tentative mechanism was proposed to describe the synthesis and dispersion of Au in LNT/aqueous solution, as seen in Figure 9. At the low concentration of 0.5 mg/mL, the reduced Au first adopted a nanobelt-like shape before 30 min and a welldispersed spherical shape after 1 h of the reaction, which was widely divergent from the nanowire structure. As shown in Figure 4a, the nanobelt appeared after a 5 min reaction, indicating that the reduction occurred very rapidly. We thus concluded that the rate of reduction by s-LNT was very prompt even at low concentrations. In addition, the triple helical structure of t-LNT was found difficult to reform due to the low s-LNT concentrations. Therefore, a dense arrangement of the locally concentrated AuNPs formed along the chain of s-LNT as a result of the interaction between AuNPs and hydroxyl groups forming nanobelt structure. This was illustrated by the UV results, as shown in Figure 4a (5, 10, 15, and 30 min). Moreover, some AuNPs aggregates were even observed within a short 30 min reaction. With continuous stirring for 60 min, the Au nanobelts and aggregates were dispersed to form individually separated spherical nanoparticles, while only scarce amounts of nanobelts or aggregates were observed (Figure 4f). Namely, the stabilization of AuNPs in water at the low concentrations of s-LNT was achieved mainly through the interaction between the surface of AuNPs and the hydroxyl groups. With high concentrations of s-LNT, AuNPs were first synthesized and stabilized through the interaction between AuNPs and the hydroxyl groups of LNT. They were then

entrapped in the hydrophobic cavity of the reformed triple helical r-LNT with increasing time.

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CONCLUSIONS In this study, a green approach was described for the synthesis and dispersion of Au in aqueous medium. Au was successfully synthesized from Au3+ using a single chain (s-LNT) from a βglucan known as LNT, which held the original conformation of triple helix. It was determined that β-glucan has certain reducibility. The reduction of Au3+ was shown to be dependent on s-LNT concentration, reaction time, and temperature. A higher β-glucan concentration and reaction temperature led to quicker formation of reduced AuNPs with uniform size and spherical shape. Prolongation of the reaction time was also found favorable for the formation of spherical AuNPs. TEM observations demonstrated that Au formed nanobelts in aqueous solution at low s-LNT concentrations and spherical AuNPs at relatively high s-LNT concentrations. AuNPs were also seen to arrange sequentially in the hydrophobic cavity of the renatured triple helical LNT (r-LNT) to form stable Au nanowire. This effect was more prominent at high s-LNT concentrations. Furthermore, both the hydrophobic interaction and the interaction between AuNPs and the hydroxyl groups of LNT were established as contributing factors in the stable dispersion of AuNPs in water. More specifically, Au3+ could be reduced and stabilized by s-LNT, and the reduced Au could be manipulated into nanobelt, spherical shape, or nanowire morphology through the s-LNT concentration, reaction time, and temperature. s-LNT could thus be used as either a reducing or stabilizing agent to prepare stable AuNPs in a water system. Neither organic nor toxic agents were used in this process, and the reduction reaction was performed in an aqueous medium. LNT also has the advantages of being a nontoxic and safe biomacromolecule with antitumor activity and immunomodulating activity. This method was thus considered environmentally friendly and safe, which is a very important factor for application in biotechnology and biomedicine.

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AUTHOR INFORMATION

Corresponding Author

*Phone/Fax: 86-27-68754188. E-mail: [email protected]. G

dx.doi.org/10.1021/bm400182q | Biomacromolecules XXXX, XXX, XXX−XXX

Biomacromolecules

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Notes

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The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We gratefully acknowledge the financial supports from the National Natural Science Foundation Grant (21274114 and 20874078), the Youth Technology Chenguang Project of Wuhan (200950431193), and the National Basic Research Program of China (973 Program, 2010CB732203).

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dx.doi.org/10.1021/bm400182q | Biomacromolecules XXXX, XXX, XXX−XXX