Dextran: Facile Synthesis and Versatile Tunability on the Antimicrobial Activity

University of Sciences, Hue University, 77 Nguyen Hue Street, Hue City, Vietnam Research and Development Center for Radiation Technology, Vietnam Atomic Energy Institute, 202A, 11 Street, Linh Xuan Ward, Thu Duc District, Ho Chi Minh City, Vietnam University of Education, Hue University, 34 Le Loi Street, Hue City, Vietnam School of Hospitality and Tourism, Hue University, 22 Lam Hoang Street, Hue City, Vietnam


Introduction
Nowadays, the antibiotic resistance is one of biggest threats to global health that encourages scientists to find new routes to develop more effective biocidal materials to overcome the challenges [1]. Nanoparticles (NPs) based on noble metals [2][3][4][5][6] have been emerged as a best alternative to antibacterial reagents due to their excellent antimicrobial activity and biocompatible nature that can be applied in biomedical implementations. Among them, Au, Ag, and bimetallic Au-Ag NPs are of great interest because of their unique optoelectronic and antimicrobial properties [7]. Since ancient time, silver has been considered as an effective bactericide [8]. The nanostructures based on silver with abundant surface-active atoms showed effective antimicrobial activity to inhibit the growth of a broad spectrum of fungi and bacteria by destroying infectious microorganism [9][10][11]. Despite its wide application, several reports indicated that the Ag NPs exhibited cytotoxic effects on some mammalian cells [4,12] and their therapeutic concentration window for the application as an antimicrobial agent should be extremely narrow [13]. The combination of Ag NPs with other noble metal could widely open their therapeutic window [12].
The Au NPs with localized surface plasmon resonance can convert to amount of heat during the process, which is useful in photothermal therapy applications such as targeting bacterial and cancer cells. The Au NPs were considered as less toxic metal and safe antibiotics [14]. However, the high cost and weak antibacterial activities are some disadvantages of the nanomaterials [12,15]. The promising strategy to improve the antibacterial activity of Au NPs and as well as reduce the toxicity of Ag NPs has been to design silver-gold bimetallic NPs. Silver-gold bimetallic NPs are attracting great attention for biomedical field because their antibacterial activity can be tuned by the composition or structure of the NPs, high stability in air, and biocompatibility [16]. Yang et al. [15] indicated that the Ag shell thickness of Au-Ag NPs influenced dramatically their antibacterial activity and biocompatibility. Ding et al. [17] reported that the Au-Ag NPs exhibited potent antibacterial effect as well as negligible toxicity to human dermal fibroblasts. Sapkota et al. [18] demonstrated that SiO 2 -Au-Ag nanocomposites exhibited not only strong antibacterial activity against Gram-positive and Gramnegative bacteria but also good biocompatibility. Thus, a facile route, which can control easily the silver content in bimetallic NPs, is of huge potential for biomedical applications.
In general, the naked noble metal-based NPs are usually not stable in solution. Their surface is usually decorated by substrates such as graphene oxide [19], carbon [20], silica [21], carbon nitride [14], or capped by organic molecules in order to stabilize them [22]. Among various capping and stabilizing agents, the dextran plays a promising role for development of noble metal NP-based novelty materials because they are not only biodegradable, environmental benignity, highly abundant, and low-cost [23] but also has better biocompatibility, lower toxicity, and slower degradation in the human body in comparison with other polysaccharides [24]. Some previous reports indicated that noble metal NPs/dextran showed significant antitumor effects [25], effective activity against different fungal and bacterial strains [26].
Xanthomonas oryzae pv. oryzae (Xoo) and Magnaporthe grisea (M. grisea, also called Magnaporthe orylzae) are known as causal agents of leaf blight and paddy blast diseases, which are the most principal diseases of rice [27,28]. Inspired by the unique chemical, physical, and biological properties of nanoscale, some nanomaterials have been investigated to prevent bacterial light and blast fungus such as Ag NPs [29,30], graphene oxide/silver NPs [31], copper NPs [32], SiO 2 -Ag nanocomposites [33], and MgO nanoflowers [34]. However, antimicrobial characteristics of M. grisea and Xoo of silver/dextran, gold/dextran, and particularly silver-gold bimetallic NP-decorated dextran are not investigated systematically.
Herein, we introduce the facile and fast strategy to synthesize Ag, Au, and Ag-Au bimetallic NP-decorated dextran that acts both reducing and capping agents. The antifungal and antibacterial effects of Ag, Au, and Au-Ag bimetallic NP-decorated dextran against M. grisea and Xoo were tested for the first time.  In a typical synthesis of Ag NPs-dextran, 10 mL of 1 mM [Ag(NH 3 ) 2 ] 2 SO 4 solution was mixed with 7 mL of NH 3 solution (~5 wt.%) for 15 min to form a homogeneous solution, and then the mixture was aged at 90°C for 15 min under magnetic stirring. The estimated concentration of silver in the suspension was 63 μg/mL. In a similar manner, the Au NPs-dextran was carried out by replacing the 10 mL of 1 mM [Ag(NH 3 ) 2 ] 2 SO 4 solution with 10 mL of 1 mM HAuCl 4 solution.
To synthesize Ag-Au bimetallic NPs-dextran, 10 mL mixtures containing 7 mL of 1 mM [Ag(NH 3 ) 2 ] 2 SO 4 and 3 mL of 1 mM HAuCl 4 or 5 mL of 1 mM [Ag(NH 3 ) 2 ] 2 SO 4 and 5 mL of 1 mM HAuCl 4 or 3 mL of 1 mM [Ag(NH 3 ) 2 ] 2 SO 4 and 7 mL of 1 mM HAuCl 4 were previously prepared, and then the as-prepared mixtures were mixed with 7 mL of NH 3 solution (~5 wt.%) for 15 min to form a homogeneous solution. After that, the mixture was aged at 90°C for 15 min under magnetic stirring to obtain Ag-Au bimetallic NP-dextran suspension. With the [Ag(NH 3 ) 2 ] 2 SO 4 : HAuCl 4 molar ratio in the mixture that is 3 : 7, 5 : 5, and 7 : 3, the respective obtained product was named as samples S3, S5, and S7. The colloidal stability of samples is about~4 weeks.

Material
Characterization. X-ray diffraction patterns were recorded by a Bruker D8 Advance X-ray diffractometer. Scanning electron microscopy (SEM) and elemental analysis and energy dispersive X-ray spectroscopy (EDS) were analyzed by a JSM-5300LV instrument. Fourier transforminfrared spectroscopy (FTIR) spectrogram was measured using a Nicolet-6700 FTIR spectrometer, with a wavelength range of 4000-500 cm -1 . Transmission electron microscopy (TEM) and high-resolution transmission electron microscopy (HRTEM) were performed using JEOL JEM 1230. The UV-vis spectra were obtained using a Jasco V-550 UV-vis spectrophotometer within the range of 350-700 nm.

Antibacterial and Antifungal
Tests. The inhibition of growth of microorganism of Ag, Au, and Ag-Au bimetallic NP-decorated dextran was carried out with bacterial Xoo and fungal M. grisea. For the antibacterial test, Modified Wakimoto's medium was previously prepared as the following: a mixture including 300 g of potato infusion, 5.0 g of peptone, 2 g of disodium phosphate (Na 2 HPO 4 ), 0.5 g of calcium nitrate (Ca(NO 3 ) 2 , 15 g of sucrose, and 17 g of agar were mixed distilled water to form 1.0 L of suspension and then sterilized by autoclaving at 125°C for 15 min. The obtained medium was used to cultivate the Xoo bacteria. The petri dishes each contained 10 mL of Wakimoto medium and 0.1 mL of each type of as-synthesized nanomaterial. For the 2 Journal of Nanomaterials nanomaterial as silver NPs/dextran, the estimated concentration of silver was used in the antibacterial test for about 6.3 μg/mL. After that, 1 mL of bacterial Xoo suspension (approximately 10 6 CFU/ml) was spread onto these petri dishes followed by incubation of these plates at 28°C for 72 h. One petri dish contained only 10 mL of Wakimoto medium but not containing the NPs has been made a similar process as a reference sample. Visible colonies were quantified after incubation. The antifungal action of Ag, Au, and Ag-Au bimetallic NPdecorated dextran was evaluated against fungal M. grisea in potato dextrose agar (PDA) medium. To prepare PDA, a mixture composed of 200 g of potato infusion, 20 g of glucose, and 20 g of agar was dissolved and makes up to 1.0 L with distilled water and then sterilized into tubes lined autoclave at 125°C for 15 min. The fresh PDA was taken in 10 mL for each petri dish and mixed with 0.1 mL of each type of assynthesized NPs. After that, 1 mL of fugal M. grisea strains (approximately 10 6 CFU/ml) was inoculated in these petri    Journal of Nanomaterials dishes. The inoculated plates were incubated at 28°C for 5 days. The size of fungus colonies was determined to calculate the inhibition efficiency of nanocomposites using the formula as suggested by Vincent [35]. Inhibition efficiency ðI, %Þ = ððC − TÞ/CÞ x 100, in which I, C, and T are the inhibition percentage, size of fungal colony in the reference sample, and the size of fungal colony in nanocomposite medium, respectively. Each test for the antimicrobial activity of these samples was repeated for three times to guarantee the uniformity.

Results and Discussions
The Ag, Au, and Ag-Au bimetallic NPs were synthesized by a fast and facile route in an aqueous phase with assistance of dextran. The dextran could act as a both reducing and stabilizing agent. The UV-visible absorptions of obtained silver, gold, and silver-gold bimetallic NPs in dextran were shown in Figure 1. As can be seen in Figures 1(a) and 1(c), the UV-visible spectra of the silver/dextran and gold/dextran exhibited bands at 424 nm and 557 nm, respectively, which 5 Journal of Nanomaterials could be indexed to the visible surface plasmon resonance band of respective Ag and Au NPs [36,37]. Compared with the spectroscopic profile of Ag/dextran and Au/dextran colloid suspensions, the UV-visible absorption bands of Ag-Au bimetallic NPs in dextran were 518, 530, and 542 nm for samples S7, S5, and S3, respectively, suggesting that a longer surface plasmon resonance wavelength could be tuned through decreasing the raw ratio of Ag/Au. The presence of only a single surface plasmon resonance band in these samples could originate to the formation of bimetallic Au-Ag NPs instead of a mixture of monometallic NPs [38,39]. The respective solutions of silver, silver-gold bimetallic, and gold NP colloidal suspension were shown inset in Figures 1(a)-1(c), respectively, as can be observed that their colors ranged from pale yellow (Ag/dextran) to dark red (Au/dextran) with intermediate hues for bimetallic samples.
The XRD patterns of the of silver and gold NPs in dextran in Figure 2 [40]. Several peaks related to crystal planes of AgCl were observed at 32.4°, 46.4°, 54.6°, and 57.7°(JCPDS card no. 031-1238) in XRD patterns of silver/dextran and silvergold bimetallic samples. The coexistence of AgCl in the synthetic process of Ag NPs is a common observance reported in literature [41,42]. The XRD patterns of silver-gold bimetallic NPs in dextran are very similar to those of the silver and gold NPs samples, which can be explained that the bimetallic samples have a similar crystal lattice structure with pristine silver and gold [43].
The morphology and particle size of silver, gold, and silver-gold bimetallic NP-decorated dextran were analyzed by SEM and TEM techniques. The SEM images (Figures 3(a)-3(e) and low magnification of TEM images ( Figure S1) indicated that the obtained silver, gold, and silver-gold bimetallic NPs in dextran are high dispersity. The higher magnification of TEM images (Figures 3(f)-3(j) of samples showed that most of NPs were spherical geometry. The particle size of silver NPs in dextran ranges from 5 to 55 nm with the average particle size of~17.5 nm ( Figure S2 (a)). In contrast to the result of Ag/dextran, the particle size of Ag-Au bimetallic NPs in dextran decreases significantly (Figures 3(c)-3(h). The average diameter of Ag-Au bimetallic NPs is~5 nm ( Figure S2 (b-d)), which is similar to that of Au NPs in dextran ( Figure S2 (e)). As can be seen in TEM image in Figure S1 (b), the sample with the highest atomic percentages of Ag in the bimetallic NPs (S7), many NPs with a diameter range of 20 to 40 nm, was observed clearly. These particles were also observed in sample S5; however, they decreased in comparison with that of sample S7 (Figure S1 (b) and (c) and Figure S2 (b) and (c)). The particle size of bimetallic NPs fabricated with the highest atomic percentages of gold (S3) range from 3 to 25 nm that have better narrow particle size distribution than that of samples S7 and S5. Furthermore, particle size distribution analysis of sample S3 concurs with the results of Au/dextran. Our findings may be explained by the higher reactivity of silver compounds; thus, the preparation of silver NPs with well-controlled dimension was more difficult than that of gold NPs [44].
The chemical composition of bimetallic products was carried out by EDS (a)-(c) attached to the SEM that was presented in Figure 4. The results indicated distinctly the main peak of Ag-Au bimetallic/dextran nanocomposite that can be assigned to Ag, Au, O, and C. After the synthetic process, the atomic ratio of Ag/Au in the bimetallic NPs changes slightly in comparison with the nominal composition (Table S1 and Table S2 and Table S3). The HRTEM images and the bimetallic NP samples (S7, S5, and S3) did not show either dark area or lighter area, demonstrating that the bimetallic NPs are alloy formulation.
FTIR spectrum of pure dextran and NP-decorated dextran samples was presented in Figure 5. From the FTIR spectrum of dextran, the major peaks of dextran were observed clearly at 3471 cm -1 , 2927 cm -1 , 1658 cm -1 , 1159 cm -1 , 1112 cm -1 , 1008cm -1 , and 909 cm -1 . Therein, the region of 3471 cm -1 was assigned to the stretching vibration of hydroxyl groups [45]. The peaks at 2927 cm -1 and 1658 cm -1 were attributed to the C-H bond and carboxyl groups, respectively. The bands at 1159 cm -1 and 1112 cm -1 were due to stretching vibrations of the C-O-C bond and C-O bond at the C-4 position of glucose, respectively [46]. The presence of peak at 909 cm -1 related to the existence of α-glycosidic bond, and the absorption peak at 1008 cm -1 was due to the great chain flexibility around the α(1 → 6) glycosidic bonds [46]. The FTIR spectra of silver, gold, and bimetallic NP-decorated dextran exhibited a similarity with that of dextran. However, there was a light shifting of bands assigning to the hydroxyl and carbonyl functional groups (Table S4), indicating that the surface of silver, gold, and silver-gold bimetallic NPs was adsorbed by these groups [47,48].  Journal of Nanomaterials The antibacterial property of the as-prepared silver, gold, and silver-gold bimetallic NP-decorated dextran was tested against bacterial Xoo strains. The optical images of Xoo colonies incubated on the blank control sample and NP/dextran samples for 72 h were shown in Figure 6. It is clearly observed that there are almost no colonies in Figures 6(a)-6(c), revealing that the silver NPs/dextran and samples S7 and S5 are effective inhibition the growth of Xoo. The colonies appeared clearly in the petri dish containing sample S3 (the bimetallic NPs with the lowest content of silver), whereas the gold NP-decorated dextran exhibited a weakly inhibited growth of Xoo. These results indicated that the antibacterial activity of bimetallic sam-ples could relate to the silver content in the NPs, which could originate the excellent and broad-spectrum antimicrobial activity of silver [49]. The decrease of the silver content in the bimetallic NPs is without significantly reducing their antibacterial properties, which could be useful for biomedical applications.
The antifungal properties of Ag, Au, and Ag-Au bimetallic NPs in dextran were tested with M. grisea by determining the size of the fungal colonies that was measured after 5 days. The optical images of colonies of the fungal growth in media with and without nanocomposite sample are shown in Figure 7. The results indicated that the silver NP-decorated dextran inhibited significantly the development of M. grisea 7 Journal of Nanomaterials (its inhibition efficiency of 69.72%). Similar to the antibacterial properties, the M. grisea antifungal characteristic of silver-gold bimetallic NPs/dextran decreased with the decrease of the silver content in the bimetallic NPs. The inhibition efficiencies of bimetallic NPs were 38.6%, 31.1%, and 18.6% for samples S7, S5, and S3, respectively (Table 1), whereas the inhibition efficiency of gold/dextran was only 4.72% that indicated that the gold/dextran has a weak effect against M. grisea ( Table 1). The results suggest that the Ag/dextran and bimetallic samples with high silver content (S7, S5) showed good antimicrobial activity against Xoo bacteria and M. grisea fungi, whereas the gold/dextran  The formation of silver-gold bimetallic NPs was known to decrease Ag dissolution behavior that reduce the overall toxicity of silver-based NPs [50]. Furthermore, the NPs were capped dextran that was biocompatibility polymer and antimicrobial activity [51]. Thus, these obtained materials could be investigated further to apply in nanomedicine.

Conclusions
In summary, the monodispersed silver, gold, and silver-gold bimetallic NPs in dextran were synthesized by a simple and fast process. The silver/dextran showed excellent antimicrobial properties against Xoo and M. gresia. The silver-gold bimetallic NPs exhibited remarkably and enhanced the inhibition of the growth of these microorganisms compared to the gold/dextran, and these samples showed a versatile tunability on the antimicrobial activity. Thus, we believe that the as-prepared nanocomposites could be considered as promising candidates for antiseptic and pharmaceutical fields.

Data Availability
The data used to support the findings of this study are included within the article and the supplementary information file.

Conflicts of Interest
The author(s) declare(s) that there is no conflict of interest regarding the publication of this manuscript.

Supplementary Materials
The supplementary materials have two figures and four tables that include the following: Figure S1 showed low magnification TEM images of Ag/dextran, sample S7, sample S5, sample S3, and Au/dextran to clearly observe the morphology and size of obtained nanocomposites. Figure S2 exhibited particle size distribution analysis of Ag/dextran, Au/dextran, and Ag-Au bimetallic nanoparticles in dextran to clearly analyze the diameter of as-synthesized nanoparticles. Tables S1-S3 exhibited elemental composition of samples S7, S5, and S3 to clearly demonstrate the chemical compo-sition of alloy nanoparticles. Table S4 showed the bands of hydroxyl and carbonyl stretching vibration of dextran, Ag/dextran, Au/dextran, and Ag-Au bimetallic/dextran to confirm the light shifting of bands assigning to the hydroxyl and carbonyl functional groups indicating the formation of silver, gold and silver-gold bimetallic nanoparticles. (Supplementary Materials)