Silver Nanoparticle Synthesis Using Monosaccharides and Their Growth Inhibitory Activity against Gram-Negative and Positive Bacteria

Using various monosaccharides as reductant, we synthesized Ag nanoparticles (NPs) in seconds employing the household microwave method described earlier. The Ag NPs containing colloidal solution showed distinctive colors with varying λmax. The sizes of the NPs formed varied significantly from 10 to 35 nm in good agreement with the localized plasmon resonance ranged from ∼300 to ∼600 nm.The antimicrobial properties of these NPs were compared in Gram-negative and positive bacteria in liquid culture. Gram-positive bacteria were highly susceptible compared to Gram-negative microbes—the additional lipopolysaccharide layer covering the peptidoglycan cell wall in the latter somewhat lessens the effect.The results indicated that larger NPs produced by glucose inhibited bacterial growth better than the smallest NPs produced by ribose.Thismay be attributed to the higher aggregation rate for larger NPs on cell wall. SEM analysis showed accumulation of NPs on cell surface and defect in budding, further supporting the cell wall interaction with Ag NPs.These observations suggested that the growth inhibition of Ag NPs is mediated by interfering with the bacterial cell wall peptidoglycan.


Introduction
The use of nanotechnology in everyday applications continues to increase due to unique chemical, optical, and mechanical properties of nanoparticles (NPs) [1] and it holds promise for multitude of potential applications in the fields of drug delivery [2], catalysis [3], cell and organelle labeling and imaging [4,5], biological sensing [6], and detection of wide range of biomolecules [7][8][9][10]. The important characteristic of NPs that separates them from their bulk counterparts is high surface area to volume ratio. Moreover, it has been shown that materials at the nanoscale level have unique chemical and physical properties compared to their bulk counterpart, and these properties are important for a variety of technological applications.
Interaction of NPs with biomolecules and its application in combating microorganisms are a new avenue for research. Increase in bactericide and antibiotic-resistant microbial strains and the toxicity of some of these agents lead to growing interest in new types of safe and cost-effective antimicrobial agents [11]. Recent studies that metal and metal oxide NPs could be used as effective bactericidal materials open the door for development of a new type of antimicrobial materials [12,13]. Among the noble-metal NPs, silver received attention due to its interesting physiochemical properties and for the well-known toxicity of its ionic form to bacteria [14]. Silver ions served as bactericide for a while in presulfa-drug era and used in dental resins composites [15] and in coatings of medical devices [16]; however, it was soon abandoned for its toxicity. Sondi and Salopek-Sondi [17] demonstrated Ag NPs as an effective bactericide against Gram-negative bacteria, E. coli. Elechiguerra et al. [18] have reported inhibition of HIV-1 transmission by size dependent interaction of Ag NPs with HIV-1. More antibacterial properties of Ag NPs have also been reported elsewhere [19,20].
For metal nanocrystals, it has been shown that the electromagnetic, optical, and catalytic properties, and biosensor or biological activities are highly influenced by shape and/or size of the NPs [21]. This has led to development of a variety of synthetic methods for better control of morphology and size. Silver of NPs of different sizes and shapes has been synthesized by various methods. The most widely used method for the synthesis of Ag NPs is the chemical reduction of silver ions in solution with the use of various reducing agents such as sodium citrate [22], glucose [23,24], formamide [25], phenylhydrazine [26], and the amino acid tyrosine [27] and cysteine [28]. Often times, the reducing agent is combined with a stabilizing agent to reduce or prevent agglomeration of the NPs synthesized [29]. Other methods described include laser ablation of a silver plate suspended in organic solvents, gamma irradiation of AgNO 3 in the presence of polyvinyl pyrrolidone [30], and electroreduction of silver ions in acetonitrile in the presence of tetrabutylammonium salts [31]. Methods involving the use of biological systems, such as fungi [32] and bacterium [33], have also been reported. The ability to control various physical attributes (size and shape) and to include functionalized groups on the surface has made NPs attractive to search for newer applications [26,34,35].
In this study, using common monosaccharides as reductant, we rapidly synthesized Ag NPs of different sizes between 10 and 35 nm employing the household microwave method described recently [24]. Comparing their antimicrobial potency against Gram-negative and positive bacteria, we found that Ag NPs were more active against Gram-positive bacteria possibly through their interaction with naked peptidoglycans forming the cell wall, while LPS layer in Gramnegative bacteria slowed this interaction. To our knowledge, a comparison of antimicrobial activity of Ag NPs between the two major types of bacteria has not been reported.

Experimental Details
2.1. NP Synthesis. Silver nanoparticles were synthesized using the household microwave method described previously by Dong et al. [24]. Briefly, aqueous solutions of 0.1 M AgNO 3 (1 mL) and 1.0 M sugar (1 mL) were mixed together in a medium size glass test tube and vortexed briefly. The tubes were heated for 8-10 seconds in a household microwave oven (J.C. Penney, model 853-5645-00-59, ∼800 watt) on the highest power setting. To speed up synthesis, up to six test tubes were microwaved at one time in an open plastic test tube holder. Monosaccharide sugars used in the reaction include ribose, fructose, sorbose, glucose, xylose, and galactose. Deionized water was added to the reaction mixtures to maintain a constant reaction volume of 2.0 mL.

UV-VIS Spectroscopy.
Immediately after microwaving, reaction mixtures were analyzed using a Cary 300 Bio UV-VIS spectrometer with Varian UV Scan Application, version 3.00 (303), and UV-VIS spectra were obtained between 300 nm and 800 nm. A 1.0 mL cuvette with 1 cm light path was used for all samples. Samples were scanned against a blank prepared of 0.05 M AgNO 3 (1 mL) and 0.50 M (1 mL) respective sugar just before use.

Photon Correlation Spectroscopy.
The hydrodynamic diameters of the synthesized NPs were determined by photon correlation spectroscopy. The target samples were prepared by diluting (1 : 50) the microwaved colloidal mixture directly into nanopure water. Alternatively, the NPs were collected by centrifugation, followed by washing twice with nanopure water. The washed NPs were resuspended in nanopure water and sonicated for 5 min to disperse aggregated NPs. Measurements were performed using a size analyzer (Brookhaven, model 90 plus). Scattered light was detected at a 90 ∘ angle through a 400 m pin hole at room temperature. The instrument was calibrated using standard reference particles. Following measurements and the values are given as the mean of 5 runs of 60 s ± standard deviation.

Scanning Electron
Microscopy. Silver nanoparticles in bacterial broths were characterized by JOEL field emission scanning electron microscope (FE-SEM) (Model JSM 7600F). The images of the samples were taken setting at the operating conditions of SEM at high voltage from 1.5 to 5 KV with wide distance (WD) from 5.00 to 7.60 mm.

Antimicrobial Activity.
Prior to antimicrobial assay, following microwaving residual silver ions that may adhere to NPs were removed by washing twice with nanopure water, each time the NPs were collected by centrifugation in a microcentrifuge at 5000 g for 2 min. The washed NPs were dried to constant mass and resuspended in nanopure water and dispersed by brief sonication.
Antimicrobial effects of Ag NPs were performed on Gram-negative, E. coli, and Gram-positive, S. epidermidis, and B. megaterium cells. Overnight cultures diluted in LB to give 1.0 × 10 7 colony forming unit (CFU) in a total culture volume of 3.5 mL LB with or without supplemented with NPs of desired concentration. These culture tubes were incubated in an orbital shaker (GYROMAX 737) at 37 ∘ C at 225 rpm for indicated period of time. Growth of the bacteria was monitored by measuring absorbance at 600 nm against LB blank.

Synthesis and Characterization of Ag NPs.
In the present study, commonly available monosaccharides were tested for their ability to synthesize Ag NPs employing the household microwave method we reported previously and to determine any effect on SPR of the NP synthesized. Aqueous solutions containing AgNO 3 and monosaccharides were mixed and microwaved for 18-0 seconds in a household microwave, all tubes at once. Monosaccharides are known as reducing sugars because of their ability to reduce Cu 2+ in alkaline solution, first described by Fehling, due to the presence of free aldehyde functional group in aldoses or -hydroxymethyl ketone in ketoses. Although the ketone functional group cannot be oxidized per se, it does so by the combination of enediol rearrangements and epimerization. The reaction is carried out in aqueous solution in the absence of any capping agent and required only a couple second of heating However, the metallic silver precipitated out of reaction mixtures after 20 h of incubation at room temperature exhibited the same color as of the colloidal mixture (Figure 1(a)), suggesting that the colors formed in the reaction mixtures were due to colloidal Ag NPs. However, it may still be possible that the Ag NPs were doped with some oxidized sugars. Ultraviolet-Visible (UV-Vis) spectroscopy is a valuable tool for structural characterization of metallic NPs. Similar to the metal surface, the electrons in nanoparticles form a conduction band surrounding the nucleus [37]. The relative excitation of these electrons near the surface of the nanoparticle by wavelength-dependent light results in Surface Plasmon Resonance [38,39]. It is well known that the optical properties of NPs depend on size, shape, surface characteristics, and other variables like doping and interaction with surrounding environment and other nanostructures. Red shift occurs with NPs as they aggregate [24]. In addition, the number of SPR peaks decreases as the symmetry of the NPs increases [39]. The UV-Vis absorption spectra taken immediately after microwaving showed a relatively sharp SPR peak at ∼ 300 nm with variable absorbance and all except ribose also showed a 2nd broader absorption band between 400-550 nm (Figure 1(b)). The latter may be attributed to the classical SPR of larger silver nanoparticles [36]. The strong SPR absorption peaks below ( Figure 1) 430 nm could be assigned to spherical particles with small diameter due to formation of small Ag n clusters. SEM (Figure 1 (A-B)) analysis exhibited spherical NPs and the sizes of the isolated NPs ranged from 10-15 nm for ribose to 20-35 nm for glucose. The sizes of the particles produced with glucose were consistent with the results reported previously [24]. The precise SPR peaks varied greatly and were 296 nm for ribose, 436 nm for xylose-both aldopentoses; 534 nm for galactose, 480 nm for glucose-both aldohexoses; 441 nm for fructose and 448 nm for sorbose-both ketohexoses.
As mentioned above, all except ribose exhibited two SPR peaks. This can be attributed to differences in size rather than shape of the NPs as we have shown previously that the broader peak increases over time with aggregation of NPs [24]. Consistent with this, SEM analysis (Figure 1(b)) revealed that the shapes of NPs produced by both glucose and ribose used as reductant were spherical. Size of the glucose reduced Ag NPs as determined by SEM (Figure 1 (A)) was ∼35 nm among the isolated NPs, but significant numbers were aggregated to form larger size. Average sizes of these NPs determined by photon correlation spectroscopy were 35 nm. SEM analysis of the ribose reduced NPs with the shortest SPR showed numerous isolated NPs with size ranged nearly 10 nm, which agreed well with the size determined by photon correlation spectroscopy ( Table 1). Sizes of the other monosaccharide reduced NPs ranged from 10 nm to 35 nm and correlated well with the SPRs. No correlation was found between either size or shape of the NPs produced with 4 ISRN Nanotechnology the functional groups (aldehyde or ketone) present in the monosaccharides nor with the C-length of the sugars.

Antimicrobial Effect.
Antibacterial tests were performed against Gram-negative bacteria, E. coli, and Gram-negative bacteria, B. megaterium, and S. epidermis in LB broth supplemented with varying concentrations of Ag NPs. Since Ag + ions show antimicrobial activity by inactivating proteins and inhibiting replication [40], we removed silver ions as much as possible by washing Ag NPs extensively with nanopure water (see methods) to avoid antimicrobial effect resulting from silver ions. Antibacterial effects were performed initially against Gram-negative E. coli with different concentration of Ag NPs. Figure 2(b) shows bacterial growth inhibition/delay, which increased with concentration of NPs used. At 50 g/mL concentration, NPs completely halted growth of E. coli (not shown) over 8 h growth periods. For comparison, effect of ampicillin on E. coli growth was performed over the same time period (Figure 2(a)). While both showed a concentration dependent effect on E. coli growth, the comparison demonstrated one major difference: Ag NPs affected growth at early hours, which was diminished gradually after peaking around 4 hrs, and caught up nearly normal growth after 8 hours. Ampicillin, however, showed a lag period of ∼2 hours, after that growth inhibition was seen increased with time. This difference in growth inhibition pattern may clearly point ISRN Nanotechnology to the different sites of action. It is well known that ampicillin acts as a competitive inhibitor of the enzyme transpeptidase, which is needed in final stage of bacterial cell wall synthesis in binary fission leading to cell lysis. That is, the site of action for ampicillin is intracellular and requires time for ampicillin to penetrate cell wall and reach the target site. The site of action for Ag NPs is most likely extracellular, the cell wall, which does not require internalization. The reason growth inhibition by Ag NPs was diminished gradually after peaking at ∼ 4 h, can be attributed to gradual decrease in the concentration of the NPs, allowing resumption of bacterial cell growth. This gradual decrease process is governed by the interaction of NPs with the intracellular materials (e.g., proteins/peptides) of the destroyed cells, causing their coagulation and removal from the liquid media as suggested earlier [17]. As expected, the extent of growth inhibition depends on the number of cells to begin with: when started with 1 × 10 6 CFU cells, the growth was drastically delayed up to 8 h in the presence of 20 g/mL NPs; on the other hand, it had very little effect when the cells were 3 times more (3 × 10 6 CFU) at the start (Figure 2(c)). These results with E. coli were consistent with those reported by Sondi and Salopek-Sondi [17], except slight difference in NPs sizes used (35 nm versus 50 nm).
The Ag NPs produced using ribose have smaller hydrodynamic diameters compared to those synthesized with glucose; thus we examined if the NP size difference has any effect on bacterial growth inhibition. Surprisingly, we found that the larger particle had better effect on the growth delay. Small particles are obviously important if endocytosis into cells [41] is necessary (Figure 2(d)). However, reports from several groups indicated that, in the submicrometer range, larger particles lead to increased efficiencies in transfection [42]. It is speculated that this might be caused, to a large extent, by increased sedimentation of larger complexes, leading to facilitated attachment and interaction to cell wall. Hence, the larger size of Ag NPs formed by glucose might be favorable for growth inhibition over smaller size NPs produced with ribose.
Next, we investigated the effect of Ag NPs on Grampositive bacteria, B. megaterium and S. epidermis. Starting with 3 × 10 6 CFU and 20 g/mL NPs both, Gram-positive bacteria especially B. epidermis showed drastic growth inhibition (Figures 3(a)-3(b)), suggesting that Ag NPs are more effective against Gram-positive than Gram-negative microbes. This difference in growth effect can be due to the difference in their cell wall architecture. The outer layer of the Gram-positive bacteria is composed of peptidoglycan, while that layer is covered by a tightly packed lipopolysaccharides layer in Gram-negative cells. It has been reported that Ag NPs directly conjugate with protein molecules and are stabilized by thiol-bearing cysteine residues [18]. While the mechanism of interaction between the NPs and the cell wall components is unfortunately still unresolved, it can be presumed that Ag NPs will interact quickly with naked peptides on the wall of Gram-positive bacteria but slowly with the wall covered by extra layer in Gram-negative bacteria. AgNPs accumulation on the cell membrane and uptake within the cell have also been reported for other bacteria such as P. aeruginosa and S. typhus [43]. SEM analysis revealed highly reflective metallic on the surface of bacteria compared to untreated cells. It also shows 6 ISRN Nanotechnology that the treated cells were significantly changed and the number of budding out cells is drastically reduced, thus preventing their growth ( Figure 4). In this study, although the growth inhibition mechanism of the Ag NPs is not fully understood and the mechanisms proposed here are disparate and indirect, they may prove to be synergetic in the action of Ag NPs in bacterial growth inhibition. Further studies are required to fully understand the mechanisms of action. The flexibility of NPs preparation methods, the multitude of functionalization techniques, and facile incorporation of NPs into a variety of media provide incentive for further research and their applications. It should be noted here that a recent report showed that antimicrobial activities of AgNPs were due to aerobic conversion of (PEG and PVP coated) AgNPs to Ag + in aqueous media [44]. However, the released Ag + can also form Ag ∘ -containing clusters through chemical reduction with the components in the media and or bacterial cell surface [43]. Whether formation of Ag + is in play in antibacterial activity observed in this study requires further investigation. However, we believe that multiple factors may govern the antibacterial activity.

Conclusion
Using common monosaccharides as reductant, Ag NPs can be synthesized in seconds employing household microwave. The Ag NPs showed distinctive colors with varying max in the range from ∼300 to ∼600 nm and the sizes of the NPs formed varied significantly from 10 to 35 nm that corresponded well with the SPR bands of Ag NPs produced. The particles were more effective in growth inhibition of Grampositive bacteria most likely mediated by their interaction with naked peptidoglycan of cell wall. The extra LPS layer covering the peptidoglycan seems to lessen the effect in Gram-negative bacteria.