A Green Approach for the Synthesis of Silver Nanoparticles Using Ultrasonic Radiation’s Times in Sodium Alginate Media: Characterization and Antibacterial Evaluation
The synthesis of silver nanoparticles (Ag-NPs) was achieved by a simple green chemistry procedure using sodium alginate (Na-Alg) under ultrasonic radiation as a stabilizer and physical reducing agent. The effect of radiation time on the synthesis of Ag-NPs was carried out at room temperature until 720 min. The successful formation of Ag-NPs has been confirmed by UV-Vis, XRD, TEM, FESEM-EDX, zeta potential, and FT-IR analyses. The surface plasmon resonance band appeared at the range of 452–465 nm that is an evidence of formation of Ag-NPs. The XRD study showed that the particles are crystalline structure in nature, with a face-centered cubic (fcc) structure. The TEM study showed the Ag-NPs have average diameters of around 20.16–22.38 nm with spherical shape. The FESEM-EDX analysis confirmed the spherical shape of Ag-NPs on the surface of Alg and the element of Ag with the high purity. The zeta potential showed high stability of Alg/Ag-NPs especially after 720 min irradiation with value of −67.56 mV. The FT-IR spectrum confirmed that the Ag-NPs have been capped by the Alg with van der Waals interaction. The Alg/Ag-NPs showed the antibacterial activity against Gram-positive and Gram-negative bacteria. These suggest that Ag-NPs can be employed as an effective bacteria inhibitor and can be applied in medical field.
In recent years, nanotechnology and its uses are among the most excited research fields with application in many industrial parts. This is due to their unique physicochemical properties and another advantages such that they can be more specific and highly sensitive material for their selective applications . Indeed, their applications have been applied in many technology areas, for instance, antimicrobial , immunology , and energy .
Synthesized metal nanoparticles are carried out by various chemical, physical, and biological methods, such as pyrolysis , chemical vapor deposition , and sol-gel ; that is, most of them use toxic reagent and are expensive. Therefore, high efforts in the synthesis of noble metal nanoparticles using environmentally friendly methods are required. These solutions generally are produced by the use of plant or fruit extracts and microorganism , which are cost-effective, time saving, and environmentally friendly .
Nanoparticles have distinguished physicochemical properties due to high surface reactivecenter, high mechanical strength, and electrical and thermal conductivity compared to the corresponding bulk material . Ag-NPs have been exhibiting tremendous applications in the field of electronics , drug delivery , antimicrobial , and so on. However, the broad methods of the synthesis of Ag-NPs have been using chemical approach impacting environmental contamination risks ; providing a new physicochemical approach for saving synthesis of Ag-NPs is important.
Ultrasonic radiation is one of the famous irradiation methods for accelerating various organic reaction [14–16]. Also, sonochemical method, since discovered, has been studied for yielding kinds of nanomaterials, especially noble metal nanoparticles, such as Ag, Au, Pt, and Pb producing nanoparticles . Sonochemical technique can produce the acoustic cavitation during irradiation process then leading to the creation of free radical [18, 19]. In the previous research, ultrasound irradiation has been involved in order to produce Ag-NPs using green algae of P. oedogonium (Mont.) Wittrock and seaweed K. alvarezii; however, using sodium alginate (Na-Alg) as the media was not reported [20, 21].
The Na-Alg is a natural carbohydrate polymer isolated from marine algae, considered to be biocompatible, nontoxic, and biodegradable. Mannuronic acid (M-block) and guluronic acid (G-block) units are the copolymer component in these algae arranged in an irregular block pattern: MM, GG, and MG blocks . Many of free hydroxyl and carboxyl groups are distributed in their backbone of the Na-Alg .
Silver and silver based compounds are highly toxic to a number of microbial cells; otherwise, they have low toxicity for human cells . Therefore, they can be an interesting part to be evaluated as the antibacterial activity. The Ag-NPs were reported recently as antibacterial agent against Escherichia coli and Staphylococcus aureus . Thereby, Alg/Ag-NPs also have been evaluated for the application as the antimicrobial test. Based on previous researches, Na-Alg has been prepared for the synthesized Ag-NPs by many different methods like green hydrothermal , microwave-assisted , and solution plasma process (SPP) .
However, there are no reports of ultrasonic radiation. Therefore, this study has proposed a green method of synthesizing Ag-NPs at room temperature by reducing AgNO3 on the Na-Alg for different ultrasonic radiation times. The Alg/Ag-NPs were optimized and characterized to study the effect of irradiation time on the surface plasmon resonance (SPR), structures, stability, and morphologies of the Alg/Ag-NPs. In addition, the application of these Alg/Ag-NPs has been also evaluated as antibacterial activity.
2. Materials and Methods
Silver nitrate (AgNO3) with a percentage of 99.89% (C0721-2284551) and sodium alginate (Na-Alg) were purchased from Bendosen and Acros (New Jersey, USA), respectively. Deionized water was used in all experiments from ELGA Lab-Water Purification System. Nutrient agar (NA) was used from Merck (Germany). All chemicals in this research were of analytical grade and without any purification.
2.2. Preparation of Silver Nanoparticles
An aqueous solution of AgNO3 (10 mM) was added to 400 mL of Na-Alg (1.2 g) in a beaker glass. Then, the Na-Alg solution was stirred until achieving the complete dissolving. Afterwards, the solution was exposed to a high-intensity ultrasound radiation ultrasonic processor (with probe-Ti horn) and placed around 2 cm from the bottom of the beaker which used the amplitude of 70% and the cycle of 0.5 from 15, 30, 60, 90, 120, 180, 240, 420, 480, 540, 600, 660, and 720 min. Later, all solutions were kept in freezer to the next analysis and application without any washing. A control was prepared without irradiation to compare the effects of ultrasound. All of this reaction was conducted under ambient temperature.
2.3. Characterization of Silver Nanoparticles
The ultrasonic radiation was utilized by Hielscer UP-200S-RN, Germany (probe 1 cm of diameter), and the pH meter by HACH sensION (MM374) was used. The Alg/Ag-NPs were characterized by UV-Visible (UV-Vis) spectroscopy (UV-1800 Shimadzu) in the range of 300–800 nm to study the formation of nanoparticles. The crystalline structure of Alg/Ag-NPs was recorded by X-ray diffraction (XRD) at a scan of 2°/min of 5–80° using Philips (X’pert, Cu-Kα). Transmission Electron Microscopy (TEM) analysis (Tecnai G2 F20 series) was carried out to examine the size and shape of the Alg/Ag-NPs. The morphology and the elemental atom of the nanoparticles were studied by Field Emission Scanning Electron Microcopy (FESEM) (JEOL-JSM-7600F) equipped with an Energy Dispersive X-ray (EDX) (XL 30 Philips Instruments). Zeta potential analyser (NanoPlus Zeta, Otsuka Electronics) determined the stability of the Alg/Ag-NPs. The Fourier transform-infrared (FT-IR) spectroscopy (NICOLET 6700) was recorded on spectrometer by ATR method and scanned range of 600–4000 cm−1.
2.4. Evaluation of Antibacterial Activity
The in vitro antibacterial evaluation of the Alg/Ag-NPs was studied using disc diffusion method, determining the inhibition zone in mm. Then, it was confirmed according to the recommended standards of the National Committee for Clinical Laboratory Standards. Four pathogens that are two Gram-positive bacteria, Bacillus subtilis B29 and Staphylococcus aureus S276, and two Gram-negative bacteria, Escherichia coli E266 (the last three purchased from IMR) and Salmonella choleraesuis ATCC10708 (purchased from ATCC), were used for the antibacterial assay. The bacteria culture was standardized to 0.5 McFarland standard which was approximately 108 cells. Afterwards, the surface of the NA was completely inoculated using a sterile swab which was steeped in the prepared suspension of each bacterium. Then, sterile paper disc (6 mm) was immersed shortly to the Alg/Ag-NPs of 60, 120, 240, 480, and 720 min after irradiation, placed on the NA, and left to incubate at 30–37°C for 18–24 hours. Streptomycin was used as the positive control in order to study the sensitivity of the bacteria and the sterile water as the negative control. After complete incubation, the diameter of the growth inhibition zones was measured including the disc. All these tests were done in triplicate.
3. Results and Discussion
A schematic illustration of this synthesized Alg/Ag-NPs method of ultrasonic radiation is presented in Figure 1.
The production of radicals of H∙ and OH∙ under ultrasonic radiation is notable and supported by Alg as the stabilizer caused this method to be a powerful technique as a green synthetic approach for synthesizing of Ag-NPs. The ultrasonic radiation is the main reducing agent in the producing Ag seed (Ag°) capped by the Alg . This equipment produces acoustic cavitation and bubble influencing the producing of local heating and high pressures (~5000°C and ~1000 atm) and the following produced free-radicals :
The mechanisms in producing Ag-NPs were firstly introduced by Nagata et al., shown in (1)–(6) . In this reaction, hydroxyl groups from Alg contributed to bearing of Ag° due to high-intensity ultrasonic radiation. Equations (7)-(8) are produced by high possibility of single silver atom to nuclei and grow to the Ag cluster as aggregation and/or disaggregation to the smaller nanoparticles . Moreover, because of high concentration of produced H+ ions, it related to decreasing of the pH which could show that before irradiation the pH was 8.72; however, after 720 min ultrasonic irradiation the pH became decreased to 6.05. The reduction of Ag+ ions was evidenced from the changing color before and after irradiation, shown in Figure 2. This positive indication of the formation of Ag-NPs will be discussed by the UV-Vis spectroscopy. The control study without irradiation did not show any different change of the color; therefore, this can indicate that ultrasound irradiation has been contributed in the producing Ag-NPs.
3.1. UV-Vis Spectroscopy Analysis
This equipment is the most widely used for structural characterization of the Ag-NPs. Figures 3 and 4 show the UV-Vis spectra after irradiation. For Figure 3, the absorption spectrum of the Alg/Ag-NPs prepared by ultrasonic irradiation shows a SPR band with a maximum of 452–465 nm. This SPR indicated the presence of spherical or roughly spherical Ag-NPs . By increasing irradiation time from 15 to 240 min, the UV-Vis peaks intensity for Alg/Ag-NPs increased. Otherwise, the shifting occurred to the left side which is related to the blue-shift from 465, 460, 463, 463, 463, 457, and 452 nm for 15, 30, 60, 90, 120, 180, and 240 min, according to Mei’s theory that blue-shift influences the decreasing of the particle size . In addition, an increasing concentration of the Alg/Ag-NPs happened due to the long time irradiation. These spectra were also supported by the previous research  that the SPR intensity could be increased by increasing time radiation.
Figure 4 shows the unique phenomenon that after 420 min of irradiation the Alg/Ag-NPs peak intensity compared with the previous period became decreased. This phenomenon was due to the agglomeration of Ag-NPs, because of their high specific surface area, thereby deteriorating their performances .
Despite that, the intensity of Alg/Ag-NPs became increased again in continuing irradiation until 720 min and associated with the increased concentration of Alg/Ag-NPs. However, no shifting occurred in the wavelength from 420 to 720 min which was represented as no change in the size of Ag-NPs . The TEM images will be clarifying this observation after ultrasonic irradiation.
3.2. XRD Pattern Study
The crystallinity of Alg/Ag-NPs has been studied by the XRD pattern. Figure 5 shows the XRD pattern of Na-Alg and the Alg/Ag-NPs. Figure 5(a) indicates the peak at 13.91° and 22.01° which corresponded to the Na-Alg structure. In addition, Figures 5(b) and 5(c) show the Alg/Ag-NPs after 240 and 720 min irradiation, respectively. These two patterns show the peaks in 38.30°, 44.37°, 64.54°, and 77.47° that could be attributed to (111), (200), (220), and (311) crystallographic planes of the face-centered cubic (fcc) structure. Based on reference database ICDD/ICSD from X’Pert High Score Plus (reference code: 01-087-0719), these peaks are specific compound to the crystalline of Ag. Therefore, this material is the Alg/Ag-NPs without any impurities .
The particle size has been calculated using Debye-Scherrer equation:where is the average crystalline particles size of Ag-NPs, is the Scherrer constant with value of 0.9, represents the X-ray wavelength (radiation/wavelength Cu -alpha is 0.154 nm), and is the Bragg angle which is , . Herein, is the full width at half maximum of the diffraction peak. It can be found that the calculated average size is 14.04 nm. This result has similarity to the XRD pattern of the Ag-NPs from waste using alkaline protease .
3.3. TEM Analysis
TEM images give valuable information about size and shape of the nanoparticles. Figure 6 represents the Alg/Ag-NPs after 60, 240, and 720 min ((a)–(c)) after ultrasonic radiations. These morphologies should be related to the different irradiation times. From all these results, the images show that the Ag-NPs are spherical in shape. However, they have different size of the Alg/Ag-NPs with the long time radiation, the size, and standard deviation being , , and nm for 60, 240, and 720 min after irradiation, respectively.
Moreover, the number of the particles became increased with the following time radiation: 124, 226, and 299 for 60, 240, and 720 min, respectively. This increasing of number was due to the increasing of the concentration of Ag-NPs. In addition, SPR for UV-Vis spectra shows that the blue-shift appearance means that the size of particles decreases. However, TEM observation shows that the particle size almost increases by following irradiation times. This phenomenon was due to the agglomeration of the Ag-NPs which also occurred to Pant et al. on their synthesized Ag-NPs on polymer matrix [29, 33].
3.4. FESEM and EDX Analysis
The FESEM images have been used to study the surface morphology of media which can be shown in Figure 7. A narrow size distribution of Ag-NPs has been anchored and well distributed on the surface of Alg as the stabilizer. It illustrates that the particles are predominantly spherical and some of them aggregate into larger particles. This aggregation was due to the unique characteristic of Ag-NPs  which has been discussed before. This condition is considered by TEM observation that the nanoparticle became increased with the increasing irradiation times and some particles grew to be agglomerated. Moreover, Balavandy et al.  have investigated with the same condition as well that Ag-NPs fully covered the surface of media.
EDX spectroscopy was used to study the elemental atom of the nanoparticles which is shown in Figure 7. The spectra reveal strong signal in the silver region and confirm the formation of Alg/Ag-NPs which originated from the biomolecules of Na-Alg as the media and also the presence of elemental Ag. Metallic silver generally shows typical optical absorption band at approximately 3.1, 3.3, and 3.4 KeV . On the other hand, the signals of O, C, and Na in the EDX spectra were recorded from the Alg. The ratios of C, O, Na, and Ag are 49.71%, 34.07%, 6.75%, 9.48% for 240 min and 43.67%, 33.36%, 6.04%, and 16.94% for 720 min, respectively. From the binding energy, no impurity is detected; therefore, this result reported that the product was composed of the high purity of Ag-NPs on the Alg as the stabilizer and it is similar to the previous research that EDX data clearly shows the elemental silver on the media .
3.5. Zeta Potential Study
Zeta potential is equipment for studying the stability of the material under dispersion condition. It can be used as an important parameter for characterizing the electrical properties of the interfacial layer . For the approximation, Smoluchowski method has been used as the method of this measurement. Figure 8 shows that zeta potential values were to be −26.16, −49.43, and −67.56 mV for Na-Alg, 240 and 720 min ((a)–(c)) after ultrasonic radiation, respectively. This negative value is related to the presence of electronegative functional group from the Alg .
Based on ASTM for the stability colloidal suspension, the value between 30 and 40 mV either positive or negative can indicate moderate stability; however, if the zeta value is more than 40 mV, it means a high stability suspension . Thereby, this Na-Alg was a moderate condition (−26.16 mV) in Figure 8(a) due to unstable suspension under water solution. In addition, after treatment under ultrasonic radiation, the zeta potential value became increased to more than −40 mV which indicated the high stability of Alg/Ag-NPs especially after 720 min irradiation (−67.56 mV). This zeta value was similar to that in the reported research by Sadeghi and Gholamhoseinpoor; however, they had studied Ag-NPs using Ziziphora tenuior (Zt) extract .
3.6. FT-IR Analysis
The FT-IR measurements were carried out to identify the functional group of the material. Figure 9(a) shows the pure Na-Alg spectra with the characteristic band of O–H stretching at 3252 cm−1 and –COO- (asymmetric) and –COO− (symmetric) at 1594, 1407, and 1297 cm−1 . An intensive band at 1026 cm−1 is representing the stretching of C–O–C group; the peaks at 949 cm−1, 885 cm−1, and 814 cm−1 belong to C–C–H stretching, C–O–C glycosidic linkage (ring breathing), and –C–C (C–C–O ring) , respectively.
On the other hand, Figure 9((b)-(c)) shows the spectra after ultrasonic irradiation for 240 and 720 min. Views of new peaks at 1338 and 1341 cm−1 are C–H and C–O–H due to ring stretching from the Alg with Ag-NPs. The comparison of FT-IR spectra between the Alg and Alg/Ag-NPs showed only minor changes in the position as well as the absorption bands. Due to the Ag-NPs, the O–H stretching vibration shifted from 3252 to 3257 cm−1 . From some other shifting and decreasing peaks intensities in FT-IR peaks, there has been an absorption mechanism as interaction between Alg and Ag-NPs. Thereby, FT-IR spectrum confirms that Ag-NPs have been capped by the lone pair electrons around the oxygen atoms in organic compound in Alg with van der Waals interaction forces .
3.7. Evaluation of Antibacterial Activity
The antibacterial activity of Alg/Ag-NPs was evaluated against Staphylococcus aureus S276, Bacillus subtilis B29, Escherichia coli E266, and Salmonella choleraesuis ATCC10708 which are common in infectious diseases. The evaluation was done on the different irradiation times that are 60, 120, 240, 480, and 720 min after irradiation for (A1), (A2), (A3), (A4), and (A5), respectively, and the results are shown in Table 1 and Figure 10. From Table 1, it can be understood that, by increasing irradiation times, the concentration of Alg/Ag-NPs became increased which is related to the UV-Vis and TEM results; then, it is responsible of increasing the antibacterial activity. The Alg/Ag-NPs have high antibacterial activity in all pathogens on both the Gram-positive and Gram-negative bacteria especially on B. subtilis B29 which is the strongest in this antibacterial test. This case on B. subtilis B29 is due to not having lipopolysaccharides in their body .
These high antibacterial activities are due to the nanosize of the Alg/Ag-NPs at ~20 nm based on TEM observation. Thereby, it could be accumulated on the bacterial membrane, caused by their permeability, and resulted in cell death; and the positive charge of Ag ions was a crucial part as well to this antibacterial activity . This process can be illustrated in a schematic illustration shown in Figure 11. The similar potential to this as antibacterial agent from Ag-NPs has been studied using olive leaf extract .
In this study, Ag-NPs have been successfully prepared in Na-Alg following a green and physical reducing method under ultrasonic irradiation. The obtained SPR peaks for Ag-NPs appeared at around 467–473 nm by using UV-Vis spectroscopy. The XRD pattern also displayed the fcc crystal structure of the Ag-NPs without any impurities. TEM results indicated that the particle size of Ag-NPs almost increased with increasing ultrasonic irradiation times to be 20.16, 21.81, and 22.38 nm for 60, 240, and 720 min. Zeta potential exhibited a high stability of Alg/Ag-NPs with value at −67.56 mV for 720 min. Furthermore, the evaluation of the antibacterial activity shows the potential due to appearing inhibition zone for both Gram-positive bacteria and Gram-negative bacteria. Therefore, it could be concluded that the ultrasonic technique using Na-Alg has a remarkable potential for synthesizing Alg/Ag-NPs which is promising for antibacterial wound dressing such as surgical devices. In addition, long time irradiation is another part that should be improved in future works.
All authors declare that they have agreed to publish this paper and that there are no competing interests.
This research was supported by the Malaysian Ministry of High Education and Universiti Teknologi Malaysia (UTM) under Fundamental Research Grant Scheme (FRGS) grant (Vot. no. 4F793). The authors also thank the Research Management Centre (RMC) of UTM for providing an excellent research environment to complete this work.
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