**2. Green nanoparticles of silver**

**1. Introduction**

150 Fourier Transforms - High-tech Application and Current Trends

Investigation of nanoparticles by different methods, especially by Fourier transform infrared (FTIR) spectroscopy, is very interesting in last years since they have a wide potential applica‐ tion in different industries [1–5]. Thus, silver nanoparticles (AgNP) of polysaccharide type and other natural products have scientific interest, but practical importance too, because of their application in pharmaceutical and cosmetic products development due to proven anti‐ microbial and antioxidant activities [6–9]. The nanoparticles are commonly synthesized by silver (or other metals) ions reduction to elementary state. But, reducing agents should also possess stabilizing properties in order to prevent aggregation [1]. Bankura and coworkers described a simple method of AgNP synthesis at room temperature from dextran, as well as their characterization and microbiological activity [6]. Pullulan‐mediated Ag nanopar‐ ticles, their synthesis, characterization, and microbiological activities are also reported [4]. Soluble starch, starch‐like polysaccharides, and chitosan are used in AgNP synthesis [5–9]. AgNP chitosan/gelatin bionanocomposites have also been studied [5]. Compounds contain‐ ing carbonyl group are relatively easy complexed with different metals [10–12]. This func‐ tional group contains some polysaccharide derivatives like carboxymethyl cellulose (CMC) or carboxymethyl dextran (CMD), which are obtained by different chemical reactions of proton exchange between OH groups of glucoside moiety and carboxymethyl groups. AgNP‐CMC

nanoparticles were prepared in weak alkaline solution by reaction of AgNO<sup>3</sup>

relation by various FTIR techniques.

reducing and capping agent. It has been established that size distribution and morphology of mentioned nanoparticles are depended on Ag: CMC weight ratio, reaction time, tempera‐ ture, and pH value of the reaction system. FTIR spectrophotometric analysis has shown that interactions between AgNP and polysaccharide have steric character [13]. Studies of com‐ posite hydrazine‐CMD and CMD magnetic Fe‐based nanoparticles [14, 15] have shown that solubility of these nanoparticles depends on pH value (NaOH), not on CMD content. This fact indicates on strong interactions of carbonyl group with magnetic nanoparticles on the surface. Hence, there are indications that carboxymethyl dextran form nanoparticles with Ag ions. CMD possess COOH group which can react with positively charged Ag ions to form complex compounds, but, it can also reduce Ag ions and stabilize the formed nanoparticles as in the case of hyaluronic acid with AgNP [16]. Since CMD has the ability to form nanoparticles, the intention of this chapter is to contribute in clarification of AgNP synthesis, physico‐chemical characterization by FTIR spectroscopic, diffraction and chromatographic methods, as well as testing of their antimicrobial activity. On the other hand, the biocomplexes based on exo‐ polysaccharides are important in treatment of biometals deficiency in human and veterinary medicine [1, 17]. Polysaccharides, oligosaccharides and their derivatives, as well as simple sugars, may be used as ligands for the synthesis of biocomplexes with different metal ions (Cu, Fe, and Zn). These biocomplexes have an important role in metal ions transporting in organism [18]. Despite a number of studies of this kind of complexes, the investigations of effect of their structure to pharmaco‐biological activity are still interesting [19]. In this respect, the presented chapter offers further progress in the investigation of cobalt complex synthesis with dextran oligosaccharide, spectroscopic characterization, and the spectra‐structure cor‐

with CMC as a

Recent investigations of nanoparticles synthesis are mostly directed to green synthetic meth‐ ods development. These methods include nontoxic reagents, synthesis procedures without problematic side products, and especially usage of biodegradable materials. Thus, chemical reduction in silver ions is the most frequently used besides photochemical or electrochemi‐ cal methods [1]. In order to reduce and stabilize the Ag nanoparticles, the polysaccharides (dextran, starch, pullulan, cellulose) and their derivatives (dextran sulfate‐DS, carboxymethyl cellulose, carboxymethyl dextran, chitosan, hyaluronic acid, heparin), and biocomposites AgNP chitosan/gelatin, are developed and improved [2–6, 13, 14, 16, 20–25]. Characterization of these particles has been carried out by UV‐Vis, FTIR spectroscopy, X‐ray diffraction (XRD), and energy‐dispersive X‐ray (EDX) methods. Electronic microscopy techniques [scanning electron microscopy (SEM) and TEM] are used for particle size determination and distribu‐ tion, as well as shape defining. It is interesting that question of interaction nature between reducing and stabilizing agents with AgNP is still opened. Some authors [13] consider that steric physical interactions are relevant, while others [2, 16] give an explanation via coor‐ dination complex of Ag ions with reducing and stabilizing agents which contain suitable functional groups (COOH, NH2 , OH, OSO<sup>3</sup> H), as in the case of Cu(II) ions complexes with carboxymethyl dextran or dextran sulfate (**Figure 1**).

**Figure 1.** Structural fragment of dextran sulfate sodium salt (DS) and carboxymethyl dextran (CMD) molecule.

Having in mind these facts, it can be assumed that dextran sulfate, which contains one or more sulfo groups in its structure, can be used as reducing and stabilizing agent for the AgNP synthesis. Also, it may be assumed that dextran sulfate forms complexes similar to CMD about what there are no literature data. Therefore, investigations in this chapter are related to the AgNP‐DS and AgNP‐CMD synthesis, their characterization by different methods and antimicrobial activity determination.

### **2.1. Synthesis procedure**

The synthesis of AgNP‐DS has been performed in a reactor at temperature of 100°C, dur‐ ing 240 min, at constant pH of 7.5 and continuously stirring. Dextran sulfate has been used as a ligand in the synthesis. The synthesis is performed by DS solution (100 cm<sup>3</sup> , 0.002 M) adding in 100 cm<sup>3</sup> of AgNO<sup>3</sup> solution (0.001 M). The complex formation has been monitored via changing of reaction solution color, from white to yellow. The AgNP‐DS complex was precipitated with 96% ethanol after cooling down the reaction mixture to the room tem‐ perature. The obtained product has been dried at105°C under vacuum during 180 min. On the other hand, the carboxymethyl dextran has been used in the case of the AgNP‐CMD complex synthesis. The synthesis has been performed by 100 cm<sup>3</sup> of AgNO<sup>3</sup> (0.001 M) solu‐ tion adding in 200 cm<sup>3</sup> of CMD ligand solution (0.002 M) at constant pH of 7.0 (adjusted by NaOH). The synthesis is performed to the defined M:L ratio (from 1:1 to 1:2) by chang‐ ing of reagents volume. The complexation has been performed at 100°C and continuously stirring during 120 min. A successful outcome of the AgNP‐CMD complex synthesis has been identified by changing of reaction solution color, from white to yellow. The reaction mixture has kept under reflux 24 h more, after that it has cooled to room temperature, and the complex AgNP‐CMD has precipitated by 96% ethanol. Final product has been dried at 105°C under vacuum during 180 min. The prepared AgNP‐DS and AgNP‐CMD complexes were characterized by different methods (FTIR, UV‐Vis, SEM, XRD, EDX) and by antimi‐ crobial activity.

### **2.2. FTIR study**

The FTIR spectra were recorded by BOMEM MB‐100 (Hartmann & Braun, Canada) FTIR spec‐ troscope and by KBr technique, at room temperature with 2 cm−1 resolution. Spectra‐structure correlation has been performed on empirical manner [2, 22], by comparing the spectra of ligands (DS and CMD) with the spectra of their complexes (AgNP‐DS and AgNP‐CMD). The appropriate FTIR spectra are shown in **Figures 2** and **3**.

The results of the complex AgNP‐DS spectral analysis are shown in **Table 1**. They show the position and assignations of bands that come from vibrations of all types of sulfo groups in DS and AgPN‐DS, as well as bands of deformation CH vibrations outside of the plane of glucopyranose unit which are characteristic for its conformation determining. As it can be seen in **Table 1**, there is a difference in the position of the νas(S–O) band which is shifted ~22 cm−1 toward lower frequencies in the AgPN‐DS spectrum (**Figure 2**), as well as the band from νas(O–S–O) which is shifted ~8 cm−1 toward higher frequencies. This difference in the bands position indicates the formation of coordination complexes between Ag ions and DS, where there is a change in conformation of sulfo groups from Eq to Ax position. The appearance of the spectrum in the area of C–CH out‐of‐plane deformational vibrations sur‐ face coupled with C–C–O, O–C–O and C–O–C vibrations depends on glucopyranose unit conformation [25–27]. When it is 4 C1 conformation, the bands at ~915 cm−1 (weak), 850 cm−1 (shoulder), and 752 cm−1 are expected in the spectrum. The results from **Table 1** show that starting DS retains the same conformation of glucopyranose unit during complex with Ag ions formation. There is a sharp intensive band at 1384 cm−1 in the FTIR spectrum of AgNP‐ DS (**Figure 2**) observed by other authors who have investigated similar complexes and given the explanation of its origin [4, 5, 13, 22]. So, in the case of AgNP‐CMD complex (**Figure 3c**), some authors consider that this band is a result of ν<sup>s</sup> (O–N–O) at O=NO2 − radicals which are formed from AgNO<sup>3</sup> agents participating in the formation of nanoparticles through the surface interactions [13].

Study of Green Nanoparticles and Biocomplexes Based on Exopolysaccharide by Modern Fourier Transform Spectroscopy http://dx.doi.org/10.5772/64611 153

**Figure 2.** FTIR spectra of dextran sulfate sodium salt (a) and AgNP‐DS complex (b).

adding in 100 cm<sup>3</sup>

tion adding in 200 cm<sup>3</sup>

crobial activity.

**2.2. FTIR study**

of AgNO<sup>3</sup>

152 Fourier Transforms - High-tech Application and Current Trends

complex synthesis. The synthesis has been performed by 100 cm<sup>3</sup>

appropriate FTIR spectra are shown in **Figures 2** and **3**.

C1

some authors consider that this band is a result of ν<sup>s</sup>

conformation [25–27]. When it is 4

are formed from AgNO<sup>3</sup>

surface interactions [13].

solution (0.001 M). The complex formation has been monitored

of CMD ligand solution (0.002 M) at constant pH of 7.0 (adjusted

conformation, the bands at ~915 cm−1 (weak), 850 cm−1

(O–N–O) at O=NO2

agents participating in the formation of nanoparticles through the

−

radicals which

of AgNO<sup>3</sup>

(0.001 M) solu‐

via changing of reaction solution color, from white to yellow. The AgNP‐DS complex was precipitated with 96% ethanol after cooling down the reaction mixture to the room tem‐ perature. The obtained product has been dried at105°C under vacuum during 180 min. On the other hand, the carboxymethyl dextran has been used in the case of the AgNP‐CMD

by NaOH). The synthesis is performed to the defined M:L ratio (from 1:1 to 1:2) by chang‐ ing of reagents volume. The complexation has been performed at 100°C and continuously stirring during 120 min. A successful outcome of the AgNP‐CMD complex synthesis has been identified by changing of reaction solution color, from white to yellow. The reaction mixture has kept under reflux 24 h more, after that it has cooled to room temperature, and the complex AgNP‐CMD has precipitated by 96% ethanol. Final product has been dried at 105°C under vacuum during 180 min. The prepared AgNP‐DS and AgNP‐CMD complexes were characterized by different methods (FTIR, UV‐Vis, SEM, XRD, EDX) and by antimi‐

The FTIR spectra were recorded by BOMEM MB‐100 (Hartmann & Braun, Canada) FTIR spec‐ troscope and by KBr technique, at room temperature with 2 cm−1 resolution. Spectra‐structure correlation has been performed on empirical manner [2, 22], by comparing the spectra of ligands (DS and CMD) with the spectra of their complexes (AgNP‐DS and AgNP‐CMD). The

The results of the complex AgNP‐DS spectral analysis are shown in **Table 1**. They show the position and assignations of bands that come from vibrations of all types of sulfo groups in DS and AgPN‐DS, as well as bands of deformation CH vibrations outside of the plane of glucopyranose unit which are characteristic for its conformation determining. As it can be seen in **Table 1**, there is a difference in the position of the νas(S–O) band which is shifted ~22 cm−1 toward lower frequencies in the AgPN‐DS spectrum (**Figure 2**), as well as the band from νas(O–S–O) which is shifted ~8 cm−1 toward higher frequencies. This difference in the bands position indicates the formation of coordination complexes between Ag ions and DS, where there is a change in conformation of sulfo groups from Eq to Ax position. The appearance of the spectrum in the area of C–CH out‐of‐plane deformational vibrations sur‐ face coupled with C–C–O, O–C–O and C–O–C vibrations depends on glucopyranose unit

(shoulder), and 752 cm−1 are expected in the spectrum. The results from **Table 1** show that starting DS retains the same conformation of glucopyranose unit during complex with Ag ions formation. There is a sharp intensive band at 1384 cm−1 in the FTIR spectrum of AgNP‐ DS (**Figure 2**) observed by other authors who have investigated similar complexes and given the explanation of its origin [4, 5, 13, 22]. So, in the case of AgNP‐CMD complex (**Figure 3c**), The infrared spectra of AgNP‐CMD products and starting CMD agent are compared with literature data and dextran spectrum because of the major bands assignment (**Figure 3**). In the CMD spectrum, vibrations of carboxymethyl groups: ν(C–O) around 1740 cm−1; deformation vibration δ(C–OH) which appears around 1250 cm−1; ν(C–O) vibration around 1150 cm−1; and deformation δ(C–O) vibration around 680 cm−1 are expected to oppose starting dextran for CMD synthesis. Stretching ν(C–O) vibration has been found in similar carboxymethyl poly‐ saccharides; after carboxymethylation of the k‐carrageenan, ν(C–O) has found at 1737 cm−1 [28], for carboxymethylated glucan at 1736 cm−1 [29], as well as at 1750 cm−1 in the spectrum of CMD [14]. As it can be seen from **Figure 3b**, the CMD spectra possess bands at 1740, 1244, 1139, and 682 cm−1 (which are marked by arrows) from CO carboxymethyl vibration of all types. The aforementioned bands are not in the range of dextran (**Figure 3a**) as it is expected. Changes in the position of the above‐mentioned bands can be a good indicator of bonds type that is eventually formed by interaction with Ag<sup>+</sup> ions [16]. Also, the changes in the area of deformation vibration of C–OH are expected. In the case of AgNP‐CMD coordination com‐

plexes formation, the frequency ν(C–O) band should be lower, or, if both O atoms of COOH groups participate in the coordination, the frequency of ν(C–O) vibration should be higher because of electron delocalization, as well as the absence of δ (C–OH) bands.

**Figure 3.** FTIR spectra of dextran (a), CMD (b), and AgNP‐CMD (c).


**Table 1.** FTIR analysis data showing various functional groups present in dextran sulfate (DS) and AgNP‐DS complex.

Speaking about carboxylate anion, delocalization of electrons causes the order of two CO bonds to be the same, so two bands (at 1600 and 1400 cm−1) are expected in CO stretching vibra‐ tion region, which are ascribed to asymmetric and symmetric C–O vibration, as it is indicated in the literature [13, 16, 28]. The similar situation is in AgNP‐CMD complex (**Figure 3c**) in the CO groups vibration area. In fact, in this area of the spectrum, there are two intensive bands (at 1603 and 1420 cm−1) which, according to its position and intensity should be attributed, νas(C–O) and ν<sup>s</sup> (C–O) vibration, indicating coordination of Ag<sup>+</sup> ions with a COOH group. In support of this is the absence of ν(C–O) and δ(C–OH) vibration bands. The appearance of spec‐ trum in the area of 1000–700 cm−1, in all three tested compounds (**Figure 3**), is very similar and according to the literature data [30, 31] suggests 4 C1 conformation of the glucopyranose unit.

### **2.3. UV‐Vis study**

plexes formation, the frequency ν(C–O) band should be lower, or, if both O atoms of COOH groups participate in the coordination, the frequency of ν(C–O) vibration should be higher

because of electron delocalization, as well as the absence of δ (C–OH) bands.

154 Fourier Transforms - High-tech Application and Current Trends

**Figure 3.** FTIR spectra of dextran (a), CMD (b), and AgNP‐CMD (c).

νs

νs

4 C1

conformation of the α‐d‐

glucopyranose ring

**Assignation DS (cm−1) AgNP‐DS (cm−1) Δν (cm−1)** νas (S‐O) 1261 1239 22

 (S‐O) 988 1060 72 νas (O‐S‐O) 824 832 8

(O‐S‐O) 585 588 3

915 915 – 850 850 – 745 752 7

**Table 1.** FTIR analysis data showing various functional groups present in dextran sulfate (DS) and AgNP‐DS complex.

Absorption spectra of starting ligand compounds (DSi CMD) as well as of final complexes (AgNP‐DS and AgNP‐CMD) are obtained by UV‐Vis spectrophotometer (Varian Cary‐100 Conc.). Spectrophotometric analysis was carried out in the range of 200–800 nm using original Cary UV‐Conc. (Varian) software. The obtained UV‐Vis spectra are presented in **Figures 4** and **5**.

**Figure 4.** UV‐Vis spectra of dextran sulfate (DS) and AgNP‐DS complex in function of the synthesis time.

Change in color from yellowish to brown, as well as careful interpretation of UV‐Vis spectra, is used for estimating of AgNP synthesis [2–10]. A strong absorption band, called SPR band (surface plasmon resonance), is expected in 370‐450 nm region in the UV‐Vis spectrum of AgNP [32]. Its exact position depends on numerous factors (the most on AgNP size), while intensity depends on their concentration [4, 21, 33]. Changes in this band position are used as a criterion of the AgNP stability, that is, aggregation of the nanoparticles during the time. As it can be seen from **Figure 4**, the existence of SPR band at 410 nm indicates the AgNP formation. However, its position is changed during synthesis with time, but after 2 h remains constant at 420 nm. The estimated particle size of AgNP‐DS based on the UV data [34] is approximately 40 nm. A change in color from yellowish to brown during the synthesis has been observed in the case of AgNP‐CMD formation. SPR band for this complex (**Figure 5**), synthesized at different molar ratio, is located at 420 nm, which is not present in the starting CMD. It is characteristic that intensity of this band is proportional to the amount of AgPN‐CMD par‐ ticles; it increases with increasing amounts of CMD, or during staying of the reaction mixture for 3 months (**Figure 5C**), which is similar to other studies [21]. Unchanged position of SPR band speaks in favor of good aggregation stability of synthesized particles. UV area below 300 nm was not investigated in the literature. However, in the UV spectra of tested com‐ pounds (**Figure 5**), there is an intense band of the formed complex at 215 nm (π→π\* transition of the carboxyl group [35]) indicating red shift effect compared to CMD. This phenomenon is an indicator of Ag ions interaction with CMD and AgNP‐CMD complex formation.

**Figure 5.** UV‐Vis spectra of CMD ligand, AgNP‐CMD = 1:1 complex (A), AgNP‐CMD = 1:2 complex (B), and AgNP‐CMD complex after 3 months (C).

### **2.4. XRD study**

Crystal structure of AgNP‐DS and AgNP‐CMD nanoparticles was determined and confirmed by X‐ray diffraction (XRD) technique. The samples were prepared by press and pull method in top‐loading specimen plate [36]. The diffractogram was measured in Bragg‐Brentano θ: 2θ geometry by a conventional powder diffractometer, Seifert V‐14, using Cu Kα radiation (λCu Kα1  = 1.5406 Å, Ni filter, generator settings: 30 kV, 30 mA). As an external standard for peak position calibration and instrumental peak broadening determination, LaB6 was used. XRD data were collected over the 2θrange of 5–90°with a step size of 0.02°, and an exposition time of 2 s per step. The obtained diffractograms are shown in **Figures 6** and **7**.

Study of Green Nanoparticles and Biocomplexes Based on Exopolysaccharide by Modern Fourier Transform Spectroscopy http://dx.doi.org/10.5772/64611 157

**Figure 6.** XRD diffraction patterns of AgNP‐DS complex.

intensity depends on their concentration [4, 21, 33]. Changes in this band position are used as a criterion of the AgNP stability, that is, aggregation of the nanoparticles during the time. As it can be seen from **Figure 4**, the existence of SPR band at 410 nm indicates the AgNP formation. However, its position is changed during synthesis with time, but after 2 h remains constant at 420 nm. The estimated particle size of AgNP‐DS based on the UV data [34] is approximately 40 nm. A change in color from yellowish to brown during the synthesis has been observed in the case of AgNP‐CMD formation. SPR band for this complex (**Figure 5**), synthesized at different molar ratio, is located at 420 nm, which is not present in the starting CMD. It is characteristic that intensity of this band is proportional to the amount of AgPN‐CMD par‐ ticles; it increases with increasing amounts of CMD, or during staying of the reaction mixture for 3 months (**Figure 5C**), which is similar to other studies [21]. Unchanged position of SPR band speaks in favor of good aggregation stability of synthesized particles. UV area below 300 nm was not investigated in the literature. However, in the UV spectra of tested com‐ pounds (**Figure 5**), there is an intense band of the formed complex at 215 nm (π→π\* transition of the carboxyl group [35]) indicating red shift effect compared to CMD. This phenomenon is

156 Fourier Transforms - High-tech Application and Current Trends

an indicator of Ag ions interaction with CMD and AgNP‐CMD complex formation.

Crystal structure of AgNP‐DS and AgNP‐CMD nanoparticles was determined and confirmed by X‐ray diffraction (XRD) technique. The samples were prepared by press and pull method in top‐loading specimen plate [36]. The diffractogram was measured in Bragg‐Brentano θ: 2θ geometry by a conventional powder diffractometer, Seifert V‐14, using Cu Kα radiation (λCu

**Figure 5.** UV‐Vis spectra of CMD ligand, AgNP‐CMD = 1:1 complex (A), AgNP‐CMD = 1:2 complex (B), and AgNP‐CMD

 = 1.5406 Å, Ni filter, generator settings: 30 kV, 30 mA). As an external standard for peak

data were collected over the 2θrange of 5–90°with a step size of 0.02°, and an exposition time

was used. XRD

position calibration and instrumental peak broadening determination, LaB6

of 2 s per step. The obtained diffractograms are shown in **Figures 6** and **7**.

**2.4. XRD study**

complex after 3 months (C).

Kα1

**Figure 7.** XRD diffraction patterns of CMD and AgNP‐CMD complex.

From the presented X‐ray diffraction patterns of AgNP‐DS (**Figure 6**) can be noticed the XRD peaks at 38.24, 44.32, 64.58, 77.59,and 81.79°. Based on literature data [4, 6], the characteristic XRD peaks could be determined as next crystallographic planes: 111, 200, 220, 311, and 222. These planes are specific for the face‐centered cubic silver crystals. This statement, along with the specified values, indicates the presence of silver nanoparticles in the synthesized AgNP‐DS complex. Similar to the previous study, the crystal structure of Ag nanoparticles was determined with complex AgNP‐CMD. Based on X‐ray diffraction patterns (**Figure 7**) and the presence of XRD peaks at 38.02, 44.50, and 64.51°, a particular crystallographic planes are as follows: 111, 200, and 220, which are specific for the cubic silver crystals. According to literature [4, 6], the XRD peak at 29.01° is characteristic of the CMD ligand.

The calculation of average AgNP size has been done from the width of reflection in the X‐ray diffraction pattern according to the Scherrer's equation (1):

$$D(\mathcal{D}\theta) \, = K\mathcal{X} \, / \, FWs \, \cos \theta \,\tag{1}$$

where *D* is the mean size of metal nanoparticles (nm); *K* is Scherrer constant (it's chosen 0.9 roughly spherical particles); *λ* is wavelength of X‐ray radiation (nm);*θ* is angle of diffraction (°); and *FW*<sup>S</sup> is specimen broadening of single peak (in radians). *FW*<sup>S</sup> is obtained according to the Eq. (2):

$$FW\_{\mathbb{S}}^{\mathbb{A}} = \, FWHM^{\mathbb{A}} - \, FW\_{\mathbb{1}}^{\mathbb{A}} \tag{2}$$

where *FWHM* is full width at half maximum of the peak; *FW*<sup>I</sup> is instrumental broadening gained from LaB6 diffractogram at the similar 2θangles; and *d* is parameter of deconvolution (here *d* is chosen as 1.5 which means that shape is partly Gaussian and partly Lorentzian). According to Scherrer's equation (1) and XRD peak at 38.24° 2θ from diffraction patterns (**Figure 7**), it is concluded that AgNP have mean crystallite size of 40 ± 4 nm.

### **2.5. SEM and EDX study**

The size and shape of AgNP‐DS and AgNP‐CMD complexes were further characterized by scanning electron microscopy (SEM) on JEOL JSM 5300 scanning electron microscope. Scanning micrographs were transformed into a PC format in order to further analyze the particles morphology. The samples for SEM analysis have been prepared by thin layer of the complex suspension overnight air drying at room temperature. Dried samples have been coated with 10‐nm‐thick film of gold in JPC JEOL‐1100 apparatus. Electron beam of 30 keV has been used. The SEM micrographs of AgNP‐DS (**Figure 8A**) showed both individual par‐ ticles, but a number of aggregates, too. Size of 10–60 nm is predominant for individual spheri‐ cal particles. Images have also indicated that obtained nanoparticles are stable, and they are not in a mutual contact. This can be ascribed to stabilization of the nanoparticles by DS as a capping agent. Aggregates of nanoparticles with poorly defined morphology and irregular structure have also been found (**Figure 8A**).

**Figure 8.** SEM images of AgNP‐DS showing the existence of individual nanoparticle and large aggregates (A) and EDX spectrum of individual AgNP‐DS (B.)

Energy‐dispersive X‐ray (EDX) spectral analysis has been performed by LINK Analytical 2000 QX microprobe assembled on a JEOL JSM 5300 scanning electron microscope. Samples pre‐ pared for SEM analyses have been used for EDX spectra measuring. EDX spectroscopy can be used for qualitative as well as quantitative assessment of silver used for the AgNP production [36]. EDX spectrum of AgNP‐DS is shown in **Figure 8B**. Strong signal comes from elemental silver, while weaker signals come from S, O, and Na (from Na salt of DS), confirming that AgNP are formed as a part of AgNP‐DS. This is consistent with an optical absorption peak appearance at approximately 3 eV (410 nm), which originates from SPR, and it is characteris‐ tic for metallic silver nanocrystals [6].

Similar to the previous complex, the SEM micrographs of AgNP‐CMD (**Figure 9A**) show single particles, but a number of aggregates as well. Particle size of 10–60 nm is dominant for individual spherical particles. SEM images showed that obtained nanoparticles are stable and not in direct contact with each other. This can be explained as stabilization effect of CMD, as a capping agent, on produced nanoparticles. But, aggregated nanoparticles with larger irregu‐ lar structure and no well‐defined morphology were also found (**Figure 9B**).

**Figure 9.** SEM images of individual spherical particles (A) and aggregated nanoparticles of AgNP‐CMD (B).

## **2.6. Antimicrobial study**

The calculation of average AgNP size has been done from the width of reflection in the X‐ray

 θ

diffractogram at the similar 2θangles; and *d* is parameter of deconvolution

= (1)

1 – *d dd FW FWHM FW <sup>S</sup>* = (2)

is obtained according to

is instrumental broadening

*D K FWs* (2 ) / cos θλ

is specimen broadening of single peak (in radians). *FW*<sup>S</sup>

(**Figure 7**), it is concluded that AgNP have mean crystallite size of 40 ± 4 nm.

where *D* is the mean size of metal nanoparticles (nm); *K* is Scherrer constant (it's chosen 0.9 roughly spherical particles); *λ* is wavelength of X‐ray radiation (nm);*θ* is angle of diffraction

(here *d* is chosen as 1.5 which means that shape is partly Gaussian and partly Lorentzian). According to Scherrer's equation (1) and XRD peak at 38.24° 2θ from diffraction patterns

The size and shape of AgNP‐DS and AgNP‐CMD complexes were further characterized by scanning electron microscopy (SEM) on JEOL JSM 5300 scanning electron microscope. Scanning micrographs were transformed into a PC format in order to further analyze the particles morphology. The samples for SEM analysis have been prepared by thin layer of the complex suspension overnight air drying at room temperature. Dried samples have been coated with 10‐nm‐thick film of gold in JPC JEOL‐1100 apparatus. Electron beam of 30 keV has been used. The SEM micrographs of AgNP‐DS (**Figure 8A**) showed both individual par‐ ticles, but a number of aggregates, too. Size of 10–60 nm is predominant for individual spheri‐ cal particles. Images have also indicated that obtained nanoparticles are stable, and they are not in a mutual contact. This can be ascribed to stabilization of the nanoparticles by DS as a capping agent. Aggregates of nanoparticles with poorly defined morphology and irregular

**Figure 8.** SEM images of AgNP‐DS showing the existence of individual nanoparticle and large aggregates (A) and EDX

diffraction pattern according to the Scherrer's equation (1):

158 Fourier Transforms - High-tech Application and Current Trends

where *FWHM* is full width at half maximum of the peak; *FW*<sup>I</sup>

(°); and *FW*<sup>S</sup>

gained from LaB6

**2.5. SEM and EDX study**

structure have also been found (**Figure 8A**).

spectrum of individual AgNP‐DS (B.)

the Eq. (2):

Agar disk diffusion method has been used for measuring of antibacterial and antifungal activity of AgNP stabilized by DS. One fungal strain (*Candida albicans* ATTC 2091) and nine bacterial strains such as Gram‐positive (*Staphylococcus aureus* ATCC 25923, *Bacillus cereus* ATCC 11778, *Bacillus luteusin haus strain*, *Bacillus subtilis* ATTC 6633, and *Listeria monocytogenes* ATCC 15313) and Gram‐negative (*Escherichia coli* ATTC 25922, *Pseudomonas aeruginosa* ATTC 27853, *Klebsiella pneumoniae* ATTC 700603, and *Proteus vulgaris* ATTC 8427) were used as an indicator strain for this analysis. Preparation of suspension was performed by already described method [37]. Direct colony method has been used for bacterial and yeast suspensions preparation, and the colonies have been taken directly from the plate and suspended in 5 cm<sup>3</sup> of sterile 0.85% saline. Turbidity of the initial suspension has been adjusted comparing with 0.5 McFarland's [38]. After this adjustment, the bacterium and yeast suspensions contained close to 108 and 106 colony‐forming units (CFU)/cm<sup>3</sup> , respec‐

tively. Initial suspension has been additionally prepared by tenfold dilution into sterile 0.85% saline. Inoculation of bacterial cell suspensions has been done to the trypton soya agar plates, while the yeast suspension to the Sabouraud maltose agar plates. Standard sterile cellulose disks of 9 mm diameter have been impregnated with different AgNP‐DS concentrations (0.25, 0.5, 1.0 mg cm‐3) and putted on surface of the inoculated plates. The plates have been incubated at 37°C for 24 h. Inhibition zones were evaluated by measuring the diameter of the zones growth (**Table 2**).


**Table 2.** Antimicrobial activity of AgNP‐DS, radial diameter of inhibition zones (mm) for tested bacterial and fungal strains.

The investigated AgNP‐DS solution has shown antibacterial activity against *S. aureus, B. cereus, B. luteus in haus strain, B. subtilis, L. monocytogenes, E. coli, P. aeruginosa, K. pneumoniae, and P. vulgaris* bacteria, which is proved by clear inhibition zones of the bacteria growth around the disks (**Table 2**). Inhibition has been observed for all analyzed bacterial strains in the 0.25 mg cm‐3 concentration of AgNP‐DS, indicating relatively low minimal inhibitory concentration against these microorganisms. For example, Dhand and coworkers [39] stated that minimal inhibitory concentrations for *E. coli* and *S. aureuss* were around 0.26 mg cm‐3. The highest inhibition zones were observed against *P. aeruginosa* and *B. luteusin haus strain*, and inhibition zones of AgNP‐DS against these microorganisms in 1.0 mg cm‐3 concentra‐ tion were 26 and 24 mm, respectively. *P. vulgaris* was the least sensitive to the AgNP‐DS (1.0 mg cm‐3) activity with zone of 15 mm. Investigation of AgNP‐DS activity in different concentrations against other bacterial strains has shown similar results with inhibition zones of 16–17 mm, 18–19 mm, and 18–21 mm for the AgNP‐DS concentration of 0.25, 0.5, and 1.0 mg cm‐3, respectively. The results for *K. pneumoniae*, *B. luteus in haus strain,* and *P. aeruginosa* are higher compared to data for AgNP‐CMD (**Table 3**). Antifungal activity against *C. albicans* was observed only in the concentration of 0.5 mg cm‐3 AgNP‐DS. Low antimicrobial activity of AgNP against *C. albicans* has been estimated for AgNP stabilized with CMD. The mechanism of AgNP antimicrobial activity can be related to silver accumu‐ lation in the membranes of bacteria, which cause cell death [40]. Silver cation can react with thiol groups and proteins in the cells; nonetheless, it can inactivate enzymes essential for the normal cell metabolism [41]. The investigated AgNP‐DS particles, in the concentration of 1.0 mg cm‐3, have shown a number of specificity concerning its antimicrobial activity. It is important that higher concentration of silver is harmful for consumer and for microbes as well, so the lower concentrations are much more applicable. The effective concentrations of AgNP, which have effect in organisms different from the control, are in the range from a few ng dm‐3 to 10 mg dm‐3; this effective concentration is depended on the organism itself as well as many other factors [42]. Having in mind these results, it can be concluded that this design of silver nanoparticles synthesis has a great potential because of their antimicrobial activity.

tively. Initial suspension has been additionally prepared by tenfold dilution into sterile 0.85% saline. Inoculation of bacterial cell suspensions has been done to the trypton soya agar plates, while the yeast suspension to the Sabouraud maltose agar plates. Standard sterile cellulose disks of 9 mm diameter have been impregnated with different AgNP‐DS concentrations (0.25, 0.5, 1.0 mg cm‐3) and putted on surface of the inoculated plates. The plates have been incubated at 37°C for 24 h. Inhibition zones were evaluated by measuring

> *B. cereus* 16 18 19 *B. subtilis* 16 17 19 *S. aureus* 17 18 19 *B. luteus haus strain* 20 21 24

> *K. pneumoniae* 16 18 19 *E. coli* 17 18 21 *P. aeruginosa* 23 24 26

**0.25 mg cm−3 0.5 mg cm−3 1.0 mg cm−3**

The investigated AgNP‐DS solution has shown antibacterial activity against *S. aureus, B. cereus, B. luteus in haus strain, B. subtilis, L. monocytogenes, E. coli, P. aeruginosa, K. pneumoniae, and P. vulgaris* bacteria, which is proved by clear inhibition zones of the bacteria growth around the disks (**Table 2**). Inhibition has been observed for all analyzed bacterial strains in the 0.25 mg cm‐3 concentration of AgNP‐DS, indicating relatively low minimal inhibitory concentration against these microorganisms. For example, Dhand and coworkers [39] stated that minimal inhibitory concentrations for *E. coli* and *S. aureuss* were around 0.26 mg cm‐3. The highest inhibition zones were observed against *P. aeruginosa* and *B. luteusin haus strain*, and inhibition zones of AgNP‐DS against these microorganisms in 1.0 mg cm‐3 concentra‐ tion were 26 and 24 mm, respectively. *P. vulgaris* was the least sensitive to the AgNP‐DS (1.0 mg cm‐3) activity with zone of 15 mm. Investigation of AgNP‐DS activity in different concentrations against other bacterial strains has shown similar results with inhibition zones of 16–17 mm, 18–19 mm, and 18–21 mm for the AgNP‐DS concentration of 0.25, 0.5, and 1.0 mg cm‐3, respectively. The results for *K. pneumoniae*, *B. luteus in haus strain,* and *P. aeruginosa* are higher compared to data for AgNP‐CMD (**Table 3**). Antifungal activity against *C. albicans* was observed only in the concentration of 0.5 mg cm‐3 AgNP‐DS. Low

**Table 2.** Antimicrobial activity of AgNP‐DS, radial diameter of inhibition zones (mm) for tested bacterial and fungal

the diameter of the zones growth (**Table 2**).

160 Fourier Transforms - High-tech Application and Current Trends

strains.

**Microbes AgNP‐DS concentration**

Fungi *C. albicans* – 16 – Bacteria G<sup>+</sup> *L. monocytogenes* 16 17 18

Bacteria G‐ *P. vulgaris* 13 14 15


**Table 3.** Antimicrobial activity of AgNP‐CMD, radial diameter of inhibitionzones (mm) for tested bacterial and fungal strains.

In order to compare antimicrobial activity of similar complexes, the results of AgNP‐CMD antimicrobial activity (radial diameter of inhibition zones) are presented in **Table 3**. The AgNP‐CMD solution exhibited antibacterial activity against bacteria *B. lutea*, *B. aureus*, *B. cereus*, *E. fecalis*, *P. aeruginosa,* and *K. pneumoniae* showing clear inhibition zones of the bacteria growth around the disk. AgNP‐CMD in the concentration of 1.0 mg cm‐3 have shown a num‐ ber of specificity concerning its antimicrobial activity. The antifungal activity of the AgNP‐ CMD has been analyzed by agar disk diffusion method. *Aspergillus* spp., *Penicillium* spp*.,* and *C. albicans* were inhibited in a concentration‐dependent manner. The radial growth inhibition zones increased with the AgNP‐CMD concentration increasing from 0.25 to 1.0 mg cm‐3. The fungus *Penicillium* spp. was more sensitive to the AgNP‐CMD comparing to the other two fungal strains.
