A completely green approach to the synthesis of dendritic silver nanostructures starting from white grape pomace as a potential nanofactory

2.1 Plant material and chemicals

White grape pomace from Malvasia di Candia cultivar was collected from local organic wineries soon after the vinification process and freeze-dried in laboratory without any pre-treatment.

All reagents used in the present study were of analytical grade. Silver nitrate, silver nanoparticles dispersion (0.02 mg mL⁻¹ in aqueous buffer, containing sodium citrate as stabilizer, 20 nm particle size), Folin-Ciocalteu reagent, 2,2-diphenyl-1-picrylhydrazyl radical (DPPH^•), 2,2′-azinobis-(3-ethylbenzothiazolin-6-sulfonic acid) (ABTS), potassium persulfate, and vanillin were purchased from Sigma-Aldrich (Milan, Italy), while the employed water was previously purified in a Milli-Q system (Millipore, Milan, Italy) and then autoclaved.

2.2 Preparation and preliminary characterization of white grape pomace extract (WGPE)

Freeze-dried white grape pomace (1.0 g) was extracted with 10 mL of autoclaved milliQ water. The mixture was exposed to microwave heating for 40 s at 700 W and then stirred at 150 rpm, at room temperature and in the dark for 30 min. The resulting crude extract was centrifuged at 7000g for 15 min at 4 °C and the obtained supernatant (WGPE) was used as reducing and capping agent in the green synthesis of AgNPs. WGPE was characterized as follow: the pH value was measured using a digital pH-meter (785 DMP, Methrom, Milan, Italy), while the total titratable acidity (TA) was measured by titration of WGPE with 0.1 N sodium hydroxide (data were expressed as meq L⁻¹). Moreover, WGPE reducing capacity was assessed by Folin-Ciocalteu reagent and expressed as mg gallic acid equivalents (GAE) L⁻¹ according to Carbone et al. (2011).

2.3 ¹H NMR metabolite profiling and FTIR spectral analysis of WPGE

In order to identify the biomolecules responsible for the reduction and stabilization of metal nanoparticles, both ¹H NMR metabolite profiling and Fourier Transform Infrared (FTIR) spectral analysis of WGPE were carried out as follows.

Concerning ¹H NMR measurements, 800 μL of WGPE were transferred in a NMR tube for the spectroscopic analysis by adding 150 µL of D₂O and 10 µL of 3-(trimethylsilyl)-propionic-2,2,3,3-d4 acid sodium salt (TSP) 10 mM solution. ¹H NMR spectra were recorded at 298 K with a Bruker (Milano, Italy) AVANCE spectrometer operating at a frequency of 400.13 MHz. The spectrum was acquired using 32 K data points over a 7211.54 Hz spectral width and adding 1 k transients. A recycle delay of 5 s and a 90° pulse of 14.5 μs were set up, with an acquisition time of 2.56 s. Saturation of residual water signal was achieved by irradiating it during the recycle delay at δ equal to 4.703 ppm. After the spectral integration, the concentration of some metabolites was calculated with respect to the internal standard TSP, assigned to the chemical shift 0.0 ppm.

Moreover, FTIR spectra of WGPE and WGPE- mediated AgNP samples were acquired on a Thermo-Scientific (mod. Is50) instrument (Thermo Scientific Inc., Madison WI), equipped with an attenuated total reflectance (ATR) ZnSe cell. Spectra were collected by dipping the solution directly on the entire ATR crystal and then left to dry. After that, a total of 128 scans at a resolution of 2 cm⁻¹ in the 4000–700 cm⁻¹ region were collected for each sample. All experiments were performed in triplicate.

2.4 Green synthesis of silver nanoparticles

Silver nanoparticles were synthesized with the assistance of microwave (MW) irradiation. The reaction conditions used for the WGPE-mediated biosynthesis of AgNPs were prior optimized (data not shown) and finally 4 mL of freshly prepared AgNO₃ aqueous solution (1 mM) were mixed with a known volume (0.5–2.0 mL) of WGPE and autoclaved milliQ water was added to reach a final volume of 8 mL, at pH 8.0 (by adding 0.1 N NaOH) in a 100 mL conical flask. The biosynthesis of silver NPs was carried out in a domestic microwave oven (Whirlpool MWD27, Italy) operating at 700 W and 2450 MHz for 40 s. The bio-reduction of Ag⁺ ions to AgNPs was followed by the visual observation of the color change of the solution from pale yellow to brownish-yellow and then confirmed by UV–vis spectral analysis (see below). Obtained colloidal suspensions using different WGPE concentrations were then cooled to room temperature in the dark for 1 h. Free biomolecules not absorbed onto AgNPs were then removed by repeated centrifugation at 15,000g for 20 min at 4 °C, followed by dispersion of the pellet in milliQ water. A calibration curve based on external standard solutions of commercial AgNPs was obtained for the quantification of biosynthesized nanoparticles. Suitable controls were maintained throughout the trials.

2.5 UV–vis and morphological characterizations of WGPE- mediated AgNPs

Preliminary characterization of WGPE-mediated silver nanoparticles was carried out using UV–vis spectroscopy (Evolution 300, THERMO Scientific, Italy). The bio-reduction of Ag⁺ ions in solution was monitored in the wavelength range of 250–600 nm, at a resolution of 1.5 nm (scan speed: 120 nm min⁻¹; data interval: 0.2 nm), and using autoclaved milliQ water as the blank.

The morphology of WGPE-mediated AgNPs was investigated by means of field-emission gun scanning electron microscopy (FEG-SEM) (Cambridge Leo Supra 35, Carl Zeiss), after sputter-coating with gold under argon atmosphere (25 mA, 120 s). Energy Dispersive Spectroscopy (EDS) microanalysis of Ag element was performed by means of INCA Energy 300, Oxford ELXII detector. The average particles diameter was determined considering randomly selected particles from SEM micrographs (ImageJ, NIH). As a reference, commercial AgNPs were also observed at SEM.

2.6 Electrochemical characterization of WGPE- mediated AgNPs

All electrochemical measurements were performed by means of a computer controlled system (AUTOLAB, model PGSTAT 12) equipped with GPES software (ECO-CHEMIE, the Netherlands). Screen Printed Electrodes (SPEs) were prepared in the Laboratory of Analytical Chemistry of the University of Rome “Tor Vergata”. SPEs were printed with a 245 DEK (Weymouth, UK) screen-printing machine. The ink used to print the working and counter electrodes was a graphite-based ink (Electrodag 423 SS from Acheson Milan, Italy), while the ink used to print the reference electrode was a silver one (Electrodag 477 SS, from Acheson Milan, Italy). A flexible polyester film (Autostat HT5, Autotype Italia, Milan, Italy) was used as a support. The diameter of the working electrode was 0.3 cm and its geometric area was 0.07 cm².

Samples of WGPE-mediated silver nanoparticles were characterized by cyclic voltammetry (CV) by direct detection of nanoparticles. Moreover, the matrix (WGPE before the addition of AgNO₃) was analyzed by CV in order to observe the possible presence of electroactive compounds that could interfere with the analysis of nanoparticles.

The CV measurements were carried out in “drop” (60 μL), diluting the sample 1:1 v/v in 0.5 M phosphate buffer + 0.1 M KCl, pH 7.4. Reported data were obtained from three independent experiments with three replicates.

2.7 In vitro antifungal activity and seed toxicity of WGPE-mediated AgNPs

The antifungal activity of WGPE-mediated silver nanoparticles was evaluated by considering the inhibitory effect on the mycelial growth of Fusarium graminearum, the causal agent of the Fusarium Head Blight (FHB). Potato Dextrose Agar (PDA; Formedium, Hunstanton, England) plates (60 mm) were prepared by adding 1 mL of four different concentrations (23.8, 36.4, 73.9 and 98.8 μg mL⁻¹, respectively) of green AgNPs to 9 mL of PDA. Media plates amended with commercial AgNPs (Sigma-Aldrich, Italy) or WGPE were also prepared.

For the bioassay, 4-mm diameter mycelia disc from 7 day-old culture was placed in the center of the PDA plates, prepared as described above. The plates were incubated at 22 °C in the dark. After four days from inoculation, the colony diameters were measured and the percentage growth inhibition was calculated in reference to the growth of the colony on PDA. Two replications were prepared for each treatment and each experiment was replicated twice.

A seed toxicity assay was also performed to test the effect of WGPE-mediated silver nanoparticles on wheat seed germination.

For this purpose, seeds of the durum wheat (T. turgidum ssp. durum) cultivar “Core” were treated with the four AgNPs doses described before. Briefly, 50 seeds were immersed in a 1% sodium hypochlorite solution for 10 min to ensure surface sterility and then were washed three times with sterile water. The treatment was performed by soaking the seeds with 3 mL of the four AgNP solutions for 1 h, at 20 °C, by 200 rpm constant shaking. Treated seeds were immediately transferred onto Petri dishes containing sterile water soaked filter paper and were incubated in a growth chamber at 20 °C. Two replications for each dose of WGPE-AgNPs were applied. Seed germination was monitored daily until 6 days after treatments, and compared to the untreated material.

2.8 Statistical analysis

Described analyses were carried out in triplicate. Statistical analysis of data expressed as mean ± standard deviation, and mean testing (Duncan’s range test) with level of significance set at p ≤ 0.05 were performed with the SPSS 17.0 software (SPSS, Inc., Chicago, Illinois), unless differently specified.

3.1 Characterization and chemical profiling of white grape pomace extract (WGPE)

The so-called “wine grape pomace”, which is the main winery by-product, is a valuable source of polyphenols, especially the one from white grape varieties (Cecchini et al., 2013). Table 1 shows the main chemical parameters of WGPE used in the present study.

Literature data highlighted the role of polyphenols in plant extracts as reducing/capping agent in the green synthesis of metallic nanoparticles (Mittal et al., 2013). However, there is a lack of information about the total phenolic content (TPC) of plant extracts used for this purpose. This is a very important information regarding the standardization of the AgNPs synthesis protocol, as TPC is generally used to measure the average reducing power of plant extracts (Huang et al., 2005). In this regard, we found only one publication reporting the average TPC of plant extract used in the biosynthesis of silver nanoparticles (Makris et al., 2007). In the present study, a WGPE with an initial standardized TPC of 294 mg/L was used in all synthesis experiments (Table 1).

To better understand the role of different plant metabolites involved in the biosynthesis of silver nanoparticles, a FTIR spectral analysis of WGPE was carried out.

As shown in Fig. 1, in the spectrum of WGPE (spectrum A) several diagnostic bands are present: the broad band at 3300 cm⁻¹ is due to the stretching of the O—H groups, while the two peaks at 3310 and 3250 cm⁻¹ account for the stretching of N—H present in the amino or amide groups. Bands in the 3000–2800 cm⁻¹ and 1400–1300 cm⁻¹ regions are associated to C—H stretching and bending, respectively. Furthermore, the bands in the 1900–1450 cm⁻¹ region are due to the stretching of the C⚌O group present in different environments (i.e., acid, ester and keto groups, in unconjugated and conjugated molecules, amide groups) as well as C⚌C stretch in aromatics. Finally, the bands at about 1260 cm⁻¹ and 1040 cm⁻¹ are due to the stretching of C—O in esters and alcohol, respectively.

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Fig. 1 FTIR spectra of (A): WGPE alone; (B): WGPE-mediated NPs obtained by adding 1.5 mL of WGPE in AgNO₃ solution; (C): WGPE-mediated NPs obtained by adding 1.0 mL of WGPE in AgNO₃ solution.

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These results are compatible with the presence, in WGPE, of molecules like polysaccharides, proteins, terpenoids, and flavonoids (Song et al., 2009). To better identify the biomolecules acting as reducing and capping agents, ¹H NMR based metabolite profiling of WGPE was also carried out. NMR spectroscopy can analyze many compounds simultaneously and provide a well-defined molecular profile in few minutes, requiring very small volumes of sample.

In Fig. 2, three main WGPE ¹H NMR spectral regions were identified as follow: (1) aromatic region (>5.5 ppm), (2) carbohydrate region (between 5.5 and 3.0 ppm), and (3) organic and amino acid region (<3.0 ppm).

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Fig. 2 400.13 MHz ¹H NMR spectrum of white grape pomace aqueous extract (WGPE).

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The metabolite assignment was achieved using literature data (Gallo et al., 2014; López–Rituerto et al., 2012; Picone et al., 2011). In the present study, 25 metabolites were unequivocally assigned (Table 2). Several phenolic compounds, as gallic acid, trans-caffeic acid, resveratrol, caftaric acid and epicatechin, were identified in the aromatic region (Gallo et al., 2014; López–Rituerto et al., 2012; Picone et al., 2011; Anastasiadi et al., 2009).

These secondary metabolites act as reducing and stabilizing agents for the bioreduction of the synthesized novel metallic nanoparticles (Kuppusamy et al., 2016). They are different types of antioxidants (Fig. 3) that reduce the metal ions by their functional groups (Malešev and Kuntić, 2007; Makarov et al., 2014) in the ionic or electrostatic interactions with the metal complexes.

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Fig. 3 Structures of the phenolic compounds identified in WGPE using ¹H NMR.

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The mechanisms of action in the complexing activity of these compounds take into consideration the hydroxyl groups: their deprotonation yields to anionic stabilized species by resonance phenomena; their stability is enhanced by the presence of a H bonding pattern (Leopoldini et al., 2011). For example, gallic acid, caffeic acid, epicatechin are excellent free radical scavengers by H atom donation (Leopoldini et al., 2011).

The presence of all these compounds in a unique natural extract, WGPE, consistently increases the antioxidant activity of the final product with respect to each single metabolite. There are not, at our best knowledge, NMR data on these model systems, which should be further investigated. In addition, using NMR spectroscopy it should be possible to study the extent of the interaction of each secondary identified metabolite (Table 2 and Fig. 3), through their quantification, in the mechanisms of antioxidant systems: from these data it will be possible, in further studies, to obtain also new information on the involved kinetic processes. In the present study, the following concentrations in the aqueous WGPE were quantified: 4.1 × 10⁻⁵ and 1.9 × 10⁻⁵ mol L⁻¹ respectively for epicatechin and gallic acid; 7.6 × 10⁻⁶ mol L⁻¹ for caffeic and caftaric acids, and finally for resveratrol a concentration of 4.8 × 10⁻⁶ mol L⁻¹ was found.

3.2 Spectral studies on WGPE-mediated AgNPs

Bio-reduction of Ag⁺ ions in solution was monitored by visual observation and confirmed by using UV–vis spectral analysis. Due to the addition of WGPE, 1 mM solution of AgNO₃ changed from colorless to yellowish brown in the experimental condition adopted (inset, Fig. 4), due to the excitation of surface plasmon resonance (SPR) vibration of NPs. The characteristic SPR peak was observed at 431.6 nm (Fig. 4). Compared to a commercial standard suspension of AgNPs (20 nm sized), SPR peak of WGPE-mediated AgNPs shifted to the red (from 394.6 to 431.6 nm), with a simultaneous broadening of the band, indicating an increase in the particle size (Bar et al., 2009). However, the SPR peak of WGPE-mediated AgNPs was below 450–470 nm, which was the classical SPR wavelength range of plant-mediated synthesis of silver nanoparticles, as reported in literature (MubarakAli et al., 2011; Gnanajobitha et al., 2013). Fig. 4 also shows another peak in the UV region (280–330 nm), which is a characteristic of polyphenol compounds (Terenteva et al., 2015). As shown in Fig. 4, the intensity of this peak declined when WGPE-mediated synthesis of AgNPs took place, indicating that polyphenols acted as reducing and capping agent, as previously described (Huang et al., 2014; Terenteva et al., 2015). The ratio AgNO₃/WGPE (v/v) strongly affected the yield of AgNPs. It is seen from the diagram in Fig. 5 that the yield of AgNPs increased almost linearly by varying the amount of WGPE added to the silver solution from 0.5 to 2.0 mL, corresponding to a final AgNPs concentration ranging from 23.8 to 98.8 μg mL⁻¹. As a consequence, when a larger volume of WGPE (2 mL) is added to a known volume of silver nitrate solution (4 mL; 1 mM), the SPR peak centred at 431.6 nm increases in intensity, pointing out an increase in the number of metallic nanoparticles formed in the reaction medium (data not shown) (Cruz et al., 2010).

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Fig. 4 Visual observations (inset) and UV–vis absorption spectra of WGPE, WGPE-mediated AgNPs, and standard AgNPs (20 nm).

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Fig. 5 Dependence of AgNPs yield on the AgNO₃/WGPE ratio.

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In the present study, the best results for AgNPs synthesis were obtained by using AgNO₃ aqueous solutions at pH 8.0, as nanoparticles formed at this pH value were more stable with no SPR peak broadening (data not shown). Khalil et al. (2014) highlighted that in the reaction the pH influences the electrical charges of biomolecules (i.e. ionization state) in the plant extract used for the green synthesis of AgNPs, affecting their capping and stabilizing abilities. In the present work, AgNPs synthesis was faster at pH 8.0, probably due to the ionization of polyphenolic compounds and other bioactive molecules (i.e. proteins) in WGPE, possessing hydroxyl and carboxyl groups, which are able to bind to metals.

Fig. 6 shows the influence of the reaction time on the intensity of SPR band of WGPE-mediated AgNPs. Data pointed out that the reduction of silver ions and optimal formation of stable AgNPs occurred within 3 h under laboratory conditions, with no significant differences between 3 and 24 h. Therefore, all further studies were carried out for 3 h. FTIR measurements were also carried out on synthetized AgNPs to identify the biomolecules present in WGPE putatively involved in the reduction and capping of green nanostructures (Fig. 1).

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Fig. 6 Dependence of SPR band intensity of WGPE-mediated AgNPs yield on the reaction time.

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The differences between spectra before (WGPE alone; spectrum A) and after NPs formation (B and C spectra) are mainly in the band envelops at 1040 cm⁻¹ and 1600–1400 cm⁻¹ (Fig. 1). This finding suggests that Ag⁺ reduction and the subsequent coordination of WGPE compounds on the surface of the nanoparticles involve the CO, OH and NH groups present in polysaccharides, proteins, terpenoids and flavonoids (Logaranjan et al., 2012; Gan et al., 2012).

3.3 Scanning electron microscopy (SEM) and EDX analysis

It is well known that the intrinsic properties of nanoparticles strongly depend on their structure. In this regard, the morphology of the produced silver based structures was analysed by observation at SEM (Fig. 7) and the presence of Ag was confirmed by EDS microanalysis (Fig. 7c, inset).

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Fig. 7 SEM micrographs of the purchased AgNPs (a and b) and of the obtained WGPE-Ag NPs (c–f) (EDS spectrum for WGPE-Ag NPs, inset of (c).

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The reported SEM micrographs show the formation of dendritic structures of different dimensions, surrounded by randomly distributed single Ag rounded nanoparticles with an average diameter of 33 ± 6 nm (Fig. 7c–f), higher with respect to the commercial standard AgNPs (average diameter of 18 ± 3 nm, Fig. 7a and b). The higher average diameter of the obtained WGPE-derived AgNPs can be ascribed to the presence of a coating based on residual WGPE components that tend to adsorb on the particles surface due to their very high specific surface area, as evident in Fig. 7f and pointed out by FTIR analysis.

Moreover, the obtained dendritic silver structures were characterized by well-defined trunk and branches on both sides of the trunk, composed of nanometric silver particles. The branches length ranged between 15 µm and 30 µm for the smaller structures and between 55 µm and 80 µm for the bigger ones, whereas the trunks were characterised by a length between 70 and 275 µm. It is evident that the dendritic fractal structures were hierarchically formed by the self-assembly of silver nanoparticles as basic building blocks.

In order to explain the underneath crystal growth mechanism and the resulting shape of the silver crystals, several mechanisms have been proposed in literature for the development of dendritic silver nanostructures in non-equilibrium conditions (Mandke et al., 2012; Duan et al., 2014) and, up to date, the underneath process has not been completely understood yet. Most of the proposed mechanisms are based on a diffusion limited aggregate (DLA) model, according to which the dendritic structure is formed from the asymmetric growth of silver nanoparticles when the growth rate is limited by the rate of diffusion of solute atoms to the interface. A layer-by-layer mechanism has been proposed: AgNPs tend to locally aggregate forming Ag nanoparticle layers and on a compact layer AgNPs can aggregate and self-assemble into small dendritic structures.

In details, in the presence of reducing agents, Ag⁺ ions convert to AgNPs, and, at the initial stage, silver nanoparticles grow along the [1 1 1] direction to form a trunk, being the growth promoted along the [1 1 1] direction. Then, the additional formed silver nanoparticles tend to attach to a silver trunk and their growth also begins along the [1 1 1] direction to form further secondary and tertiary branches. As the reaction proceeds with time, all the trunks, branches and leaves grow and finally interconnect to each other, leading to the formation of a dendritic structure (Wen et al., 2006; Mandke et al., 2012).

It has been demonstrated that a strong anisotropic growth contributes to the evolution of Ag nanostructure into a thermodynamic stable dendritic structure and that AgNP dendritic structures are formed at the interface when Ag ions are reduced in the presence of surfactants.

Several authors highlighted that the formation of silver hierarchical dendritic structure strictly depends on the reduction process (Alam et al., 2014), the reaction time (Lin and Kim, 2010), the concentration of the AgNP precursor (Baruwati et al., 2009), and the strength of the reducing agent (Alam et al., 2014).

In details, long reaction times lead to the obtainment of thick and brittle dendritic structures (He et al., 2010), becoming the obtained side branches of the dendritic structures less well defined, since more silver nanoparticles tend to aggregate each other into the spaces between the side branches as the reaction went on; higher AgNP precursor concentration leads to large dendritic nanostructures. According to Alam et al. (2014), a strong reducing agent (e.g. NaBH₄) is not able to induce the dendritic nanostructure formation and fast reduction processes lead to silver nanoparticles formation instead of dendritic structure, even if the main reason has not been well understood yet.

Furthermore, the obtained structures depend on the used process and reducing agent. For example, several authors obtained dendritic structures, using PVP as stabilizing and reducing agent by means of sonoelectrochemistry and microwave irradiation processes, demonstrating that PVP leads to the formation of side branches and leaves and promotes the formation of finer, more hierarchical microstructures (Agrawal et al., 2008; Tang et al., 2009).

Alam et al. (2014) reported that it was possible to obtain dendritic structures using disodium citrate sesquihydrate (C₆H₆Na₂O₇·3/2H₂O) as reducing agent, whereas using sodium tetra hydroborate (NaBH₄) Ag nanoparticles are formed instead of dendritic structures (Alam et al., 2014).

Basing on these previous reports and of our results, it is possible to conclude that MW power and the presence of organic components within the used WGPE play pivotal roles in the formation of Ag dendrites.

3.4 Potential applications of WGPE-AgNPs

A preliminary evaluation on the potential applications of synthetized green nanostructures in biosensing and agriculture was carried out. At this regard, WGPE-AgNPs were investigated on the basis of their electrochemical and antifungal properties and results are discussed below.

3.4.1

3.4.1 Electrochemical characterization of WGPE-mediated AgNPs

Among different silver nanostructures (e.g. nanospheres, nanorods, nanosheets, and nanowires), the dendritic ones attracted considerable attention due to their high electrical conductivity and the high specific surface area that make them particularly suitable for application in the field of optoelectronics and sensors. In this regard, to evaluate the potential application of green denditric structures in sensoring field, the electrochemical properties of biosynthesized silver nanostructures were examined using cyclic voltammetry (CV). Results showed that the WGPE-AgNPs exhibit similar redox properties as solution-synthesized silver nanoparticles by modifying the Brust method or other reported in literature (Welch and Compton, 2006; Giovanni and Pumera, 2012).

Fig. 8 shows typical voltammograms of cyclic scan originating from −0.8 to +0.05 V, for blank (only buffer) and WGPE-nanostructures in solution at three different concentrations (23.8, 36.4, and 73.9 μg mL⁻¹). The CV of blank matrix (WGPE diluted 1:1 (v/v) in 50 mM phosphate buffer, pH 7.4) did not show any peak, index of absence of electroactive compounds in the analyzed extract.

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Fig. 8 Cyclic voltammetry of WGPE-mediated AgNPs (blank matrix: cyan line; AgNPs at 23.8 μg mL⁻¹: green line; AgNPs at 36.4 μg mL⁻¹: blue line; AgNPs at 73.9 μg mL⁻¹: red line).

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The different dilutions of WGPE-AgNPs showed the characteristic CV profile of traditional AgNPs. There is an apparent feature of electrochemical dissolution (stripping) of silver nanostructures on the electrode surface, at ∼−200 mV, and reduction of the generated silver ions in the oxidative part of cycle from the solution at ∼+100 mV. As it can be seen from the voltammograms, the behavior of WGPE-mediated nanoparticles significantly differs, depending on the concentration of the nanoparticles, with a broadening of the peak at high concentrations, indicating a probable aggregation.

Giovanni and Pumera (2012) established that the electrochemical behavior of various silver nanoparticles significantly differs, depending on the size of the nanoparticles. WGPE-AgNPs showed a voltammetric profile, for potential peak values and for shape of both peaks, similar to the traditional AgNPs with size higher than 40 nm (Giovanni and Pumera, 2012), probably due the presence of organic components within the used WGPE coating agent.

Fig. 9 shows typical voltammograms after ten consecutive cyclic scans for WGPE-AgNPs. As it can be seen from the voltammograms, the potential of oxidation peak decreased upon increasing the number of scans. This reflects the fact that, during consequent reduction steps, lower and lower amount of Ag⁺ ions is available and smaller and smaller AgNPs are re-deposited and consequently re-oxidized, which is a characteristic behavior of Ag-NPs (Ivanova and Zamborini, 2010).

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Fig. 9 Overlay cyclic voltammetry of WGPE-mediated AgNPs (one cycle: red line; after 10 cycles: black line).

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The interaction between metal nanoparticles (MNPs) and biomolecules has led to new intriguing approaches for the immunoassays, using the electrochemical detection to obtain a high sensitivity (Welch and Compton, 2006). In this regard, the electroactive properties of WGPE-mediated AgNPs suggest the possibility to use them as an enzymeless electrochemical biosensor through the functionalization of AgNPs with a bioreceptor to recognize specific analytes. In this case, AgNPs should be the support of the bioreceptor and, at the same time, the transducer in order to detect the specific analyte (AgNPs) possess the dual role of electrochemical markers in electrochemical measurements and backing material for the bioreceptor immobilization (El-Ansary and Faddah, 2010; Nidzworski et al., 2014).

3.4.2

3.4.2 Antifungal activity of WGPE-mediated AgNPs

The intensive use of chemical pesticides during the last decades caused serious human and environmental hazard. Consequently, the interest towards the discovery of green formulations as alternative to the conventional plant protection products is increasing. In this contest, silver nanoparticles of either simple nature or combined with natural compounds could represent an innovative approach to plant disease control. The in-vitro antibacterial and antifungal activity of silver nanoparticles also obtained from plant extract has been demonstrated against various bacteria and fungi (Li et al., 2005; Kim et al., 2007; Jo et al., 2009; Kim et al., 2012; Thombre et al., 2012). To the best of our knowledge, this is the first time that the antifungal activity of different concentrations of WGPE-mediated AgNPs is tested against F. graminearum, the most important causal agent of the Fusarium Head Blight (FHB) in wheat and barley. F. graminearum has major economic impacts in the agriculture industry and production losses worldwide have been estimated to be as much as 50%. To evaluate the potential fungistatic activity of dendritic nanostructures, the radial growth of fungal mycelium was measured after 4 days (Fig. 10) and the growth inhibition percentage was calculated. Moreover, the antifungal activity of green Ag nanostructures against F. graminearum was investigated in comparison to that of a commercial suspension of standard AgNPs (20 nm) and WGPE. Results pointed out that F. graminearum isolate was mild sensitive to the green nanostructures and no reduction of the growth was achieved in a dose-response manner. Specifically, about 20%, 24%, and 19% of growth inhibition was recorded using 36.4, 73.9, and 98.8 µg mL⁻¹ of WPGE-mediated AgNPs, respectively; whereas the lowest level of inhibition (16%) was obtained on PDA treated with 23.8 µg mL⁻¹ of nanoparticles (Fig. 10). In contrast, commercial standard AgNPs, AgNO₃ solution (1 mM), and raw WPGE did not exert any growth inhibition activity. These preliminary findings highlight the potential of synthetized green nanostructures as a promising eco-friendly alternative to the use of chemicals for controlling plant fungal pathogens. Further research should be conducted to clarify the antifungal action mechanism of these novel green structures also taking into consideration different fungal species and strains.

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Fig. 10 Effect of WGPE-mediated AgNPs, AgNPs standard, AgNO₃ and WGPE on mycelia growth of Fusarium graminearum. Colony diameter (cm) 4 days post inoculation. Controls are media plate without additions.

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To the best of our knowledge, in fact, there are no literature data about the antifungal activity of green silver dendrimers with which compare our results. To explain our results we can speculate that dendrimers possess an architecture that allows the organization of metal nanoparticles on their surface and/or in their interior. As a consequence, the accessibility of Ag becomes a limiting factor for a successful fungistatic action of synthetized nanostructures. Besides, an unsuitable dispersion in water of WGPE-mediated nanostructures could be responsible of the observed results as a function of different WGPE-AgNP concentration used.

To be applicable in open field, however, the assessment of a possible phytotoxic effect of WGPE-AgNPs is necessary. In this regard, and considering the limited studies on the phytotoxicity of nanoparticles (Lin and Xing, 2007), the effect of synthetized green nanostructures on durum wheat seed germination was also investigated. After 6 days of incubation, 95% of the treated seeds had germinated and developed roots and shoot. No differences of germination rate were observed with respect to control seeds treated with water, indicating that wheat seed germination was not affected by the treatment with the nanostructures, even applied at high concentrations (Fig. 11). In this study, the lack of toxicity of AgNPs on wheat seed germination, also at the highest concentration exposure, indicates a potential use of these silver nanostructures in crop plant protection.

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Fig. 11 Durum wheat seed germination after 6 days of incubation. A) AgNPs at 23.8 μg mL⁻¹; B) AgNPs at 36.4 μg mL⁻¹; C) AgNPs at 73.9 μg mL⁻¹; D) AgNPs at 98.8 μg mL⁻¹; E) control seeds treated with distilled water.

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