**3. Results and Discussion**

### *3.1. AgNPs Synthesis and Characterizations*

The synthesis of AgNPs by wet chemical reduction is a useful method to obtain spherical nanoparticles with tuned sizes and opportune capping agen<sup>t</sup> [60]. In this work, AgNPs have been prepared by the reduction in aqueous solution of silver nitrate with sodium borohydride as strong reducing agent. Two different capping agents have been chosen: Cit, crucial to induce a high hydrophilic behavior, and L-cys, to induce selective interaction with the environment.

In fact, it is well known that the amino group can easily interact with the chemical environment and in particular with Hg<sup>2</sup>+ ions [10]. In order to avoid the excessive presence of L-cys, leading to nanoparticles aggregation, the molar ratio Au/Cit/L-cys = 1/4/2 was chosen. The obtained AgNPs scheme is shown in Figure 1a. After careful purification, AgNPs have been characterized by means of UV-Vis, FTIR and XPS spectroscopies, by DLS and TEM studies. UV-Vis spectra have been carried out together with DLS measurements in water, as reported in Figure 1b,c. The UV-Vis spectrum showed the typical LSPR band, at λ max = 400 nm, confirming the nanodimensions. DLS measurements in water showed hydrodynamic diameter <2RH> = 8 ± 1 nm, as expected. Moreover, FTIR investigations showed the presence of Cit and L-cys on the particles, as reported in Figure 1d.

When the ATR-FTIR spectrum of AgNPs bifunctionalized with Cit and L-cys was compared with reference spectra of these capping agents Figure 1d, it is immediately eVident the absence of the band at ~1582 cm<sup>−</sup><sup>1</sup> (νas(COO−)) in AgNPs spectrum [66]. This could be interpreted as eVidence that most carboxylate groups of the citrate and cysteine are attached to the surface of the AgNPs. In fact, similar results was observed by Frost et al. [67], who studied the ATR-FTIR spectra of citrate-capped AgNPs and Au-NPs; only for AgNPs, they observed the disappearance of the νas(COO−) peak and interpreted this result in terms of adsorption geometry of the citrate molecule, with all the three carboxylate groups interacting with the AgNPs surface: the νas(COO−) lying parallel to the AgNPs surfaces results in an infrared-inactive transition. Moreover, in the AgNPs spectrum is present a broad peak at ~1547 cm<sup>−</sup><sup>1</sup> that could be due to the overlapping between the N-H bending of cysteine and the residual carboxylate groups not attached to the AgNPs surface. The functionalization with Cit and L-cys can be also supported by the presence in the AgNPs spectrum of a broad peak centered at 1379 cm<sup>−</sup><sup>1</sup> indicating the COO− symmetric stretching vibrations; the peak position is slightly shifted to lower wavenumber compared to the free carboxylate anion, as already eVidenced by Frost et al. [68]. The main contributions to the symmetric and asymmetric stretching vibration of the carboxylate group are obviously mainly due to the citrate molecule, that is present in higher concentration on the AgNPs; however, it seems reasonable to hypothesize a similar reactivity for the different carboxylate groups. The broad peak centered at 3300 cm<sup>−</sup><sup>1</sup> is attributed to the stretching vibrations of the hydroxyl group (ν(O–H)) (data not shown) probably coming from water adsorbed onto nanoparticles surface. We were not able to detect any S-H band around 2550 cm<sup>−</sup>1, that would indicate the presence of free L-cys on the AgNPs surface [68,69]. The intensity of this peak in the spectrum of L-cys is actually rather low (see Figure S2 in Supplementary Material), however, XPS results confirm the absence of S-H groups.

To support FTIR data, AgNPs were also investigated by NEXAFS spectroscopy. C k edge spectra of AgNPs were recorded at normal, magic and grazing incidence; however, no angle-dependent effect was detected; therefore, the C k-edge spectrum of AgNPs is shown in Figure 2. The main feature in the spectrum is the 1s→π\* consisting of two peaks located at 287.7 and 288.8 eV, with a shoulder at 285.5 eV. According to literature, the 1s→π\* transition related to the C=O bond in the carboxylate of L-cys is expected at 288.6 eV [70]; similar values are expected for carboxyl groups [71]. Moreover, L-cys is expected to show a 1s→σ\* peak related to the C-S bond at about 287.3 eV. Therefore, we can assign this complex band to overlap between <sup>π</sup>\*C=O and σ\*C–S transitions. The broad bands located at about 294 and 300 eV are related to 1s→σ\* transitions of C–H and C=O bonds respectively [72]. The overall spectrum yields prove of successful capping of the AgNPs surface by both Cit and L-cys.

**Figure 1.** (**a**) Scheme of bifunctionalized hydrophilic silver nanoparticles (AgNPs); (**b**) UV-Vis spectrum in water of AgNPs, with local surface plasmon resonance (LSPR) band centred at 400 nm; (**c**) DLS measurements in water: <2RH> = 8 ± 1 nm; (**d**) ATR-FTIR spectra of bifunctionalized AgNPs (Top) and the capping agents, Cit (Center), and L-cys (Bottom).

**Figure 2.** C k-edge NEXAFS spectrum of AgNPS recorded at 20◦ incidence angle.

Moreover, the TEM studies carried out on diluted samples show AgNPs with narrow size distribution (TEM Ø 5 ± 2 nm) but with a certain tendency to aggregation, which could be due only to drying (see Figure S1). DLS measurements show a monodisperse population of NPs in solution These data are in agreemen<sup>t</sup> with DLS data. It can be noticed that the dimensions obtained from DLS are greater than those obtained from TEM images, since DLS estimated the hydrodynamic radius < 2RH > of the particles in the aqueous environment and it is the Z-average value, which is the mean diameter weighted over the scattered light intensity. Therefore, the DLS data are not directly comparable with dimensions obtained from TEM images, as reported in the literature [7,56]. Anyway, the data confirmed in both cases the nanodimension of the AgNPs.

Measurements of Ag concentration in fresh and marine aqueous media showed an almost absent Ag+ ions release from AgNPs (Table 1) as opposed to what observed in the literature for other types of AgNPs [45–49,53,55].


**Table 1.** Ag+ concentrations (expressed as μg/L) in freshwater and marine waters with algal medium solution (CTRL), as well as algal medium solutions with AgNPs (500 μg/L) and with AgNO3 (7 μg/L).

In fact, Ag+ concentrations in both TG 201 medium (freshwater) and F/2 medium (marine water) with 500 μg AgNPs/L were low (up to 0.4 μg/L) and comparable with those measured in controls (up to 0.36 μg/L; Table 1). However, it is to be noted that in the presence of AgNPs, Ag+ levels slightly increased after 72 h, from 0.19 to 0.4 μg/<sup>L</sup> in freshwater, and 0.15 to 0.37 μg/<sup>L</sup> in marine water. Since AgNPs dissolution was demonstrated to be independent from particles aggregation state [73], the reason for the lack of ion release is probably to be found in the Cit/L-cys coating.
