*3.2. AgNPs Sensing Tests*

Sensing tests were made by checking the optical absorption spectra of the contaminated systems with respect to the reference AgNPs water solution (see also Figure S3). In Figure 3a the absorption spectra of AgNPs in water solutions without (reference solution) and with different concentrations of Hg<sup>2</sup>+ ions in the range 1 to 10 ppm, are reported. An eVident red shift of the band peak energy together with an increase of the intensity and a broadening of the band with the increasing concentration of ions can be appreciated. The same type of measurement has been performed for all the 16 ions listed in the experimental part for concentration from 10 ppm down to the lowest concentration of 1 ppm. Figure 3b shows the red shift of the absorption band of all the metal ions tested at the concentration of 2.5 ppm. A significant change has been detected only for Hg<sup>2</sup>+ ions while for all the other ions the absorption bands remain almost constant within the error bars.

**Figure 3.** (**a**) Absorption spectra of AgNPs water solutions at room temperature and pH = 6.5 without and with different concentrations of Hg<sup>2</sup>+ listed in the figure; (**b**) calibration curve as a function of Hg<sup>2</sup>+ concentration; (**c**) redshift of the absorption band maximum in presence of all the metal ions tested at concentration of 2.5 ppm.

The limit of detection (LOD) of the nanosensor for the analysis of Hg<sup>2</sup>+ was determined using calibration curves. The LOD can be estimated as three times the standard deviation of the blank signal, obtaining a value equal to 0.6 ppm. The obtained LOD was found to be comparable with other AgNPs systems and the experimental results in the determination of Hg<sup>2</sup>+ obtained by some other methods are listed in Table 2 for comparison [8,10,40,74–77].


**Table 2.** Comparison between dimension and limit of detection (LOD) of some metal NPs used as a colorimetric sensor for the detection of Hg<sup>2</sup>+.

*3.3. SR-XPS Characterization of AgNPs and AgNPs-Hg<sup>2</sup>*+

Synchrotron radiation-induced X-ray photoelectron spectroscopy (SR-XPS) measurements allowed to probe the interaction between AgNPs and Hg<sup>2</sup>+ ions. Spectra were collected at C1s, N1s, O1s, Ag3d, S2p and Hg4f core levels (all BE (eV), FWHM (eV), relative intensity values and proposed signals assignments are reported in Supplementary Materials file, Table S1). All the individuated spectral components confirm AgNPs stability, and Cit and L-cys capping e fficiency. C1s spectrum has three components at 285.00, 286.46 and 288.45 eV BE, respectively associated with aliphatic carbons (mainly impurities, that are always observed in samples prepared in air by liquid solutions); O1s spectra, reported in Figure 4c, also show three di fferent kinds of oxygen atoms, respectively belonging to carbonyl (C=O) functional groups (532.00 eV BE), hydroxyl moieties (-OH, 533.00 eV BE) and physisorbed water (small contribution at about 534.5 eV BE). The atomic ratio between C=O and –OH is C=O/–OH = 1/1.4, very close to the C=O/–OH = 1/1.3 that is theoretically expected for a Cit/L-cys stoichiometry = 2/1, as in the synthetic procedure reported in Materials and Methods. N1s spectrum was also collected, showing a single component centred at 400.24 eV, as expected for amine-like nitrogens. Indeed, C1s, O1s, and N1s data analysis confirm the molecular stability of the AgNPs, also after interaction with Hg<sup>2</sup>+ ions.

**Figure 4.** SR-XPS spectra collected on AgNPs-Hg<sup>2</sup>+ aggregates at (**a**) Ag3d; (**b**) S2p; (**c**) O1s and (**d**) Hg4f core levels.

Generally speaking, the most indicative signals for the surface-structure analysis of metal nanoparticles capped with thiols are M (Au4f, Ag3d) and S2p core levels; for this purpose, Ag3d and S2p spectra are reported in Figure 4 and will be here discussed in detail.

Ag3d spectra Figure 4a are asymmetric at high BE, a common feature in capped nanoparticles [20,27], indicating that at least two di fferent kinds of silver atoms compose the nanoparticle: the spin-orbit pair at lower BE values (Ag3d5/2 = 368.08 eV) is associated with metallic silver at the NPs core; the signal at higher BE, of very small intensity (about 9% of the whole signal) is due to positively charged silver atoms at the NP surface, interacting with the capping molecule [20,27]. S2p spectra are also composite, showing two spin orbit pairs of very similar intensity (atomic percents are 54.4% lower BE–45.6% higher BE, as reported in Table S1 in the Supplementary Material); interestingly, the two signals are both indicative for sulphur atoms covalently bonded to silver, but with two di fferent hybridizations: the spin-orbit pair at lower BE (S2p3/2 = 161.05 eV) is indicative for S-Ag bonds with sp hybridized sulphur; the signal at higher BE (S2p3/2 = 162.09 eV) suggests S-Ag bonds with sp3 S atoms [78]. It is noteworthy that no physisorbed thiol moieties appear (R-SH S2p3/2 signals are expected around 163–164 eV BE); this finding is in excellent agreemen<sup>t</sup> with the hypothesis made by A Majzik et al. [61] based on 1H NMR studies and suggesting that in metal nanoparticles stabilized by mixed Cit and L-cys capping agents the L-cys molecules preferentially tend to directly bond the metal surface, inducing the most part of Cit molecules to form a shell around the first L-cys layer, in a "layer-by-layer"-like arrangement. It is noteworthy that XPS data do not allow excluding that some Cit molecules could intercalate between L-cys and directly interact with Ag atoms at the NP surface, as eVidenced by IR spectroscopy results. The last signal reported in Figure 4d is the Hg4f spectrum. As eVidenced in the figure, two spin-orbit pairs can be individuated; the first signal (Hf4f7/2 BE = 100.10 eV) can be associated with metallic Hg atoms [79]; the components at higher BE (Hg4f7/2 = 100.85 eV) are consistent with literature data reported for Hg<sup>2</sup>+ ions in oxides or coordination compounds [72].

The occurrence of a signal indicative for metallic Hg is in excellent agreemen<sup>t</sup> with the findings reported in [68], where for AgNPs stabilized by Cit molecules and interacting with Hg(II) ions, a direct interaction between Hg and Ag atoms at the nanoparticle surface was envisaged. To better understand the chemistry and geometry of Hg(II)–AgNPs interaction, XAS experiments at the Hg LIII-edge are in programme. Actually, X-ray absorption experiments will allow to directly probe the local coordination chemistry of the metal ion, providing information complementary to the SR-XPS data and allowing for a precise description of the Hg coordination site.
