*2.7. Ag Nanostars Colloid: Preparation and Application on the Cross Sections*

AgNSs were synthesized using the method proposed by A. García-Leis et al. [14], using analytical grade reagents and water purified with a Milli-Q system (Millipore). Four solutions were required:


Next, 500 µL of the NaOH and 500 µL of the hydroxylamine solutions were mixed in a flask and stirred at 670 rpm with a magnetic stirrer for one minute. Then, 9 mL of the AgNO<sup>3</sup> solution were added and the mixture kept under stirring for 5 min. Afterwards, 100 µL of the citrate solution were dropped in the flask, stirring for approximately 15 min, i.e., until it developed a dark green color. The reduction of Ag<sup>+</sup> ions was achieved by hydroxylamine, but this process, at room temperature, is very slow. In order to make the long arms of these spiky nanoparticles grow faster, citrate was added. The complete growth of the star-shaped NPs, starting from a spherical faceted morphology, takes more or less 48 h. Therefore, the colloidal solution of AgNSs was concentrated by centrifuging three times at 8000 rpm in a 1.5 mL Eppendorf centrifuge tube from 500 µL of colloid. After each centrifugation step, 400 µL of supernatant were removed and replaced by the same quantity of colloid. Before centrifuging, nanoparticles which got stuck at the bottom, were redispersed. After this procedure, 2 µL of the concentrated AgNSs dispersion were deposited on the cross section by means of a micropipette. Once completely dried, the sample was ready to be analyzed with the µ-Raman spectrometer. slow. In order to make the long arms of these spiky nanoparticles grow faster, citrate was added. The complete growth of the star-shaped NPs, starting from a spherical faceted morphology, takes more or less 48 h. Therefore, the colloidal solution of AgNSs was concentrated by centrifuging three times at 8000 rpm in a 1.5 mL Eppendorf centrifuge tube from 500 µL of colloid. After each centrifugation step, 400 µL of supernatant were removed and replaced by the same quantity of colloid. Before centrifuging, nanoparticles which got stuck at the bottom, were redispersed. After this procedure, 2 µL of the concentrated AgNSs dispersion were deposited on the cross section by means of a micropipette. Once completely dried, the sample was ready to be analyzed with the µ-Raman spectrometer. **3. Results and Discussion**

*Heritage* **2020**, *3* FOR PEER REVIEW 5

stirred at 670 rpm with a magnetic stirrer for one minute. Then, 9 mL of the AgNO<sup>3</sup> solution were added and the mixture kept under stirring for 5 min. Afterwards, 100 µL of the citrate solution were

Next, 500 µL of the NaOH and 500 µL of the hydroxylamine solutions were mixed in a flask and

### **3. Results and Discussion** *3.1. Synthesis and Characterization of AgNSs*

### *3.1. Synthesis and Characterization of AgNSs* The macroscopic optical properties and TEM image of the AgNSs are shown in Figure 2, while

The macroscopic optical properties and TEM image of the AgNSs are shown in Figure 2, while Figure S1 shows the extinction spectra of the colloidal suspension of AgNSs [16]. This colloid presents a dark green color (Figure 2A) and, as shown by TEM analysis, is indeed composed by a monodisperse suspension of AgNSs (Figure 2B). A typical AgNS presents a central core of around 50 nm diameter and a variable number of pods arranged in an octahedral structure reaching an overall dimension of approximately 200 nm. The growth starts from spherical seeds of Ag reduced by hydroxylamine, then, in the following 48 h, the pods start taking shape thanks to the citrate ions in the presence of NaOH, which directs the growth of the branches [14,15]. Figure S1 shows the extinction spectra of the colloidal suspension of AgNSs [16]. This colloid presents a dark green color (Figure 2A) and, as shown by TEM analysis, is indeed composed by a monodisperse suspension of AgNSs (Figure 2B). A typical AgNS presents a central core of around 50 nm diameter and a variable number of pods arranged in an octahedral structure reaching an overall dimension of approximately 200 nm. The growth starts from spherical seeds of Ag reduced by hydroxylamine, then, in the following 48 h, the pods start taking shape thanks to the citrate ions in the presence of NaOH, which directs the growth of the branches [14,15].

**Figure 2.** (**A**) Photograph showing the macroscopic appearance of the colloidal suspension of Ag **Figure 2.** (**A**) Photograph showing the macroscopic appearance of the colloidal suspension of Ag nanoparticles; (**B**) relevant TEM image of the nanoparticles obtained.

nanoparticles; (**B**) relevant TEM image of the nanoparticles obtained.

The UV-Vis-NIR extinction spectrum of the colloidal dispersion of AgNSs (see Figure S1)

The UV-Vis-NIR extinction spectrum of the colloidal dispersion of AgNSs (see Figure S1) presents features in agreement with the previous literature [15–17], namely an absorption peak with maximum extinction at 370 nm, followed by a progressive increase in extinction in the NIR region, starting approximately for λ > 550 nm. Further details on the general characterization of the AgNSs synthesized here are described elsewhere [16]. *Heritage* **2020**, *3* FOR PEER REVIEW 6 starting approximately for λ > 550 nm. Further details on the general characterization of the AgNSs synthesized here are described elsewhere [16].

### *3.2. Cross-Sections Analysis: Bluish Background (CS7), an Exemplificative Study 3.2. Cross-Sections Analysis: Bluish Background (CS7), an Exemplificative Study*

The preliminary analyses were fundamental to plan the sampling areas (see SM). Indeed, mapping the more recently retouched areas of over painting was necessary to avoid them during the sampling. Seven cross-sections were sampled from the painting and the *predella*, and six from the frame. Each one was taken in order to answer specific questions about the materials employed in every layer of the stratigraphy at those precise points (Figure 3). For the sake of brevity, only the most important and relevant results are presented (see SM). The preliminary analyses were fundamental to plan the sampling areas (see SM). Indeed, mapping the more recently retouched areas of over painting was necessary to avoid them during the sampling. Seven cross-sections were sampled from the painting and the *predella*, and six from the frame. Each one was taken in order to answer specific questions about the materials employed in every layer of the stratigraphy at those precise points (Figure 3). For the sake of brevity, only the most important and relevant results are presented (see SM).

**Figure 3.** Sampling map of the numbered cross sections (CS) in the (**a**) panel and (**b**) frame (CS5 is an **Figure 3.** Sampling map of the numbered cross sections (CS) in the (**a**) panel and (**b**) frame (CS5 is an erratic sample).

erratic sample). The analysis of CS7, sampled in the bluish background behind the Virgin Mary and the clients, is the most exemplificative in the context of the present study. Figure 4a shows the exact area where CS7 was sampled and, once embedded in a polyester resin block, it was analyzed using 50× magnification (Figure 4b) and optical microscope in reflected light (Figure 4c). At high magnifications (50×), all the layers composing the painting are visible. Starting from the bottom, we can find the ground layer, made of gypsum, which is necessary to make the support even and homogeneous, in this case, the wood panel. Then, it is possible to note the paint layer made up of a mixture of blue and white grains. The peculiar feature is that this sequence is repeated: a new ground and paint layer The analysis of CS7, sampled in the bluish background behind the Virgin Mary and the clients, is the most exemplificative in the context of the present study. Figure 4a shows the exact area where CS7 was sampled and, once embedded in a polyester resin block, it was analyzed using 50× magnification (Figure 4b) and optical microscope in reflected light (Figure 4c). At high magnifications (50×), all the layers composing the painting are visible. Starting from the bottom, we can find the ground layer, made of gypsum, which is necessary to make the support even and homogeneous, in this case, the wood panel. Then, it is possible to note the paint layer made up of a mixture of blue and white grains. The peculiar feature is that this sequence is repeated: a new ground and paint layer were added on the original ones in more recent times. Apparently, they may be composed of the same materials, but this must be investigated by means of elemental and molecular analyses.

were added on the original ones in more recent times. Apparently, they may be composed of the same materials, but this must be investigated by means of elemental and molecular analyses. First of all, the cross section was examined by SEM. The analysis of data in Figure 5 confirms the presence of four layers: the 1st ground (I) (around 150 µm), characterized by light elements

(darker areas); the 1st paint layer (II) (≈70 µm) composed by grains of quite light elements in a heavier matrix (brighter areas); the 2nd ground layer (III) (≈50 µm) and the 2nd paint layer (IV) (≈15 µm), both containing mainly light elements with some brighter spots which indicate the presence of heavy elements. EDS analysis provided important information on the elemental composition of the layers. EDS spectra inserted in Figure 5 confirm the presence of Ca and S in the 1st ground layer, which agree with the likely use of gypsum (CaSO4). The peak of Cu in the grains and the one of Pb in the matrix of the 1st paint layer suggest the presence of a blue copper pigment such as azurite [Cu3(CO)2(OH)2] mixed with a white lead pigment, probably lead white [(PbCO3)2·Pb(OH)2]. Ca and S are detected also in the 2nd ground layer, indicating again the presence of gypsum. Finally, the 2nd paint layer is mainly characterized by the presence of Ti, Si, Al, Na, S, Cu, and some Cr. The presence of Ti suggests the use of titanium white, TiO2, a modern pigment introduced in 1920 [30].

Additional EDS spectra were recorded, focusing on the bluish grains present in the 2nd paint layer, revealing the presence of S, Ba, Cr, and Co (see Figure 6). The detection of S and Ba can agree with the presence of barite, BaSO4. This natural occurring mineral has been synthetically produced from the beginning of the nineteen century to be extensively employed both as filler in the formulations of colors [31] and used in the preparation of TiO<sup>2</sup> [30]. Cr and Co could be attributed to the presence of a bluish chromium-based pigment: cobalt chromite, CoCr2O<sup>4</sup> (PB36, cobalt chromite blue-green spinel) often substitutes the historically genuine cerulean blue (PB35, cobalt stannate), the latter being introduced in 1860 as a pigment [32]. Unfortunately, no Raman or SERS spectral evidence allowed to confirm this supposition. *Heritage* **2020**, *3* FOR PEER REVIEW 7

**Figure 4.** (**a**) The sampling area of CS7 is marked on the picture; (**b**) CS7 analyzed using the stereomicroscope, a transversal sample (1–2 mm) embedded in a resin block showing all the layers the painting is composed of; (**c**) magnification of the cross section (objective of the microscope 50×) **Figure 4.** (**a**) The sampling area of CS7 is marked on the picture; (**b**) CS7 analyzed using the stereomicroscope, a transversal sample (1–2 mm) embedded in a resin block showing all the layers the painting is composed of; (**c**) magnification of the cross section (objective of the microscope 50×) showing from the bottom to the top the original ground and paint layer and a more recent coat composed of a new ground and paint layer.

showing from the bottom to the top the original ground and paint layer and a more recent coat

First of all, the cross section was examined by SEM. The analysis of data in Figure 5 confirms the presence of four layers: the 1st ground (I) (around 150 µm), characterized by light elements (darker

composed of a new ground and paint layer.

elements. EDS analysis provided important information on the elemental composition of the layers. EDS spectra inserted in Figure 5 confirm the presence of Ca and S in the 1st ground layer, which agree with the likely use of gypsum (CaSO4). The peak of Cu in the grains and the one of Pb in the matrix of the 1st paint layer suggest the presence of a blue copper pigment such as azurite [Cu3(CO)2(OH)2] mixed with a white lead pigment, probably lead white [(PbCO3)2·Pb(OH)2]. Ca and S are detected also in the 2nd ground layer, indicating again the presence of gypsum. Finally, the 2nd paint layer is mainly characterized by the presence of Ti, Si, Al, Na, S, Cu, and some Cr. The presence of Ti suggests

the use of titanium white, TiO2, a modern pigment introduced in 1920 [30].

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**Figure 5.** SEM image of CS7 and EDS spectra recorded on the I, II, and IV layers (the EDS spectrum of the III layer is comparable with that of layer I). **Figure 5.** SEM image of CS7 and EDS spectra recorded on the I, II, and IV layers (the EDS spectrum of the III layer is comparable with that of layer I).

Additional EDS spectra were recorded, focusing on the bluish grains present in the 2nd paint layer, revealing the presence of S, Ba, Cr, and Co (see Figure 6). The detection of S and Ba can agree with the presence of barite, BaSO4. This natural occurring mineral has been synthetically produced from the beginning of the nineteen century to be extensively employed both as filler in the formulations of colors [31] and used in the preparation of TiO<sup>2</sup> [30]. Cr and Co could be attributed to

**Figure 6.** EDS spectrum performed on blue grain of the 2nd paint layer of CS7. In order to obtain information at a molecular level, micro-Raman spectroscopy was applied. Figure 7 shows the Raman spectra recorded analyzing defined points on the magnified cross section, indicated in Figure 7a. The two ground layers were confirmed to contain gypsum, whose spectrum presents the typical band at 1009 (strong) cm−<sup>1</sup> (Figure 7b) [33]. Comparison with literature spectra [33] proved that the 1st paint layer contains azurite, as indicated by the bands at 1578 (weak), 1423 (medium), 1096 (m), 832 (w), 770 (m), 401 (s), 247 and 83 (m) cm−<sup>1</sup> (Figure 7c), and lead white, whose

bands are detected at 1055 (s) and 401 (m) cm−<sup>1</sup> (Figure 7d).

allowed to confirm this supposition.

In order to obtain information at a molecular level, micro-Raman spectroscopy was applied. Figure 7 shows the Raman spectra recorded analyzing defined points on the magnified cross section, indicated in Figure 7a. The two ground layers were confirmed to contain gypsum, whose spectrum presents the typical band at 1009 (strong) cm−<sup>1</sup> (Figure 7b) [33]. Comparison with literature spectra [33] proved that the 1st paint layer contains azurite, as indicated by the bands at 1578 (weak), 1423 (medium), 1096 (m), 832 (w), 770 (m), 401 (s), 247 and 83 (m) cm−<sup>1</sup> (Figure 7c), and lead white, whose bands are detected at 1055 (s) and 401 (m) cm−<sup>1</sup> (Figure 7d).

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The results of the Raman analyses performed on the 2nd paint layer are exposed below. The presence of titanium white (PW6) in the form of anatase, was confirmed by the detection of its typical bands at 640 (m), 510 (m), 397 (m) and 143 (s) cm−<sup>1</sup> (Figure 8) [32]. **Figure 5.** SEM image of CS7 and EDS spectra recorded on the I, II, and IV layers (the EDS spectrum of the III layer is comparable with that of layer I).

The Raman characterization of the bluish pigment, due to the scarcity of grains and the tiny thickness of the layer, is particularly challenging. The Raman spectrum recorded on this layer allowed us to identify ultramarine blue: sodium polysulphide-aluminosilicate (PB29, Na6-10Al6Si6O24S2-4, which is responsible for the Raman band at 548 cm−<sup>1</sup> (s) (the bands in gray belong to titanium white) (Figure 9) [34]. This attribution is supported by the detection of Si, Na, Al, S in the EDS spectrum reported in Figure 5IV. Natural ultramarine is a mineral called *lazurite*, a complex sulfur-containing sodium aluminosilicate based on a body-centered cubic lattice. Synthetic ultramarine was synthesized in 1828 by Jean Baptiste Guimet in Paris and then rapidly adopted by artists [35]. Note that natural and synthetic ultramarine blue provide comparable spectral signatures and cannot be distinguished by Raman analysis. Additional EDS spectra were recorded, focusing on the bluish grains present in the 2nd paint layer, revealing the presence of S, Ba, Cr, and Co (see Figure 6). The detection of S and Ba can agree with the presence of barite, BaSO4. This natural occurring mineral has been synthetically produced from the beginning of the nineteen century to be extensively employed both as filler in the formulations of colors [31] and used in the preparation of TiO<sup>2</sup> [30]. Cr and Co could be attributed to the presence of a bluish chromium-based pigment: cobalt chromite, CoCr2O<sup>4</sup> (PB36, cobalt chromite blue-green spinel) often substitutes the historically genuine cerulean blue (PB35, cobalt stannate), the latter being introduced in 1860 as a pigment [32]. Unfortunately, no Raman or SERS spectral evidence allowed to confirm this supposition.

**Figure 6.** EDS spectrum performed on blue grain of the 2nd paint layer of CS7. **Figure 6.** EDS spectrum performed on blue grain of the 2nd paint layer of CS7.

presents the typical band at 1009 (strong) cm−<sup>1</sup>

bands are detected at 1055 (s) and 401 (m) cm−<sup>1</sup>

(medium), 1096 (m), 832 (w), 770 (m), 401 (s), 247 and 83 (m) cm−<sup>1</sup>

In order to obtain information at a molecular level, micro-Raman spectroscopy was applied. Figure 7 shows the Raman spectra recorded analyzing defined points on the magnified cross section, indicated in Figure 7a. The two ground layers were confirmed to contain gypsum, whose spectrum

[33] proved that the 1st paint layer contains azurite, as indicated by the bands at 1578 (weak), 1423

(Figure 7d).

(Figure 7b) [33]. Comparison with literature spectra

(Figure 7c), and lead white, whose

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**Figure 7.** (**a**) Optical micrograph of CS7 (50×); (**b**–**d**) Raman spectra on CS7 revealing: (**b**) in the 1st and 2nd ground layers, at point 1, the spectral features typical of gypsum; in the 1st paint layer, (**c**) at point 2 the features of azurite, and (**d**) at point 3, of lead white.

bands at 640 (m), 510 (m), 397 (m) and 143 (s) cm−<sup>1</sup>

and cannot be distinguished by Raman analysis.

point 2 the features of azurite, and (**d**) at point 3, of lead white.

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**Figure 7.** (**a**) Optical micrograph of CS7 (50×); (**b**–**d**) Raman spectra on CS7 revealing: (**b**) in the 1st

and 2nd ground layers, at point 1, the spectral features typical of gypsum; in the 1st paint layer, (**c**) at

The results of the Raman analyses performed on the 2nd paint layer are exposed below. The

(Figure 8) [32].

**Figure 8.** (**a**) Optical micrograph (50×) and (**b**) Raman spectrum recorded at point 4 on the 2nd paint layer of CS7, showing the spectral features of titanium dioxide.

**Figure 8.** (**a**) Optical micrograph (50×) and (**b**) Raman spectrum recorded at point 4 on the 2nd paint layer of CS7, showing the spectral features of titanium dioxide. The Raman characterization of the bluish pigment, due to the scarcity of grains and the tiny thickness of the layer, is particularly challenging. The Raman spectrum recorded on this layer During the analysis of the blue layer, we noticed that some spots gave strong fluorescence while others did not. Indeed, the spectrum in Figure 9 refers to the non-fluorescent grains. Comparing the results obtained with the composition of the most common contemporary color tubes, it was clear that the presence of synthetic ultramarine alone was unlikely since this pigment is often mixed with synthetic organic dyes [36]. Normal Raman spectroscopy did not provide spectra useful to solve this diagnostic issue because of the fluorescence, typically generated by some organic dyes and pigments (Figure 10a). For this reason, we opted to employ the SERS technique using AgNSs as signal enhancers and fluorescence quenchers, in order to amplify the Raman spectrum of the fluorescent grains. After addition of the Ag nanostars [14], the spectrum shown in Figure 10b was collected.

titanium white) (Figure 9) [34]. This attribution is supported by the detection of Si, Na, Al, S in the

EDS spectrum reported in Figure 5IV. Natural ultramarine is a mineral called *lazurite*, a complex

sulfur-containing sodium aluminosilicate based on a body-centered cubic lattice. Synthetic

ultramarine was synthesized in 1828 by Jean Baptiste Guimet in Paris and then rapidly adopted by

artists [35]. Note that natural and synthetic ultramarine blue provide comparable spectral signatures

(s) (the bands in gray belong to

The fluorescent background is now dramatically lowered and a defined spectrum emerges being characterized by detectable bands at 1566 (m), 1514 (s), 1438 (w), 1400 (w), 1379 (m), 1349 (s), 1303 (m), 1142 (w), 1104 (s), 1002 (m), 720 (m), 679 (s), 649 (m), and 585 (w) cm−<sup>1</sup> . Comparison with literature data indicate that these features corresponds to those of copper alpha-phthalocyanine (PB 15:2) [37–39], a synthetic pigment. A detailed comparison of the experimental and the reference bands of PB 15:2 is reported in Table S1 in Supplementary Materials. Interestingly, the presence of PB 15:2 agrees with the presence of the Cu signals in the EDS spectrum in Figure 5II. Organic phthalocyanines and their metal complexes were synthesized at the beginning of the twentieth century and are widely used as blue and green pigments until the present time [40]. Note that the band at 211 cm−<sup>1</sup> is produced by the interaction of Ag with ions present in the colloidal solution of nanoparticles [41]. *Heritage* **2020**, *3* FOR PEER REVIEW 12

**Figure 9.** (**a**) Optical micrograph (50×) and (**b**) Raman spectrum recorded at point 5 on the 2nd paint layer of CS7, showing the spectral features of ultramarine blue.

**Figure 9.** (**a**) Optical micrograph (50×) and (**b**) Raman spectrum recorded at point 5 on the 2nd paint

others did not. Indeed, the spectrum in Figure 9 refers to the non-fluorescent grains. Comparing the

results obtained with the composition of the most common contemporary color tubes, it was clear

that the presence of synthetic ultramarine alone was unlikely since this pigment is often mixed with

synthetic organic dyes [36]. Normal Raman spectroscopy did not provide spectra useful to solve this

diagnostic issue because of the fluorescence, typically generated by some organic dyes and pigments

(Figure 10a). For this reason, we opted to employ the SERS technique using AgNSs as signal

enhancers and fluorescence quenchers, in order to amplify the Raman spectrum of the fluorescent

grains. After addition of the Ag nanostars [14], the spectrum shown in Figure 10b was collected. The

fluorescent background is now dramatically lowered and a defined spectrum emerges being

characterized by detectable bands at 1566 (m), 1514 (s), 1438 (w), 1400 (w), 1379 (m), 1349 (s), 1303

data indicate that these features corresponds to those of copper alpha-phthalocyanine (PB 15:2) [37–

39], a synthetic pigment. A detailed comparison of the experimental and the reference bands of PB

15:2 is reported in Table S1 in Supplementary Materials. Interestingly, the presence of PB 15:2 agrees

with the presence of the Cu signals in the EDS spectrum in Figure 5II. Organic phthalocyanines and

their metal complexes were synthesized at the beginning of the twentieth century and are widely

used as blue and green pigments until the present time [40]. Note that the band at 211 cm−<sup>1</sup>

produced by the interaction of Ag with ions present in the colloidal solution of nanoparticles [41].

. Comparison with literature

is

(m), 1142 (w), 1104 (s), 1002 (m), 720 (m), 679 (s), 649 (m), and 585 (w) cm−<sup>1</sup>

During the analysis of the blue layer, we noticed that some spots gave strong fluorescence while

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**Figure 10.** (**a**) Raman spectrum of the blue organic dye in 2nd paint layer of CS7, which resulted **Figure 10.** (**a**) Raman spectrum of the blue organic dye in 2nd paint layer of CS7, which resulted completely fluorescent; (**b**) SERS spectrum of the blue dye, recorded after AgNSs deposition, showing the spectral features of copper phthalocyanine.

### completely fluorescent; (**b**) SERS spectrum of the blue dye, recorded after AgNSs deposition, showing **4. Conclusions**

scientific approach in the field of cultural heritage.

the spectral features of copper phthalocyanine. **4. Conclusions** The overall results obtained with this investigation on the Madonna della Misericordia of the Parma National Gallery are summarized in Table S1 in Supplementary Materials. In particular, the careful analysis of the cross section CS7 from the Madonna della Misericordia*,* combined with general The overall results obtained with this investigation on the Madonna della Misericordia of the Parma National Gallery are summarized in Table S1 in Supplementary Materials. In particular, the careful analysis of the cross section CS7 from the Madonna della Misericordia, combined with general visual and instrumental investigation on the painting, revealed at least two moments of execution. In particular, referring to the blue area from which CS7 was sampled, a probably older paint layer characterized by the presence of azurite and lead white was discerned. Over this, separated by a gypsum ground overlayer, a second layer of paint was found which contains modern components such as titanium white, copper phthalocyanine blue, and barite. These findings can indicate that the painting was realized in the 19th century and heavily remodeled during the restoration works of

visual and instrumental investigation on the painting, revealed at least two moments of execution. In particular, referring to the blue area from which CS7 was sampled, a probably older paint layer

gypsum ground overlayer, a second layer of paint was found which contains modern components such as titanium white, copper phthalocyanine blue, and barite. These findings can indicate that the painting was realized in the 19th century and heavily remodeled during the restoration works of the 20th century. This dating is in substantial opposition to the temporal collocation reported in the inventory of the gallery which attributed the painting to the 15th century [28]. The extensive study of this work of art led to particularly interesting results: the scientific together with the stylistic analyses enabled to achieve a more accurate and precise dating, underlining the importance of adopting a

From an analytical diagnostic viewpoint, this study demonstrates the real-world applicability

and practical usefulness of AgNSs as highly effective agents to achieve SERS detection of Raman signals for identifying pigments such as copper phthalocyanine, whose fluorescence hampers their detection by means of regular Raman spectroscopy. The one-pot, easy synthesis of AgNSs, together the 20th century. This dating is in substantial opposition to the temporal collocation reported in the inventory of the gallery which attributed the painting to the 15th century [28]. The extensive study of this work of art led to particularly interesting results: the scientific together with the stylistic analyses enabled to achieve a more accurate and precise dating, underlining the importance of adopting a scientific approach in the field of cultural heritage.

From an analytical diagnostic viewpoint, this study demonstrates the real-world applicability and practical usefulness of AgNSs as highly effective agents to achieve SERS detection of Raman signals for identifying pigments such as copper phthalocyanine, whose fluorescence hampers their detection by means of regular Raman spectroscopy. The one-pot, easy synthesis of AgNSs, together with their easy applicability on cross-sections or other kinds of samples, make their use highly practicable as SERS enhancers for real world cultural heritage diagnostics.

**Supplementary Materials:** The following are available online at http://www.mdpi.com/2571-9408/3/4/74/s1.

**Author Contributions:** Conceptualization, M.S.Z., I.A., P.U.; formal analysis, M.S.Z., N.K., P.U.; investigation, M.S.Z., N.K.; methodology, M.S.Z., I.A., N.K., P.U.; project administration, I.A., P.U.; supervision, I.A. and P.U.; validation, M.S.Z., I.A., P.U.; visualization, M.S.Z., P.U.; writing—original draft preparation, M.S.Z., I.A., P.U.; writing-review and editing, M.S.Z., I.A., N.K. and P.U. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Acknowledgments:** The Monumental Complex of the Pilotta and the National Gallery of Parma, in particular, the museum director Simone Verde and Maria Cristina Quagliotti of the administrative staff are sincerely thanked for the possibility of analyzing the painting Madonna della Misericordia. Philip N. Bartlett, University of Southampton, UK, is thankfully acknowledged for providing access to Raman and SERS instrumentation and for useful suggestions for improving the quality of the manuscript. We thank Danilo Bersani, University of Parma, for useful discussion on the Raman spectra. At University Ca' Foscari of Venice, we acknowledge the assistance of Eleonora Balliana and Elisabetta Zendri for the preparation of the cross-sections and Patrizia Canton for TEM analysis.

**Conflicts of Interest:** The authors declare no conflict of interest.
