**1. Introduction**

Nowadays the scientific approach plays a key role in the conservation-restoration of cultural heritage, which is necessary not only to sort out conservation issues but also to answer diagnostic inquiries. The analysis of the composition of a work of art follows a precise analytical protocol which starts from the visual analysis (optical microscopy in visible and UV light, IR reflectography), then it involves the use of elemental and molecular non-destructive techniques (X-rays fluorescence spectroscopy), scanning electron microscopy (SEM), attenuated total reflection-Fourier transform infrared (ATR-FTIR) spectroscopy, Raman spectroscopy, up to eventually resorting to micro-destructive ones (gas chromatography and mass spectrometry, high performance liquid chromatography). Indeed, since they are composite materials, works of art must be studied in their three-dimensionality in order to take into account all the layers underneath the superficial one. For this reason, transversal micro-sample, the so-called cross-section, once embedded in resins or salts, represent the ideal condition for an exhaustive analysis. Cross-sections should contain all the layers of which the work of art is composed: support, ground layer, priming, paint layer, varnish, and glaze. The complexity of the layering and the variety of material employed (binders, pigments, dyes, varnishes) depend on several factors: the period and place of execution, the artist, the restoration interventions, etc. Cross-sections can tell us the story of a work of art: by performing analyses directly on each layer, we can map the materials employed and organize the layers in a reliable sequence [1,2].

Chemical information on the composition of the layers of a cross-section can be obtained by analytical techniques characterized by spatial resolution capabilities. Typically, localized elemental information is obtained by SEM combined with electron dispersive spectroscopy (EDS) while molecular data are obtained by using vibrational spectroscopies combined with optical microscopy, such as micro-FTIR or micro-Raman spectroscopy.

In particular, Raman spectroscopy represents one of the most employed techniques in diagnostics thanks to the following advantages: non-destructiveness, efficient in situ performance, high spatial resolution (up to 1 µm), presence of Raman bands in the "fingerprint" region of the spectra, capability of discriminating between different crystalline structures, and to resolve weak Raman signals of the analyte from water and glass [3,4]. On the other hand, Raman spectroscopy presents some disadvantages which can hamper its widespread use. Raman scattering is weak (spectroscopic cross-section of 10−<sup>28</sup> cm<sup>2</sup> /mol), as a result, it can be obscured by the much more intense fluorescence (spectroscopic cross-section of 10−<sup>16</sup> cm<sup>2</sup> /mol) emitted by organic molecules with conjugated electronic structures, such as the colorants. The low sensitivity of Raman spectroscopy and the presence of fluorescence can hinder the detection of dyes, in particular, when present at low concentration levels.

Surface enhanced Raman spectroscopy (SERS), observed for the first time by Fleischmann, Hendra, and McQuillan in 1973 [5], helps to overcome these shortcomings. This technique takes advantage of the optical properties of nanostructured metal surfaces, in particular silver, which locally enhance the electromagnetic field, at the same time quenching the fluorescence. As a consequence, a dramatic enhancement of the Raman signal is detected for molecules in close contact with the nanomaterial so facilitating their detection [6].

The SERS technique has been employed in the field of cultural heritage (see ref. [2] for a recent review), in particular, for the analysis of colorants directly on fibers, micro-fragments, and cross-sections [7–9]. Typically, the materials chosen for this purpose are spherical silver nanoparticles, prepared as colloidal dispersion by using the Lee–Meisel method [10,11]. Recently, other nanomaterials have shown higher effectiveness in producing significant SERS effects, including anisotropic nanostructures such as silver nanostars (AgNSs) [12–15]. Due to their stellate shape composed of a central core and many pods, these particular nanoparticles are expected to absorb photons in different spectral regions, namely around 370–380 nm and in the near infrared region and, when excited with red-light laser beams, AgNSs show Raman enhancement factors in excess to 10<sup>6</sup> [16–18]. Gold and silver nanostars (Au/AgNSs) can be obtained employing different reagents and reduction methodologies. Among the different synthetic methods proposed for the preparation of AgNSs [13,16–25], the one-pot method recently proposed by A. García-Leis et al. [14,15], looks particularly attractive for its feasibility and reproducibility. It involves the reduction of the metallic precursor (AgNO3) by a reducing agent (hydroxylamine) in the presence of a capping agent (trisodium citrate) and additive (NaOH). Here, we propose for the first time, as far as we know, the use of AgNSs prepared with the above method, to detect dyes in painting cross-sections. The efficiency of AgNSs as SERS substrates has been tested in our lab on other kinds of samples such as thiols adsorbed on metals, including metal nanofibers [15], and lake pigments, indicating the superiority of AgNSs over spherical silver nanoparticles [26].

As a case example, the methodology was applied to cross-sections from the Madonna della Misericordia, an altarpiece kept by the National Gallery of Parma, Italy. The study took advantage of recent restoration intervention on the paint, with the goal to obtain information on the history of this work of art, in particular, to identify original pictorial layers and the role of previous restoration or even repainting interventions. In this study, Raman spectroscopy was applied together with other

## *Heritage* **2020**, *3*

diagnostic tools, demonstrating that SERS with the help of AgNSs is effective in detecting organic and metalorganic dyes at trace levels, which are not detectable by means of "regular" Raman spectroscopy. and metalorganic dyes at trace levels, which are not detectable by means of "regular" Raman spectroscopy. A case study is exposed in this paper, illustrating how AgNSs can be applied to a cross section

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

diagnostic tools, demonstrating that SERS with the help of AgNSs is effective in detecting organic

A case study is exposed in this paper, illustrating how AgNSs can be applied to a cross section of a real painting for diagnostics purposes, confirming that this kind of SERS substrate can be effective for the sensitive detection of organics and metallorganics employed in the field of cultural heritage. of a real painting for diagnostics purposes, confirming that this kind of SERS substrate can be effective for the sensitive detection of organics and metallorganics employed in the field of cultural heritage.

### **2. Materials and Methods 2. Materials and Methods**

### *2.1. The painting "Madonna della Misericordia" from the National Gallery of Parma 2.1. The painting "Madonna della Misericordia" from the National Gallery of Parma*

The Madonna della Misericordia (Inv. 450) at the National Gallery of Parma is an oil on panel painting, 192 × 82 cm, which represents Virgin Mary housing under her mantle the clients, two ladies and two gentlemen, kneeling at her left and right, respectively. It is completed by a painted frame characterized by two lateral *paraste*, a central rose and a *predella* decorated by three saints (Figure 1). The Madonna della Misericordia (Inv. 450) at the National Gallery of Parma is an oil on panel painting, 192 × 82 cm, which represents Virgin Mary housing under her mantle the clients, two ladies and two gentlemen, kneeling at her left and right, respectively. It is completed by a painted frame characterized by two lateral *paraste*, a central rose and a *predella* decorated by three saints (Figure 1).

**Figure 1.** Pictures showing the Madonna della Misericordia (**a**) at the entrance of Parmigianino and Correggio's display rooms in the 19th century and (**b**) in the picture gallery where it has been **Figure 1.** Pictures showing the Madonna della Misericordia (**a**) at the entrance of Parmigianino and Correggio's display rooms in the 19th century and (**b**) in the picture gallery where it has been displayed until 2018.

displayed until 2018. This painting arrived at the Gallery in 1868, coming from the Cappuccini's monastery in Borgo Santa Chiara, Parma [27]. The director of the Gallery, Corrado Ricci, attributed this painting to an anonymous painter belonging to the school of Cremona of the 15th century [28]. The painting underwent important restoration interventions (insertion of wedges into the panel to level it *sverzatura*—which is the main cause of cracks on the pictorial layer, numerous re-paintings, etc.) carried out in 1896 by G. Frenguelli [28] and in 1951 by L. Arrigoni [29]. The present study was This painting arrived at the Gallery in 1868, coming from the Cappuccini's monastery in Borgo Santa Chiara, Parma [27]. The director of the Gallery, Corrado Ricci, attributed this painting to an anonymous painter belonging to the school of Cremona of the 15th century [28]. The painting underwent important restoration interventions (insertion of wedges into the panel to level it—*sverzatura*—which is the main cause of cracks on the pictorial layer, numerous re-paintings, etc.) carried out in 1896 by G. Frenguelli [28] and in 1951 by L. Arrigoni [29]. The present study was performed during a restoration intervention of the painting performed in 2018 and 2019.

### performed during a restoration intervention of the painting performed in 2018 and 2019. *2.2. UV-Visible Absorption Spectroscopy*

apparatus. The Ag colloids samples were diluted 1:3 in water.

*2.2. UV-Visible Absorption Spectroscopy* UV-Visible absorbance spectra were recorded with a Perkin-Elmer Lambda 40 spectrophotometer equipped with a Peltier-Elmer PTP6 (Peltier temperature programmer) UV-Visible absorbance spectra were recorded with a Perkin-Elmer Lambda 40 spectrophotometer equipped with a Peltier-Elmer PTP6 (Peltier temperature programmer) apparatus. The Ag colloids samples were diluted 1:3 in water.
