*2.2. SERS Implementation*

SERS has been induced by using home-made Gold nanoparticles (GNP) onto conventional microscope slides. A colloid of GNPs has been obtained by a conventional citrate reduction method [15,16]. A 0.01% HAuCl4 solution was reduced by 1% sodium citrate with vigorous stirring at near boiling temperature. The amount of sodium citrate was defined in order to obtain GNPs with controlled size with particle diameter in the range of 20–50 nm. Preliminary tests have shown that the GNPs with a diameter equal to ∼27 nm give optimum SERS features [16].

Particle size, preparation stability and Raman signal of the resulting preparations have been investigated by means of absorption spectroscopy, Transmission Electron Microscop (TEM), and Dynamic Light Scattering (DLS). In particular, absorption spectroscopy was used to get information on the plasmon resonance peak and to study the stability of the preparations [17,18]. TEM and DLS were employed to estimate the size of the GNPs. Absorption spectra of the prepared GNPs were recorded by UV–VIS spectroscopy by a two-beams spectrophotometer (LS25, Perkin Elmer, Waltham, MA, USA).

Hydrogen tetrachloroaurate (HAuCl4), and trisodium citrate were purchased from Sigma-Aldrich (Sigma-Aldrich Co., St. Louis, MO, USA). All chemicals were used as received. Rhodamine 6G were purchased from Sigma-Aldrich (St. Louis, MO, USA).

### *2.3. SERS Measurements*

A small amount of samples to be analyzed was dropped on the dried residual of the GNP colloid, and quickly measured. SERS measurements were performed by using a Jobin–Yvon system from Horiba Scientific ISA (Osaka, Japan), equipped with a TriAx 180 monochromator, a liquid nitrogen cooled charge-coupled detector and a 1800 grooves/mm grating (final spectral resolution: 4 cm−1). The spectra were recorded in air at room temperature using a 17 mW He–Ne laser source (wavelength 632.8 nm). Accumulation times in the 30–180 s range were used. The laser beam was focused to a 2-μm spot size on the sample through an Olympus microscope equipped with 100× optical objective. Raman spectroscopy measurements were also done for comparison, by using conventional microscope slides as substrates. Acquisition times and modalities were similar to those used for SERS measurements.
