**5. Sample preparation**

As discussed in the previous paragraph, the field enhancement strongly decreases with the distance from the metallic surface, as consequence delivering the substance under study in close proximity of the metal surface is a prerequisite for measuring SERS signals. Therefore, the samples were analyzed as evaporated films prepared by dropping on the SERS active surface a controlled volume of a solution of CNTs or graphene in ultra-pure water (1 mg/mL). The solvent spreads across the surface selectively evaporates leaving a dried film, which is clearly visible under the optical microscope coupled with the spectrometer (see **Figure 3**).

In our procedure, the substrates were used as-received without any pre-treatment and the solvent evaporated in air without any heating.

We examined the dried film morphology under high-resolution scanning electron microscope (HR-SEM), using a Leo 1525 hot cathode field emission microscope, with a resolution of 1.5 nm at 20 kV.

The SERS activity depends either from the substrate morphology either from the number of sites in the surface that are accessible to the molecular adsorption. In fact, the SERS signal from molecules on the second monolayer and beyond is reduced because the SERS effect depends on the distance between the nanostructure and adsorbed molecule, as shown in **Figure 4**. Surface-Enhanced Raman Spectroscopy Characterization of Pristine and Functionalized Carbon… http://dx.doi.org/10.5772/intechopen.74065 209

have the correct geometry for surface enhancement, as occurred by using substrates with random nanosized roughening or nanoparticle separation and sharp metallic features, produced with the previous techniques. Usually, the SERS substrates are gold coated because, although

We used three variations of SERS active gold substrates: an ordered array of inverted squarebased pyramids, (KlariteTM, D3 Technologies Ltd3), an electrochemically nano-roughened surface (Nanova), and an array of nanopillars, 50–80 nm in width, 600 nm in height, fabricated by the Department of Micro and Nanotechnology of the University of Denmark [21–23]. We measured the EFs of all the above cited substrates by measuring the Raman signal of the same molecule, in this way only electromagnetic enhancement due to the geometry of the used structure was considered, avoiding the effect of chemical enhancement [20, 24]. The obtained results are reported in **Table 1**. The regularity of nanostructure guaranteed a uniform enhancement factor across the surface and a high reproducibility of Raman signal in spite of the dif-

As discussed in the previous paragraph, the field enhancement strongly decreases with the distance from the metallic surface, as consequence delivering the substance under study in close proximity of the metal surface is a prerequisite for measuring SERS signals. Therefore, the samples were analyzed as evaporated films prepared by dropping on the SERS active surface a controlled volume of a solution of CNTs or graphene in ultra-pure water (1 mg/mL). The solvent spreads across the surface selectively evaporates leaving a dried film, which is clearly

In our procedure, the substrates were used as-received without any pre-treatment and the

We examined the dried film morphology under high-resolution scanning electron microscope (HR-SEM), using a Leo 1525 hot cathode field emission microscope, with a resolution

The SERS activity depends either from the substrate morphology either from the number of sites in the surface that are accessible to the molecular adsorption. In fact, the SERS signal from molecules on the second monolayer and beyond is reduced because the SERS effect depends on the distance between the nanostructure and adsorbed molecule, as shown in **Figure 4**.

visible under the optical microscope coupled with the spectrometer (see **Figure 3**).

the gold has a smaller EF than silver, it is more resistant against oxidation in air.

**Substrate EF** KlariteTM 1.1 × 106 Nanova 8.0 × 105 Technical University of Denmark 8.0 × 108

**Table 1.** Enhancement factors of gold coated substrates with different geometry.

ferences among the EFs.

208 Raman Spectroscopy

of 1.5 nm at 20 kV.

**5. Sample preparation**

solvent evaporated in air without any heating.

**Figure 3.** (a) Sequential steps of the drop and dry technique for films deposition and (b) top view of dried film.

When the number of analyte molecules is too large, a sample layer is formed covering the molecules with surface-enhanced Raman signal. To this respect, it is of prime importance that the substrate geometry provides enhancement region accessible for molecular adsorption.

**Figure 4.** Variation in the intensity of surface-enhanced Raman signal with the amount of analyte mass probed by the laser.
