**3. Results**

#### *3.1. Response of the SPR-Bare Platforms to the Refraction Index Variation*

The response of the SPR platforms, not derivatized with MIP (SPR-bare), here considered, has been checked as previously proposed [36]. In particular, the resonance wavelengths of the platform in liquid media of different RI, measured by an Abbe refractometer, were registered. As an example, the spectra of water–glycerol solutions with different RI are reported in Figure 2a. The response curve Δ*λ* (referring to pure water) against RI is shown in Figure 2b.

**Figure 2.** (**a**) Transmission spectra of the MIP-bare platform in water–glycerol solutions with different refractive index (RI). (**b**) Variation of the resonance wavelength with the RI of the overlying liquid.

The sensitivity (δΔ*λ*/δRI) is significantly different from zero in the whole RI range, and it increases at increasing RI (Figure 2b). Nevertheless, for short RI ranges, the curve can be assumed to be linear. At increasing RI, the peak is shifted to higher wavelength values and becomes larger and flatter, making the acquisition of the minimum less accurate and precise. For this reason, the best RI of the dielectric layer in contact with the resonant surface must be a compromise between the highest sensitivity and precision.

#### *3.2. MIP Layer Analysis*

The SEM image reported in Figure 3 shows the gold surface modified with MIP polymerized in situ, where the presence of polymer aggregates of small particles can be seen. This SEM image is relative to a sensing platform in which the MIP layer has been obtained by spinning the prepolymeric mixture at high velocity (about 1000 rpm). It shows that at the micro level, the MIP film does not entirely cover the gold surface, so in some way it reproduces the surface derivatized with a bioreceptor, i.e. a large biomolecule.

Figure 4 reports the comparison between the SPR spectrum in water of a bare platform (SPR-bare) with that of the same platform modified with multiple layers of MIP (SPR-MIP). The MIP made the RI of the region overlying gold higher than pure water, as proven by the resonance wavelength shift towards higher values (red shift). The SPR spectra in Figure 4 are relative to a set of sensors prepared by depositing successively one, two, or three layers of MIP on equal platforms, as reported in Table 1. Water (*n* = 1.332 RIU) was the liquid over the platform. The example in Figure 4 shows that the resonance wavelength shift (Δ*λ*) with respect to that of pure water depends on the amount of MIP deposited. The resonance wavelengths are reported in Table 1.

**Figure 3.** SEM images of different points at the surface of the sensing part.

**Figure 4.** Transmission spectra of C1 (SPR-MIP) platform, with one, two, or three MIP layers, in water, normalized on the spectrum of the corresponding platforms in air.

**Table 1.** Resonance wavelength of sensors with multiple MIP layers. Formation of each MIP layer: 40 μL of prepolymeric mixture spun at 300 rpm. Normalization on the corresponding platform in air.


By increasing the number of MIP layers, the resonance wavelength increases, and a shift to higher wavelengths (red shift) is observed (from 663.0 nm to 718.3 nm). Even with three layers deposited (C1-3), the resonance wavelength (718.3) is lower than the maximum wavelength useful for measurement.

In order to verify if the sensor with three layers of MIP is still sensitive to the RI of the overlying liquid, the SPR transmission spectra in water–glycerol solutions with different refractive indices, positioned like a drop over the MIP (bulk solution), are reported in Figure 5.

**Figure 5.** Comparison of the spectra of an SPR-MIP sensor (C1-3) in water–glycerol solutions with different refractive indices and the spectrum of an SPR-bare platform in water.

The resonance in water of the SPR-MIP platform is at 715 nm (see Figure 4), with a shift of 115 nm compared to the resonance of SPR-bare in water, indicating that the amount of MIP layer is relatively large. However, a shift to higher wavelengths is observed when liquids with RI higher than water substitute water. This behavior demonstrates that the MIP layer is not so thick as to completely include the plasmonic wave, as schematically illustrated in Figure 1b,c.

#### *3.3. Sensor Response in Water and in Wine-Mimicking Solution*

Water is a well-suited solvent for measurements with the platforms here described since its RI matches the operative range for the proposed SPR sensors [36]. The spectra can be recorded directly in the sample, since 2-FAL standards in water are easily prepared. When the 2-FAL concentration increases, the SPR wavelength is shifted to higher values, as seen in Figure 6a. As an example, a standardization curve of 2-FAL in water is shown in Figure 6b, and is obtained by reporting the wavelength variation Δ*λ* vs. 2-FAL concentration. The experimental data are well fitted by Equation (1), as expected when the sorption takes place according to the Langmuir model. A plateau corresponding to Δλmax is obtained for concentrations higher than 1 mg·L−<sup>1</sup> due to the receptor's sites saturation. The parameters, evaluated by a non-linear regression method, are *K*aff, 9.4 <sup>L</sup>·mg<sup>−</sup><sup>1</sup> (9.0 10<sup>5</sup> <sup>M</sup>−1) and Δ*λ*max = 3.3 (0.447) nm. The sensitivity at low concentration (i.e., in the linear part of the curve) is 31.6 nm·mg<sup>−</sup>1·L. The saturation was reached at about 1 mg·L−1.

The response of the sensor to 2-FAL concentration in wine-mimicking solution is reported in Figure 6c,d. In this case, the spectra were recorded in water, after equilibration with the sample and washing, according to the method of the solvent exchange.

**Figure 6.** (**a**) SPR spectra of the SPR-MIP sensor at different 2-furaldheide (2-FAL) concentrations in water; (**b**) SPR wavelength shift vs. concentration of 2-FAL in a water solution and the calculated continuous curve; (**c**) SPR spectra of the SPR-MIP sensor at different 2-FAL concentrations in wine-mimicking solution; (**d**) SPR wavelength shift vs. concentration of 2-FAL in wine-mimicking solution and the calculated continuous curve. Vertical bars in (**b**,**d**) correspond to the standard deviations (error bars).

The dose–response curve (Δ*λ* vs. 2-FAL concentration) in wine-mimicking solution followed the Langmuir model (see Equation (1)) and the parameters obtained are: *K*aff = 75.6 <sup>L</sup>·mg<sup>−</sup><sup>1</sup> and Δ*λ*max = 3.3 (0.247) nm. The sensitivity at low concentration is 254.9 nm·mg<sup>−</sup>1·L, and the limit of detection (LOD) is 0.004 mg·L−1**.** The saturation was reached at about 0.6 mg·L−1.

The affinity constant is about 8 times higher in wine-mimicking solution than in pure water; consequently, the LOD is lower than in water.

#### *3.4. Determination of 2-FAL in a White Wine Sample*

The sample considered was a white wine in a tetra pack container (WW) purchased in a local supermarket, in which a concentration of 2-FAL of 0.1 ppm was found by an HPLC method. The SPR spectra were obtained as reported in the experimental part, using water as the liquid in which the spectra were finally recorded (bulk liquid). This method had to be applied since the exact RI of the white wine is unknown. Figure 7 shows the transmission spectra recorded in water of the considered WW sample, and with some standard additions of 2-FAL.

**Figure 7.** SPR spectra obtained by the SPR-MIP sensor in wine sample.

The variation of *λ*ris from the blank (water) to the WW (2.8 nm) is due to the adsorption of 2-FAL on the MIP during the incubation with the sample. There is only a very slight variation of *λ*ris in response to the standard additions of 2-FAL to the sample. This behavior indicates that the imprinted sites are almost saturated by the 2-FAL originally present in the WW considered. In fact, the concentration calculated from Equation (1), using the parameters evaluated in wine-mimicking solution, is 0.107 mg <sup>L</sup>−1, near to the approximate saturation concentration, in acceptable agreemen<sup>t</sup> with the value found by the HPLC method (0.1 mg <sup>L</sup>−1).
