## 3 Results
### Investigation of the Binding of 2-FAL to MIP by an SPR Sensor  
The receptor layer binding properties are often investigated by SPR measurement based on prism (Kretschmann configuration). Compared to this \"classical\" configuration, the D-shaped POP based SPR platform has the advantage of not requiring an expensive dedicated apparatus. A schematic view of the sensor is reported in Figure 6.  
The possibility of performing measurements in a particularly convenient way for practical application, i.e., in a few \(\upmu\)L drop, is offered by the shape of the optical platform here proposed, which presents a flat surface and can be easily maintained in a horizontal position. As an example some spectra obtained with the D-shaped POP-MIP-SPR sensor are reported in Figure 7. They were acquired in 75%water-25%ethanol solution at different concentration of 2-FAL, by \begin{table}
\begin{tabular}{l c c c c} \hline \hline  & \(K_{\rm A}\) (M\({}^{-1}\)) & \(\mathbf{k_{\rm CR}}\) (\(\upmu\)A) &
\begin{tabular}{c} **Sensitivity (\(\upmu\)A/M) at** \\ **Low Concentration** \\ \end{tabular} & **LOD (M) 1** \\ \hline Site with high affinity (sites 2) & 2(1) \(\times\) 10\({}^{5}\) & 2(2) & 6(2) \(\times\) 10\({}^{5}\) & 7.6 \(\times\) 10\({}^{-7}\) \\ Sites with low affinity (sites 1) & 3.1(9) \(\times\) 10\({}^{2}\) & 51(1) & 1.60(2) \(\times\)10\({}^{4}\) & \\ \hline \hline \end{tabular}  
* LOD obtained as 3\(\upmu\)s of the ordinate at the origin of the dose response curve by SPC-MIPS divided by the sensitivity at low concentration.  
\end{table} 
Table 1: Binding parameters of 2-FAL on MIP5 determined by the sensor SPC-MIP5 by Scatchard plot, and sensing parameters. Figure 6: SPR sensor based on D-shaped POP (D-shaped POP-MIP-SPR). contacting a drop of sample with the MIP receptor layer for 5 min before the spectrum registration. One resonance dip is present at around 500 nm, depending on the concentration of 2-FAL. The resonance wavelength shift with increasing concentration of 2-FAL is clearly seen in the inset. The response is obtained for 2-FAL concentrations much lower than those for which an electrochemical response was acquired. The sorption isotherm at about 500 nm for 2-FAL is shown in Figure 8. The continuous curve was calculated with the parameters obtained by the Hill fitting [12]: K = 1.1(3) \(\times\) 10\({}^{7}\) (M\({}^{-1}\)); sensitivity at low concentration = 5(2) \(\times\) 10\({}^{7}\) (nm \(\times\) M\({}^{-1}\)), n = 1. Figure 8: Sorption isotherm of 2-FAL on MIP, obtained from the resonance wavelength shift observed in Figure 7.  
Figure 7: Normalized spectra (against the bare optical platform in air) of the D-shaped POF-MIP-SPR sensor in 75%water-25%ethanol, at different concentrations of 2-FAL in the range 1 \(\times\) 10\({}^{-6}\) M to 6 \(\times\) 10\({}^{-4}\) M.  
A good fit of the data is obtained by the Hill model, which can be of great help for quantification purposes. However from the Scatchard plot (Equation (7)) it is clearly seen that two different complexes are formed in the concentration range 1.0 \(\times\) 10\({}^{-7}\) M-1.2 \(\times\) 10\({}^{-5}\) M as shown in Figure 9. The binding parameters of the two sites involved in the combination with 2-FAL in the considered concentration range are reported in Table 2.  
The association constant (\(K_{\text{A2}}\)) of 2-FAL for the sites at low affinity is not significantly different from that obtained by the electrochemical platform at a similar concentration range. For this reason these sites are named \"sites 2\" as sites 2 detected by the electrochemical sensor. In addition sites with much higher affinity are detected too by the SPR sensor (sites 3). The maximum signal is very similar for the two kinds of interaction sites, despite of the fact that the sensitivity is much lower. The same kind of sensor prepared using NIP instead of MIP did not give any resonance wavelength shift at the same 2-FAL concentration range. This indicates that the sites at high affinity have also a high selectivity.