## 5 Lacase-Based Sensor Development
Stacked films of MBTH in hybrid Nafion/sol-gel silicate and lacase in chitosan have been adopted for the preparation of a biosensor that is particularly sensitive to catechol, in comparison with other examined analytes, such as guaiaicol, o-cresol, and m-cresol, that have also been considered by the authors. In Figure 13, the response curve of the biosensor is reported for increasing concentrations of catechol in phosphate buffer solution at pH 6.0. As is evident, the linearity range is equal to a 0.5-8.0 mM catechol concentration. A detection limit equal to 0.33 mM is evaluated. The detection limit has been estimated according to Refs. [128; 129]. The same approach used by [PERSON] et al. [123] has been adopted for preparing a biosensor to determine the concentration of hydroquinone, resorcinol, and catechol. Lacases and phenol reaction products show useful optical signals in the UV and visible range. These signals have been adopted for biosensing applications since the compounds produced by the lacase interaction with different phenols are characterized by different optical absorption spectra. The occurrence of these different spectra increases the specificity of lacase-based biosensors [42], and they have been used to determine hydroquinone, resorcinol, and catechol concentrations, respectively (see Figure 14). In Figure 14, the absorption spectra of lacase solutions in the presence of the three phenol above-mentioned compounds are presented: (a) for hydroquinone, (b) for resorcinol and (c) catechol. For developing useful biosensors, lacase was immobilized in sol-gel matrices as described in Refs. [130; 131]. On the right side of Figure 14, the calibration curves of sol-gel immobilized lacase at increasing concentrations of resorcinol and catechol are shown. The hydroquinone-lacase reaction is characterized by a slow response time that prevented the use of this sensing scheme that was successfully used for resorcinol and catechol. Linear ranges up to 1.4 and 0.2 mM and an LOD of 4.5 and 0.6 mM have been obtained for resorcinol and catechol, respectively. Larger linear ranges, significant sensitivities, and good LODs are obtained with this biodevice in comparison with other biosensors using lacase from _Tametes versicolor_, as can be seen from Table 4 in Ref. [131]. This biosensor was  
Figure 13: Calibration curve and linear range (inset) of the optical biosensors proposed by [PERSON] et al. for catechol detection (reprinted from Ref. [127] under an open access condition). also used with tap water samples added with known amounts of catechol and resorcinol for testing biosensing performances with real samples. [PERSON] et al. [132] developed a lacase-polyacrylamideamide sensor exploiting the absorption and fluorescence properties of the enzyme and characterized by a linear range of 0.11-2.5 mM and a limit of detection (LOD) of 100 uM for phenols. This biosensor was also challenged with wastewater samples. Table 1 reports the results obtained for an enzyme solution (equal to 1.56 \(\times\) 10\({}^{-5}\) M) and the lacase immobilized in a polyacrylamide film working at pH 6 when the absorption properties of lacase are exploited. In Table 1, the performances of the working parameters are defined as  
\[\Delta Abs=Abs_{\lambda max,t}-Abs_{\lambda max,0} \tag{1}\]  
\[V_{0}=\frac{\Delta Abs_{\lambda max}}{\Delta t}=\frac{Abs_{\lambda max,t}-Abs _{\lambda,0}}{t} \tag{2}\]  
where \(Abs_{\lambda max,0}\) and \(Abs_{\lambda max,t}\) represent the absorbance at a time \(t\) or 0 at the working wavelength. \begin{table}
\begin{tabular}{c c c c c c} \hline \hline  &
\begin{tabular}{c} **Linear** \\ **Range (M)** \\ \end{tabular} & **Slope** & **\% RSD** & **LOD (M)** & **LOQ (M)** \\ \hline \(\Delta Abs\)a & \(\begin{array}{c}9.79\times 10^{-6}\\ -7.50\times 10^{-4}\end{array}\) & \(4.20\times 10^{3}\) & \(1.07\) & \(2.94\times 10^{-6}\) & \(9.79\times 10^{-6}\) \\ \hline \hline \end{tabular}
\end{table}