## 5 Lacase-Based Sensor Development

Table 1: Working characteristics for lacase solution and lacase-polyacrylamideamide sensor when \(\Delta Abs\) and \(V_{0}\) parameters are employed. Data are given in Ref. [132]. Figure 14: In the left of the Figure see panels (**a**–**c**). (**a**) Absorption spectra of hydroquinone and a solution of lacase and hydroquinone; (**b**) absorption spectra of resorcinol and a solution of lacase and resorcinol; (**c**) absorption spectra of catechol and a solution of lacase and catechol. The (**a**,**b**) panels on the right show the calibration curve of the optical biosensors based on sol–gel immobilized lacase at increasing concentrations of resorcinol and catechol (reprinted from Ref. [131] under open access conditions). [PERSON] et al. [133] used lacase from _Cerrena unicolor_ immobilized in low-temperature co-fired ceramics (LTCC) by physical adsorption to develop a valuable optical biosensor for monitoring of phenolic species in water in a continuous way. LTCC technology is a consolidated technique that is generally employed in the industry. LTCC materials show very good physical and chemical properties, and three-dimensional structures can be readily fabricated. In a single LTCC multilayer substrate, optoelectronic components and systems for testing micro- and nanoliter volumes of samples can be incorporated [134; 135]. The various steps necessary for fabricating the biosensor and for optimizing the different working parameters are described by [PERSON] et al. [133]. Scanning electron microscopy has been employed to investigate the morphology of the lacase layer deposited on chemically modified supports. Sensor performances have been investigated in a flow-through system by exploiting the optical absorbance changes that occur when various concentrations of standard lacase assay substrate 2,20-azino-bis (3-ethylbenzothazoline-6-sulfonic acid)-ABTS have been used. The contribution of various sensor working parameters (flow rate, optical source characteristics, and reproducibility) was studied. In their paper, [PERSON] et al. [133] demonstrated that optical sensors with excellent working parameters (sensitivity, precision, and linearity) and simplicity of construction can be fabricated by using LTCC technology. In Figure 15, the linear range obtained by considering the absorbance changes versus different concentrations of ABTS is shown. The R\({}^{2}\) coefficient for the linear range is close to 1 (R\({}^{2}\) = 0.975), indicating good accuracy of the fabricated sensor in a concentration range varying from 57 to 171 uM. \begin{table}
\begin{tabular}{c c c c c c} \hline  &
\begin{tabular}{c} **Linear** \\ **Range (M)** \\ \end{tabular} & **Slope** & **\% RSD** & **LOD (M)** & **LOQ (M)** \\ \hline \(V_{0}\)1  & 1.36 \(\times\) 10\({}^{-4}\) \\  & \(-\)5.00 \(\times\) 10\({}^{-3}\) & & & & \\ \hline \(\Delta\Delta\text{Abs}\)2  & 1.09 \(\times\) 10\({}^{-4}\) \\  & \(-\)2.50 \(\times\) 10\({}^{-3}\) & & & \\ \hline \end{tabular}
\end{table} 
Table 1: _Cont._  
Figure 15: The linear range for an LTCC sensor exploiting the changes in absorbance due to solutions with different concentrations of ABTS (measurement conditions: flow rate = 25 uL/min, LED current: 15 mA) (reprinted with permission from Ref. [133]). An innovative optical biosensor for determining the catechol concentration has recently been presented by [PERSON] et al. [136]. Laccase from _Trametes versicolor_ is bonded to anionic polyamide 6 (PA6) porous microparticles integrated into a Pebax MH1657 polymer binder that includes MBTH that gives a colored product when it interacts with the o-benzoquinone produced by the enzymatic reaction of catechol. The catechol concentration was determined by evaluating the changes in absorbance at 500 nm. In Figure 16, the calibration plot obtained for the developed biosensor is reported, and an LOD equal to 11 \(\mu\)M and a linear range up to 118 \(\mu\)M have been obtained.