## 5 Lacase-Based Sensor Development
The sensor was also tested using water samples from rivers and springs spiked with different concentrations of catechol, and it shows a recovery rate varying in the 97-108% interval. [PERSON] et al. [137] developed an innovative optical biosensor using a laccase-based chimeric protein. Laccase from _Pleurotus ostreatus_ was combined with hydrophbin layers to improve the immobilization efficiency of laccase. The chimeric protein was immobilized into multi-well plates without any purification step and used for monitoring two phenolic compounds: L-DOPA, which is a relevant compound in the pharmaceutical field; and caffeic acid, which is present in food [137]. The calibration curves for these two compounds have been constructed using changes in the absorption at 420 nm using ABTS as a substrate and the analytes as competing inhibitors in the ABTS oxidation due to laccase in indirect assays. The working parameters of these optical biosensors are reported in Table 2.  
The biosensor was also challenged with spiked real samples: L-DOPA in plasma samples and caffeic acid in different beverages, such as tea and fruit juices. In both cases, high efficiency in analyte quantification was obtained [137]. (b) Sensors based on fluorescence spectroscopy \begin{table}
\begin{tabular}{c c c c} \hline \hline
**Analyte.** & **Sample** &
\begin{tabular}{c} **Linearity Range** \\ **(\(\mu\)M)** \\ \end{tabular} & **LOD (\(\mu\)M)** \\ \hline L-DOPA & Buffer & 5–1000 & 3 \\ \hline \hline  & Plasma & 10–1000 & 2 \\ \hline Caffeic acid & & 50–4000 & 1 \\ \hline \hline \end{tabular}
\end{table} 
Table 2: Working parameters of the optical biosensors developed by [PERSON] et al. in Ref. [137]. Figure 16: Calibration plot for catechol concentration determination. In the inset, the linear range is shown (reprinted with permission from Ref. [136] under open access conditions). The first attempt to exploit the fluorescence properties of lacase has been ascribed to [PERSON] et al. [138] in 1993. They aimed to investigate the performance of a solid-state fiber-optic oxygen sensor and design a new approach to enzymatic sensing using flow-injection analysis. The authors developed a simple and robust lacase-based sensing system that is useful for practical applications. In Figure 17, the scheme of the [PERSON]'s enzyme sensor is reported. The sensor was used for determining the concentration of different phenolic substances (Hydroquinone, phenol, 4-chlorophenol, 2,3-dichlorophenol, and 2,3,4-trichlorophenol) and real commercially available samples (Lipton \"Yellow Label\" tea (UK), Duncans \"Double Diamond Leaf Tea\" (India), Yun Nan black tea (China), \"C.T.C\", granulated tea (India), and Caykur Rize tea (Turkey)). The [PERSON] et al. paper [138] reported the calibration curve for catechol and the quantitation of the polyphenol content in the different above-mentioned teas. The working characteristics of the fiber-optic flow-cell lacase sensor are summarized in Table 1 of Ref. [138] and reported in Table 3.  
[PERSON] et al. [139] developed a significant approach for the optical detection and quantification of different polyphenol compounds (catechol, gallic acid, hydroquinone, hydroxyhydroquinone, phloroglucinol, pyrogallol, and resorcinol) that can be present in beverages. The described biosensor takes advantage of the inhibition of the green indocyanine fluorescence induced by polyphenols in the presence of lacase and gold nanoparticles with positive charges. Lacase fluorescence rapidly decreases when the fluorophore is added. The decrease can be ascribed to the catalytic effect of the enzyme on fluorophore oxidation. The fluorescence decrease effect is slower proportionally to the concentration of the present polyphenols. Figure 18 shows the experimental apparatus used, which includes a device for the rapid mixing of reagents. A solution of lacase and gold nanoparticles was placed in one of the syringes; in a second syringe, the sample was placed in a solution with indocyanine green. A third syringe was used for flow control.