## 5 Lacase-Based Sensor Development
The fluorescence was excited at 764 nm and is collected at 806 nm using a spectrofluorometer, which allows us to follow the time course of the fluorescence on the time scale of seconds. The spectrofluorometer software used for the treatment of kinetic measurements was adopted for acquiring and processing data. To quantitatively determine the analyte, the authors used the difference in the necessary time to observe a decrease in the fluorescence signal in the presence and absence of the analyte, as shown in Figure 19. In this figure, the slope of the curve obtained in the presence and the absence of the polyphenol has practically the same value, indicating that the oxidation kinetics do not influence each other. The present biosensor allowed catechol to have an LOD of 0.01 mM and a linear range of 0.08-5 mM and has been used for the determination of the concentration of polyphenols in beverages such as tea and fruit juices. Figure 17: Schematic drawing of [PERSON]’s enzyme sensor. Modified from Ref. [138]. \begin{table}
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The proposed system suffers from the limit of enzyme consumption being higher compared to the normal technological use of enzymes, which provides for their immobilization; however, the consumption is limited. Furthermore, they use positively charged gold nanoparticles to modulate the timing of enzyme kinetics, by exploiting the nanoparticle's ability to reduce enzyme activity. [PERSON] et al. [140] proposed an optical biosensing scheme with a different use of the lacase enzyme. They exploited the conversion mechanism from polyphenols in mono/polyquinones induced by lacase for quenching CdTe quantum dot fluorescence. The resulting mono/polyquinones can quench quantum dot fluorescence as a result of broad spectral absorption due to multiple excitonic states resulting from quantum confinement effects and by a supposed charge transfer process from quinones to QDs. This process, illustrated in Figure 20, was used for detecting and quantifying polyphenols that can detect these analytes in the 1 ng/mL-100 mg/mL range with an LOD equal to 1 ng/mL. The authors observed that quenching of QDs fluorescence is linear with polyphenol con  
Figure 19: Kinetic curves have been acquired for the lacase–indocyanine green system alone (1) and in the presence of AuNPs (2), gallic acid (3) and AuNPs + gallic acid (4). [laccase] = 0.1 U mL–1, [indocyanine green] = 5.2 μm mol L–1, [galllic acid] = 2 μm mol L–1, temperature = 20 \({}^{\circ}\)C, pH 7.5 and [Tris] = 25 mmol L–1 (reprinted with permission from Ref. [139]). Figure 18: Schematic of the instrumental system: S1 and S2, driving syringes containing lacase + AuNPs and indocyanine green + polyphenolic compound, respectively; S3, stopping syringe; FD, spectrofluorometer; C, observation cell; T, thermostat; PC, computer (reprinted with permission from Ref. [139]). centration, so a differential quenching response provided sensitive \"fingerprint\" detection of individual polyphenols [140]. The developed sensor has been also applied to real samples obtained by a water solution of plant extract spiked with various polyphenols. The added quantities have been recovered in a very efficient way, as shown in Table 2 of Ref. [115]. The authors also demonstrated that there are no interference effects due to metal ions like aluminum, copper, zinc, iron, etc., which can be present in small quantities in plant extracts. Recently, [PERSON] et al. [141] developed a fluorescence-based assay for evaluating the total polyphenol concentration in beverages using lactose from _Trantes versicolor_ and carbon nanodots (CD). Using this bioconjugate, it was possible to monitor the enzymatic reaction thanks to the decrease in fluorescence signal due to the quinone quenching effect. This method has been successfully applied for the determination of the total concentration of polyphenols (represented as gallic acid concentration) in wine, fruit juice, and rice leaf extract spiked samples.