# Metallic Nanoparticles Obtained via \"Green\" Synthesis as a Platform for Biosensor Construction
## 3 Results and Discussion
### Biosensor Evaluation  
#### 3.2.1 Laccase-Based Amperometric Biosensors  
The laboratory prototypes of laccase-based ABSs were developed as described in Section 2.3, including a control electrode (laccase/GE) and a modified one with green-synthesized Pd (laccase-gPdNPs/GE). Both ABSs were tested using two laccase substrates: bisphenol A (BP) and ABTS. Figure 4 shows electrochemical characteristics of the laccase-based ABSs, namely chronoamperograms (Figure 4a) and calibration curves (Figure 4a,b) of the responses to increasing concentrations of BP, for both bioelectrodes--control (laccase/GE) and modified with green-synthesized Pd (laccase-gPdNPs/GE). According to the graphs presented in Figure 4b, maximal current responses on BP at substrate saturation (\(I_{max}\)) and the apparent Michaelis-Menten constant (\(K_{M}\)\({}^{app}\)) to BP as a laccase substrate were estimated for both ABSs. As shown in the calibration curves (Figure 4b), the control ABS has a higher \(I_{max}\) and a 2.5-fold lower \(K_{M}\)\({}^{app}\) compared to the modified bioelectrode (laccase-gPdNPs/GE). Following the obtained chronoamperograms for the ABSs, the calibration curves for BP determination were plotted as illustrated in Figure 4c. The linear ranges for BP determination and sensitivities were calculated for both bioelectrodes. Analytical characteristics of the developed ABSs  
Figure 3: Characteristics of gPdNPs synthesized using the culture liquid of a 3-day yeast culture: (a) and (b) fluorescence images and (c) FTIR spectra. are summarized in Table 1. As shown, the linear range for BP determination is 1.4-fold larger for lactose-gPdNPs/GE than for lactose-GE. The sensitivity of the ABS was calculated, taking the slope (B) of the calibration curve and the surface area of the electrode used (7.3 mm\({}^{2}\)) into account. As shown in the Table 1, lactose-gPdNPs/GE is 1.7-fold less sensitive to BP than the control ABS. The developed lactose-based ABSs were tested with another lacase substrate, ABTS, as a non-phenolic redox mediator. Figure 5 presents electrochemical characteristics, namely chronoamperograms of the responses to increasing concentrations of ABTS (Figure 5a) and calibration curves (Figure 5b,c) for the developed lactose-based ABSs. I\({}_{\text{max}}\) and \(K_{M}\)\({}^{app}\) to ABTS were estimated for both ABSs (Figure 5b). As shown in the calibration curves (Figure 5b), the control ABS has a 1.3-fold higher \(I_{max}\) and a 1.2-fold lower \(K_{M}\)\({}^{app}\) compared to lactose-gPdNPs/GE. It is known from the literature that the \(K_{M}\) of ABTS for free lactose from _Trametes versicolor_ in solution is 0.29 mM. With the ABS, the \(K_{M}\)\({}^{app}\) for ABTS was similar (Table 1). Figure 4: Chronoamperograms (**a**), dependence of the amperometric response on increasing concentrations of BP (**b**) and calibration curves (**c**) for lactose-based bioelectrodes: control (1) and modified with gPdNPs (2). Conditions: working potential \(-100\) mV vs. Ag/AgCl (reference electrode), 50 mM acetate buffer, pH 4.5. Following the chronoamperograms, calibration curves for ABTS determination of the developed ABSs were plotted as illustrated in Figure 5c. The linear ranges for ABTS determination and sensitivities of both bioelectrodes were calculated. Analytical characteristics of the developed ABSs are summarized in Table 1. Laccase-gPdNPs/GE has a 1.5-fold smaller sensitivity, but a 1.2-fold broader linear range compared to control laccase-GE. Therefore, the tendency of laccase-based-ABSs is as follows: electrode modification with gPdNPs results in lower sensitivity and higher LP for different substrates.