# Metallic Nanoparticles Obtained via \"Green\" Synthesis as a Platform for Biosensor Construction
## 3 Results and Discussion
### Biosensor Evaluation
#### 3.2.2 AO-Based ABSs  
AO is an oxidoreductase with a wide substrate specificity. The enzyme has the highest affinity to methanol (100%) and is less sensitive to ethanol (50%), propanol (15%), formaldehyde (12%) and butanol (6%) [71]. We have previously reported the development of bi-enzyme AO/HRP-based ABSs sensitive to primary alcohols [71; 72; 73; 74; 75]. AO preparations were isolated from the selected mutant strain _O. polymorpha_ C-105 [75] or mutated AO with a decreased affinity toward substrates [73]. In the current research, we tested the characteristics of bioelectrodes based on yeast AO and green-synthesized Pd (AO/HRP-gPdNPs/GE) compared to a control electrode without using NPs--(AO/HRP-GE). Both ABSs were tested on methanol.  
Analytes that are structurally similar to methanol, namely ethanol and formaldehyde, also give significant amperometric signals. Figure SI.2 (Supplementary Information) presents the results of the selectivity test for AO/HRP-gPdNPs/GE. It can be seen that the intensity of the amperometric signal of ethanol is 40% and the signal of formaldehyde is ca. 15% of the methanol signal. This result is not surprising, in light of the above-mentioned ability of AO to oxidize these analytes. The results of the stability test for the AO-based bioelectrodes (Figure SI.3, Supplementary information) demonstrate that AO/HRP-gPdNPs/GE immersed in 50 mM phosphate buffer, pH 7.5, for 3 days had a higher storage stability than the unmodified graphite electrode with immobilized alcohol oxidase and horseradish peroxidase (AO/HRP/GE).  
Figure 6 presents electrochemical characteristics of AO/HRP-based ABSs, namely calibration curves (Figure 6a,b) of responses to increasing methanol concentrations. According to Figure 6a and Table 2, the control AO-based ABS has 2.9-fold higher \(I_{max}\), 1.4-fold higher \(K_{M}\)\({}^{app}\) and 3.7-fold higher sensitivity to methanol compared to the modified bioelectrode. It is worth mentioning that both \(K_{M}\)\({}^{app}\) values are higher compared to the \(K_{M}\) of mM in solution (0.58 mM) [58]. The advantages of the constructed AO/HRP-gPdNPs/GE are as follows: a 1.6-fold broader linear range of methanol determination and higher stability than the control ABS. When compared to AO/HRP-EDP/GE [71], AO/HRP-gPdNPs/GE demonstrates a similar linear range, a lower \(I_{max}\) (25-fold) and lower sensitivity (4.7-fold), but a higher \(K_{M}\)\({}^{app}\) (1.3-fold).  
Therefore, in our research we proved the possibility of using gNPs as enzyme carriers for construction of ABSs. It was shown that gNPS-based bioelectrodes, although less sensitive, have a broader linear range and prolonged storage stability compared to control ABSs. The achieved characteristics of these ABSs are desirable for enzymes with analytical importance that have a very high affinity for their substrates, for example laccase and AO. In this case, lower sensitivity is not a critical parameter for the ABSs. Use of gNPs-modified bioelectrodes enables simplifying and increasing the precision of the analytical procedure by avoiding additional dilutions of tested samples.