# Metallic Nanoparticles Obtained via \"Green\" Synthesis as a Platform for Biosensor Construction
## 3 Results and Discussion  
### Characterization of the Green-Synthesized Nanoparticles  
Several mono- and bi-metallic gNPs were obtained as a result of the reduction of inorganic salts/acids (see Section 2.1) by the yeast culture liquid (see Section 2.3). Incubation of the reaction mixture (RM) was carried out under the conditions we selected for obtaining gAuNPs [56]. The kinetics of the formation of different gNPs in RM were studied for 5 days visually as well as by UV-Vis. The colors of the different RMs changed after 2 days incubation at 37 \({}^{\circ}\)C. Incubation with yeast extracellular metabolites caused the yellow-colored solutions of 0.5 mM HAuCl\({}_{4}\), K\({}_{2}\)CrO\({}_{4}\), PdCl\({}_{2}\) and H\({}_{2}\)PtCl\({}_{6}\) to change to dark red for gAuNPs, light green for gCr\({}_{2}\)O\({}_{3}\)NPs, dark green for gPdNPs, and colorless for gPtNPs. The UV-Vis optical spectra of the produced gNPs are presented in Figure 1.  
The characteristic peaks of the obtained gNPs are identical to the peaks of the plasmon resonance of the described inorganic NPs obtained by chemical synthesis. For example, the spectrum of gPdNPs has a peak at 390 nm (Figure 1), which is specific for PdNPs [18; 61]. A peak of gAuNPs in the range 525-590 nm is specific for chemically-synthesized AuNPs of different sizes, according to data reported in the literature [8; 12; 13; 19; 38; 42; 43; 48] as well as our results [56; 62]. The color, size and shape of AuNPs and gAuNPs are dependent on the NPs' synthesis conditions: culture liquid of a 1-day culture incubated for 3 days with HAuCl\({}_{4}\) produces a dark blue solution of small gAuNPs with a peak at 525 nm. Under the same conditions, a 3-day culture generates a brown-red solution of gAuNPs with a peak at 550 nm that corresponds to larger AuNPs. The pellet after centrifugation of the last solution looks like a film of pure Au. It was demonstrated that using 1 L of waste (culture liquid of a 3-day yeast culture) produces approximately 1 mg gAuNPs [56].  
Some of the obtained gNPs, namely gAuNPs and gPdNPs, exhibited low toxicity to yeast cells and did not significantly inhibit laccase and AO when added in moderate concentrations. These gNPs, as prospective carriers for enzyme immobilization, were characterized by scanning electron microscopy  
Figure 1: UV-Vis absorption spectra of gNPs synthesized using the culture liquid of a 3-day yeast culture.  
coupled with X-ray microanalysis (SEM-XRM). Figure 2 demonstrates the overall morphology of the formed particles (triangular, tetrahedral and monodispersed). The XRM data proved the formation of Au\({}^{0}\)-containing (Figure 2a) and Pd\({}^{0}\)-containing (Figure 2b) products. An AFM study showed that the triangular gAuNPs had an average thickness of 10 \(\upmu\)m [56].  
GPdNPs were chosen for construction of amperometric biosensors (ABSs) and for further characterization (Figure 3 and Figure SI.1, Supplementary information). The fluorescence images demonstrate that the obtained NPs shine brightly at two filters and emit blue (Figure 3a) and green (Figure 3b) light. Infrared spectroscopy of the gPdNPs (Figure SI.1) demonstrated that they contained organic compounds (Figure 3c). The large bands centered at 3369, 1645 cm\({}^{-1}\) (amide C=O stretching vibration) and 1560 cm\({}^{-1}\) (amide C-N stretching and N-H bending vibrations) suggest the presence of  
Figure 2: Characteristics of gNPs synthesized using the culture liquid of 3-day yeast culture: SEM images with X-ray spectral characteristics of (**a**) gAuNPs and (**b**) gPdNPs.  
amide groups. The 3369 and 1059 cm\({}^{-1}\) bands were assigned to O-H stretching and C-O stretching modes of the hydroxyl function. Proof for the presence of methyl (-CH\({}_{3}\)) and methylene (-CH\({}_{2}\)-) groups was obtained by absorption peaks at 2963 and 2930 cm\({}^{-1}\).  
The FTIR results proved that the gNPs are products of an interaction of metallic NPs generated in the RM with organic molecules from the culture liquid which contains metabolites of living cells.