# Metallic Nanoparticles Obtained via \"Green\" Synthesis as a Platform for Biosensor Construction
## 4 Conclusions  
In our research, we demonstrated the possibility of developing reagent-less enzyme-based amperometric biosensors using gNPs. Extracellular metabolites of the thermotolerant yeast _Ogataea polymorpha_ were shown to be a promising cost-effective tool for obtaining gNPs of noble and transition metals. Several gNPs were synthesized and characterized. Yeast AO and fungal lacasse were immobilized on the surfaces of graphite electrodes, modified by the most effective enzyme carrier, gPdNPs. Analytical characteristics of the developed bioelectrodes were studied and compared. It was shown that bioelectrodes modified with gPdNPs have lower sensitivities to their substrates, but broader linear ranges of their detection and higher storage stabilities, than unmodified control electrodes. Such bioelectrode characteristics are desirable for enzymes which have a very high sensitivity for their substrates, because before testing, the corresponding samples must be very dilute, which is a problem during on-line control of real samples.  
The advantage of the \"green\" approach is rapid growth of the yeast cells with a high-yield of pure gNPs surrounded by organic compounds that provides the anti-aggregative effect on metallic gNPs. Use of waste yeast cultivation liquid may afford an essential economic effect of green synthesis of NPs, which can be useful as carriers of enzymes and cells in biosensors and biofuel cells.  
**Supplementary Materials:** The following are available online at [[http://www.mdpi.com/2076-3417/9/4/720/s1](http://www.mdpi.com/2076-3417/9/4/720/s1)]([http://www.mdpi.com/2076-3417/9/4/720/s1](http://www.mdpi.com/2076-3417/9/4/720/s1)).  
**Author Contributions:** Conceptualization: G.Z.G. and N.Y.S.; methodology: N.Y.S.; investigation: O.M.D., R.Y.S., M.D.L. and A.E.; resources: M.V.G.; data curation: G.Z.G. and M.N.; writing--original draft preparation: G.Z.G., O.M.D., and N.Y.S.; writing--review and editing G.Z.G. and M.N.; supervision: M.V.G.; project administration: G.Z.G.; funding acquisition: M.V.G. and M.N.  
**Funding:** This work was partially funded by NAS of Ukraine (The program \"Smart sensor devices of a new generation based on modern materials and technologies\"), by the Ministry of Education and Science of Ukraine (projects #0116U004737 and 0118U000297) and by the Research Authority of the Ariel University, Israel.  
\begin{table}
\begin{tabular}{c c c c c c} \hline \hline
**Bioelectrode** & \(\mathbf{K_{M}}^{\textit{APP}}\)**, **mM** & \(\mathbf{I_{max}}\)**, **\(\mathbf{\mu}\)A** & \
\begin{tabular}{c} **Linear Range**, \\ **up to mM** \\ \end{tabular} &
\begin{tabular}{c} **Sensitivity**, \\ **A\(\cdot\)M\({}^{-1}\)-m\({}^{-2}\)** \\ \end{tabular} & **LOD 2, mM** \\ \hline AO/HRP/GE & 0.88 \(\pm\) 0.05 & 0.45 \(\pm\) 0.03 & 0.5 & 52 & 0.005 \\ AO/HRP-gPdNPs/GE & 0.64 \(\pm\) 0.02 & 0.16 \(\pm\) 0.01 & 0.8 & 14 & 0.011 \\ AO/HRP-EDP 1/GE [71] & 0.48 & 4 & 1 & 66 & ND 3 \\ \hline \hline \end{tabular}  
* EDP—Electrodeposition paint; 2 LOD—limit of detection; 3 ND—ot determined.  
\end{table}
Table 2: Analytical characteristics of the developed methanol-sensitive amperometric biosensors.  
Figure 6: Graphs of the response as a function of increasing concentrations of methanol (**a**) and the calibration curves (**b**) for bioelectrodes: control AO/HRP/GE (1) and graphite electrode, modified by gPdNPs with immobilized alcohol oxidase and horseradish peroxidase AO/HRP-gPdNPs/GE (2). Conditions: working potential \(-\)50 mV vs Ag/AgCl (reference electrode), 50 mM phosphate buffer, pH 7.5.  
**Acknowledgments:** We acknowledge [PERSON] (Institute of Cell Biology) for technical support and [PERSON] (Institute of Cell Biology) for experimental assistance.  
**Conflicts of Interest:** The authors declare no conflicts of interests.