Metallic Nanoparticles Obtained via “ Green ” Synthesis as a Platform for Biosensor Construction

Novel nanomaterials, including metallic nanoparticles obtained via green synthesis (gNPs), have a great potential for application in biotechnology, industry and medicine. The special role of gNPs is related to antibacterial agents, fluorescent markers and carriers for drug delivery. However, application of gNPs for construction of amperometric biosensors (ABSs) is not well documented. The aim of the current research was to study potential advantages of using gNPs in biosensorics. The extracellular metabolites of the yeast Ogataea polymorpha were used as reducing agents for obtaining gNPs from the corresponding inorganic ions. Several gNPs were synthesized, characterized and tested as enzyme carriers on the surface of graphite electrodes (GEs). The most effective were Pd-based gNPs (gPdNPs), and these were studied further and applied for construction of laccaseand alcohol oxidase (AO)-based ABSs. AO/GE, AO-gPdNPs/GE, laccase/GE and laccase-gPdNPs/GE were obtained, and their analytical characteristics were studied. Both gPdNPs-modified ABSs were found to have broader linear ranges and higher storage stabilities than control electrodes, although they are less sensitive toward corresponding substrates. We thus conclude that gPdNPs may be promising for construction of ABSs for enzymes with very high affinities to their substrates.

In many studies, the size and morphology of gNPs are considered as their basic parameters.Scanning electron microscopy (SEM) can afford information on the size, distribution and shape of the gNPs.However, the drying and contrasting processes alter the characteristics of the tested NPs and lead to imaging faults or artifacts [32].Transmission electron microscopy (TEM) is a high-tech approach in the characterization of gNPs, and is more powerful and competitive than SEM.It can be used to determine the particle size, dispersion and aggregation in aqueous environment with a high spatial resolution that can reveal details of crystal structure.Since many gNPs are irregular in shape and exhibit a tendency to form large particles, it is difficult to conclude definitely whether their final form was caused by polydispersity or agglomeration.The most suitable method for characterization of gNPs is a dynamic light scattering (DLS) technique, which can provide data on sphere hydrodynamic diameters of homogeneous NPs in an aqueous phase [44].However, this method cannot provide information on particles with a heterogeneous size distribution.Therefore, the diversity and ambiguity of gNPs are the reason why it is necessary to use multiple techniques, for instance a combination of TEM and DLS, for valid characterization of gNPs [32,44].
In our previous study, we reported on green synthesis of AuNPs by the yeast Ogataea polymorpha [56].BioAuNPs of different sizes (20-40 nm) and shapes, from triangular nanoprisms to nearly spherical and hexagonal ones, were obtained using both the yeast culture and extracellular metabolites of the culture liquid after removal of the cells.The optimal conditions (inorganic ion concentrations and incubation times) required for extracellular synthesis of AuNPs were chosen.The size, structure and fluorescence of the NPs were characterized using scanning, atomic force, transmission and fluorescence microscopy [56].
The aim of the current research was to obtain various NPs via green synthesis using the yeast O. polymorpha, to characterize these gNPs and to study their possible application in biosensorics on models of amperometric bioelectrodes based on laccase and alcohol oxidase for assays of phenolic compounds and alcohols, respectively.

Green Synthesis of Nanoparticles
The recombinant yeast strain O. polymorpha NCYC495-pGAP1-HsARG1 (leu2car1 Sc:LEU2) [59] was used as a tool for the green synthesis of gNPs.The cells were cultivated in Erlenmeyer flasks under aeration in a circular shaker (200 rpm) at 30 • C, for 48 h as described earlier [56].The cells were centrifuged at 10,000× g for 5 min, and the supernatant, i.e., the culture liquid, was used for synthesis of gNPs.For this purpose, the culture liquid was incubated with 0.5 mM solution of the corresponding inorganic salt/acid (see Section 2.1) for up to 5 days at 37 • C. Each experiment was performed in triplicate.
gNP formation was monitored daily, both visually and with UV-visible absorption spectroscopy (Shimadzu UV1650 PC spectrophotometer, Japan).The generated gNPs were also tested using FM and SEM.The resultant gNPs were collected either by centrifugation (using the Eppendorf 5424R centrifuge at 20,000× g for 1 h) followed by homogenization of aggregated gNPs in water, or by the freezing-thawing method [55].The sample was frozen at −20 • C in a narrow upright capillary-like vessel.gNPs were concentrated to the top and to the bottom of the ice-whisker and the fractions of concentrated gNPs were gathered after slow defrosting, and kept at −20 • C until used.

Characterization of the Synthesized NPs
Optical UV-visible spectra for characterizing the process of gNP formation from inorganic ions were obtained using a UV1650 PC spectrophotometer (Shimadzu, Kyoto, Japan).
A scanning electron microscope (SEM-microanalyser REMMA-102-02, Sumy, Ukraine) was used for morphological analyses of the samples.The special cover film on the samples with a Butvar solution B-98 (Sigma, St. Louis, MO, USA) in 1.5% chloroform was formed using an ultrasound method.The distance from the last lens of the microscope to the sample (WD) ranged from 17.1 mm to 21.7 mm; the accelerator voltage was in the range of 20 to 40 eV; zooms were from 2500 to 10,000.
An inverted fluorescence microscope (FM, Axio Lab.A1., Carl Zeiss, Esslingen, Germany) with excitation by a mercury vapor arc lamp (X-Cite series 120Q) was used for sample imaging.The filters set was composed of fluorescein isothiocyanate (green) and 4 ,6-diamidino-2-phenylindole (blue).In order to prepare the tested sample, a 10 µL aliquot of a water-diluted probe was placed on a glass slide and covered with a cover slip (12 mm diameter).
The Infrared spectra of gPdNPs were acquired by a Nexus 470 Thermo-Nicolet FTIR spectrometer (Thermo Fisher Scientific, Dardilly, France) using a single bounce attenuated total reflectance accessory (Spectra-Tech Thunderdome, Thermo Fisher Scientific) with a germanium crystal.Background was systematically recorded before the data acquisition of each sample (spectra from 4000 to 690 cm −1 , 256 scans, and resolution 4 cm −1 ).Spectra were obtained after depositing a drop on the crystal and after evaporation of water.
A semi-quantitative express method was used for studying the effect of the synthesized gNPs on alcohol oxidase activity.10 µL aqueous NP solutions were dropped into the cells of a 96-well microplate and supplemented with 10 µL chitosan solution (1 mg mL −1 ) in PB.After 15 min incubation for solid film formation, 15 µL AO solution (0.5 U mL −1 ) in PB were dropped into the cells containing gNPs immobilized in chitosan films.200 µL o-dianizidine-based substrate for assay of AO activity were added to each cell, and after 2 min incubation at room temperature, the supernatants were transferred into glass tubes.The cells were washed twice with 200 µL PB, and the supernatants were combined with the initial supernatants in glass tubes.300 µL of concentrated HCl were then added to each tube.As a control, AO solution was put into cells supplemented with chitosan, but without gNPs.AO activity was determined spectrophotometrically [60] and calculated as % relative to the control sample.

Apparatus
The amperometric biosensors (ABSs) were evaluated using constant-potential amperometry in a three-electrode configuration with an Ag/AgCl/KCl (3M) reference electrode, a Pt-wire counter electrode and a working graphite electrode (GE).Graphite rods (type RW001, 3.05 mm diameter) from Ringsdorff Werke (Bonn, Germany), sealed in glass tubes by means of epoxy glue and used as working electrodes, were polished with emery paper for decreasing their size.Amperometric measurements were carried out using a potentiostat CHI 1200A (IJ Cambria Scientific, Burry Port, UK) connected to a personal computer and performed in a batch mode under continuous stirring in an electrochemical cell with a 20 mL volume at 25 • C.

Immobilization of the Enzymes onto Electrodes
In order to construct the enzyme-based amperometric biosensors, GEs were previously modified by dropping 5 µL gPdNPs (or not modified for control electrodes).After formation of solid films, aliquots of a freshly prepared enzyme solution (see Section 2.2) were dropped.A GE with an enzyme only, but without gPdNPs, was used as a control.For construction of the laccase-based ABS, 5 µL laccase solution was dropped onto the surfaces of bulk GEs and gPdNPs-modified GEs.For construction of the AO-based ABS, 5 µL HRP solution was dropped onto the surface of both GEs (control and modified).After formation of solid films, 7 µL of AO solution were dropped on the electrodes.After drying for 10 min at room temperature, the layers of enzymes on the electrodes were covered with supporting polymers.
Several fixing methods were tested, including a dialysis membrane, solutions of Nafion, chitosan and polyethyleneimine.The main principle for choosing the most effective covering film was the maximal amperometric output of the bioelectrode on the correspondent substrate and the stability of the bio-recognition layer on the surface of the GE with the enzyme.Experimentally, the best composition was selected: 10 µL of the mixture, which contains 1% chitosan and 0.5% polyethyleneimine solutions (1:1, v/v), were dropped on the surfaces of laccase-and AO-based electrodes.The prepared bio-functionalized electrodes were rinsed with corresponding buffers (see Section 2.2) and kept in these buffers with 0.1 mM EDTA at +4 • C until used.The electrodes were washed with corresponding buffer solutions before and after each measurement in order to stabilize the basic signal.

Statistical Analysis
Statistical analysis of the measurement results and the correlations between the values of the results obtained by different analytical methods was performed using Origin 8.0 and Microsoft Excel.

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 • 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.
Appl.Sci.2019, 9 FOR PEER REVIEW 5 washed with corresponding buffer solutions before and after each measurement in order to stabilize the basic signal.

Statistical Analysis
Statistical analysis of the measurement results and the correlations between the values of the results obtained by different analytical methods was performed using Origin 8.0 and Microsoft Excel.

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 °C.Incubation with yeast extracellular metabolites caused the yellow-colored solutions of 0.5 mM HAuCl4, K2CrO4, PdCl2 and H2PtCl6 to change to dark red for gAuNPs, light green for gCr2O3NPs, 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 1day culture incubated for 3 days with HAuCl4 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 coupled with X-ray microanalysis (SEM-XRM).Figure 2 demonstrates the overall morphology of the formed particles (triangular, tetrahedral and monodispersed).The XRM data 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 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 µm [56].
(a) (b)  GPdNPs were chosen for construction of amperometric biosensors (ABSs) and for further characterization (Figure 3  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.

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 (Figures 4a and 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 (Imax) and the apparent Michaelis-Menten constant (KM 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 Imax and a 2.5-fold lower KM 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 are summarized in Table 1.As shown, the linear range for BP determination is 1.4-fold larger for laccase-gPdNPs/GE than for laccase-GE.The sensitivity of the ABS was calculated, taking the slope 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.

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 are summarized in Table 1.As shown, the linear range for BP determination is 1.4-fold larger for laccase-gPdNPs/GE than for laccase-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, laccase-gPdNPs/GE is 1.7-fold less sensitive to BP than the control ABS.
(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, laccase-gPdNPs/GE is 1.7-fold less sensitive to BP than the control ABS.The developed laccase-based ABSs were tested with another laccase substrate, ABTS, as a nonphenolic 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 laccase-based ABSs.Imax and KM 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 Imax and a 1.2-fold lower KM app compared to laccase-gPdNPs/GE.It is known from the literature that the KM of ABTS for free laccase from Trametes versicolor in solution is 0.29 mM.With the ABS, the KM app for ABTS was similar (Table 1).The developed laccase-based ABSs were tested with another laccase 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 laccase-based ABSs.I 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 laccase-gPdNPs/GE.It is known from the literature that the K M of ABTS for free laccase from Trametes versicolor in solution is 0.29 mM.With the ABS, the K M app for ABTS was similar (Table 1).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 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.
Numerous biosensors based on laccase from T. versicolor have been constructed [63][64][65][66][67][68][69][70].Different methods of enzyme immobilization on multi-walled carbon nanotube electrodes were tested [63,64].All tested bioelectrodes were rather sensitive to ABTS, had a limited linear range (up to 0.060 mM) and a low I max (up to 11 µA).Laccase from T. pubescens immobilized on an electrode [66] had a 2-fold higher sensitivity and a similar K M app value, but a 2.5-fold lower top of the linear range compared with the currently proposed laccase-gPdNPs/GE (Table 1).K M app values of laccase from T. versicolor immobilized on control or modified graphite electrodes are comparable to the K M of laccases from other fungi [67].It should be noted that laccase can communicate with the electrode through direct electron transfer, mediated by enzyme substrates.The similarity of the K M obtained for different substrates with free laccases in solutions and immobilized on the electrode surface confirms that the physical binding of the enzyme on electrodes does not have a fundamental effect on the native structure of the enzyme.
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.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.

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 laccase 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 (a)  1 EDP-Electrodeposition paint; 2 LOD-limit of detection; 3 ND-ot determined.

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 laccase 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.

Figure 1 .
Figure 1.UV-Vis absorption spectra of gNPs synthesized using the culture liquid of a 3-day yeast culture.

Figure 1 .
Figure 1.UV-Vis absorption spectra of gNPs synthesized using the culture liquid of a 3-day yeast culture.

Figure 2 .
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.

Figure 2 .
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.

Figure 3 .
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.

Figure 3 .
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.

Table 1 .
Analytical characteristics of the developed laccase-based amperometric biosensors.

Table 1 .
Analytical characteristics of the developed laccase-based amperometric biosensors.

Table 2 .
Analytical characteristics of the developed methanol-sensitive amperometric biosensors.

Table 2 .
Analytical characteristics of the developed methanol-sensitive amperometric biosensors.