Silver Nanowires as Electron Transfer Mediators in Electrochemical Catechol Biosensors

The integration of nanomaterials as electron mediators in electrochemical biosensors is taking on an essential role. Due to their high surface-to-volume ratio and high conductivity, metallic nanowires are an interesting option. In this paper, silver nanowires (AgNWs) were exploited to design a novel catechol electrochemical biosensor, and the benefits of increasing the aspect ratio of the electron mediator (nanowires vs. nanoparticles) were analyzed. Atomic force microscopy (AFM) studies have shown a homogeneous distribution of the enzyme along the silver nanowires, maximizing the contact surface. The large contact area promotes electron transfer between the enzyme and the electrode surface, resulting in a Limit of Detection (LOD) of 2.7 × 10−6 M for tyrosinase immobilized onto AgNWs (AgNWs-Tyr), which is one order of magnitude lower than the LOD of 3.2 × 10−5 M) obtained using tyrosinase immobilized onto silver nanoparticles (AgNPs-Tyr). The calculated KM constant was 122 mM. The simultaneous use of electrochemistry and AFM has demonstrated a limited electrochemical fouling that facilitates stable and reproducible detection. Finally, the biosensor showed excellent anti-interference characteristics toward the main phenols present in wines including vanillin, pyrogallol, quercetin and catechin. The biosensor was able to successfully detect the presence of catechol in real wine samples. These results make AgNWs promising elements in nanowired biosensors for the sensitive, stable and rapid voltammetric detection of phenols in real applications.


Introduction
Phenolic compounds are relevant in the food industry due to their antioxidant properties and health benefits. They are also responsible for some important organoleptic characteristics of foods and affect their antioxidant properties [1]. Flavonoids are an important class of phenolic compounds contained in both wines and grapes. Anthocyanins and flavanols are the two main groups of flavonoids present in red wines. They have a strong influence on the organoleptic properties of wines such as color, astringency, and bitterness. In addition, they are very active chemically and are involved in several chemical reactions during winemaking and aging. The assessment of certain polyphenols (such as catechol and catechol derivatives) is crucial to improve the quality of wines. The assessment of phenolic compounds in wines is usually carried out using chromatographic or spectroscopic techniques [2]. Electrochemical biosensors are an alternative, due to their selectivity, portability, and low cost. They can overcome the limitations of traditional methods [3], and their performance can be improved using nanomaterials as electron mediators [4].
In the last few years, a number of electrochemical biosensors combining phenol oxidases with a variety of materials, such as polymeric matrices [5,6] or nanomaterials,

Preparation of AgNWs
AgNWs were synthesized following a modification of the polyol method, consisting in the reduction of a silver precursor (AgNO 3 ) in the presence of EG as the reducing agent. PVP was used as a surfactant to facilitate the AgNW dispersion and CuCl 2 was added to promote the growth of AgNWs and to prevent the presence of free Ag + ions in the solution through the formation of AgCl products [34].
Even though the polyol process is an efficient method, AgNWs are typically synthesized within a certain range of lengths and diameters, depending on various factors such as the reaction time, temperature, velocity of addition of the metallic precursor and the surfactant, and use of a mediated agent.
In this work, the process was optimized to obtain homogeneous AgNWs. In detail, 0.5 mL of CuCl 2 (1.5 × 10 −4 M, in EG) was added to 5 mL of EG previously heated in a round bottom flask and maintained under stirring (200 rpm) and reflux (the temperature was kept constant at 160 • C during the procedure). After 5 min, 5 mL of PVP (0.36 mM in EG) and 2.5 mL of AgNO 3 (0.12 M in EG) were pumped dropwise simultaneously over 10 min (special attention to the pump speed of each component must be paid in order to obtain homogeneous nanowires: flow rates were 0.25 mL/min for AgNO 3 and 0.5 mL/min for PVP). The reaction was maintained for 1 h until the silver precursor was completely reduced. The AgNWs were separated from the Ag nanoparticles that might have appeared by centrifugation (2000 rpm for 20 min). Seven washing cycles with ethanol were carried out until the yellow color of the supernatant (corresponding to the presence of AgNPs) was no longer observed (these AgNPs were used in the experiments carried out for comparison purposes). Finally, the purified AgNWs were resuspended in ethanol and kept in darkness at room temperature.

Preparation of AgNWs-Tyr Modified Electrode
AgNWs were deposited onto ITO substrates by adding 50 µL AgNWs suspension (2 g/L in ethanol) using the spin coating method (120 s at 1000 rpm). Then, 50 µL of Tyr (5 g/L in PBS 0.01 M, pH 7.0) was drop-casted onto the electrode surface. The electrode was then exposed to glutaraldehyde vapors (25% aqueous solution) for 10 min. After drying overnight at room temperature, the biosensor was washed with a PBS 0.01 M solution.

Characterization of AgNWs
AgNWs were prepared using a modified polyol method that allowed us to obtain nanowires with homogeneous sizes. Figure 1a shows the UV-Vis in an absorption spectrum of the purified AgNWs resuspended in ethanol. The spectrum showed a peak at 402 nm, accompanied by a shoulder at 355 nm, which are considered the AgNWs fingerprint [39]. TEM images (Figure 1b) revealed homogeneous metallic nanostructures with an average diameter of 400 ± 20 nm and 10 ± 0.05 µm in length. The XRD pattern is shown in Figure 1c.

Characterization of AgNWs
AgNWs were prepared using a modified polyol method that allowed us to obtain nanowires with homogeneous sizes. Figure 1a shows the UV-Vis in an absorption spectrum of the purified AgNWs resuspended in ethanol. The spectrum showed a peak at 402 nm, accompanied by a shoulder at 355 nm, which are considered the AgNWs fingerprint [39]. TEM images (Figure 1b) revealed homogeneous metallic nanostructures with an average diameter of 400 ± 20 nm and 10 ± 0.05 μm in length. The XRD pattern is shown in Figure 1c. As Ag arranges in the fcc structure, the five sharp peaks, displayed at 2θ angles  of 38°, 44°, 64°, 77° and 82°, correspond to   The resistivity of a thin film of AgNWs (deposited on ITO by means of spin coating), was tested by means of the four point probe test. The resistivity (ρ) was calculated using Equation (1): where V is the voltage measured between the inner probes, I is the intensity flowing through the circuit (between the outer probes), and t is the film thickness (which is equal to 1 if the thickness is negligible). Results are shown in Figure 2. As expected, the presence of AgNWs increased the conductivity of the electrode. This effect could be advantageous in sensing applications. The resistivity of a thin film of AgNWs (deposited on ITO by means of spin coating), was tested by means of the four point probe test. The resistivity (ρ) was calculated using Equation (1): where V is the voltage measured between the inner probes, I is the intensity flowing through the circuit (between the outer probes), and t is the film thickness (which is equal to 1 if the thickness is negligible). Results are shown in Figure 2. As expected, the presence of AgNWs increased the conductivity of the electrode. This effect could be advantageous in sensing applications.

Characterization of the AgNWs-Tyr Biosensors: Spectroscopy and AFM
A buffer solution of the enzyme was drop-casted onto the AgNWs film to obtain the AgNWs-Tyr biosensor. UV-Vis, FTIR and AFM analysis were performed to characterize the structure of the biosensor and confirm the immobilization of the enzyme. The UV-Vis spectrum of the biosensor clearly shows the features corresponding to the AgNWs, along with a characteristic absorption peak at 280 nm due to the presence of the amino acid tyrosine (Figure 3a). The adsorption of tyrosinase was corroborated by the FTIR spectrum (Figure 3b), where one band at 1100 cm −1 associated to the presence of hydroxyl groups, and two characteristic bands at 1550 and 1650 cm −1 (corresponding to the presence of the

Characterization of the AgNWs-Tyr Biosensors: Spectroscopy and AFM
A buffer solution of the enzyme was drop-casted onto the AgNWs film to obtain the AgNWs-Tyr biosensor. UV-Vis, FTIR and AFM analysis were performed to characterize the structure of the biosensor and confirm the immobilization of the enzyme. The UV-Vis spectrum of the biosensor clearly shows the features corresponding to the AgNWs, along with a characteristic absorption peak at 280 nm due to the presence of the amino acid tyrosine (Figure 3a). The adsorption of tyrosinase was corroborated by the FTIR spectrum (Figure 3b), where one band at 1100 cm −1 associated to the presence of hydroxyl groups, and two characteristic bands at 1550 and 1650 cm −1 (corresponding to the presence of the enzymatic Amide II and I, respectively) could be observed [40].

Characterization of the AgNWs-Tyr Biosensors: Spectroscopy and AFM
A buffer solution of the enzyme was drop-casted onto the AgNWs film to obtain the AgNWs-Tyr biosensor. UV-Vis, FTIR and AFM analysis were performed to characterize the structure of the biosensor and confirm the immobilization of the enzyme. The UV-Vis spectrum of the biosensor clearly shows the features corresponding to the AgNWs, along with a characteristic absorption peak at 280 nm due to the presence of the amino acid tyrosine (Figure 3a). The adsorption of tyrosinase was corroborated by the FTIR spectrum (Figure 3b), where one band at 1100 cm −1 associated to the presence of hydroxyl groups, and two characteristic bands at 1550 and 1650 cm −1 (corresponding to the presence of the enzymatic Amide II and I, respectively) could be observed [40]. AgNWs were also characterized using AFM. As observed in Figure 4, AgNWs deposited onto the ITO glass showed a length of ~10 μm and a width of ~400 nm. The AFM image of Tyr deposited on the ITO-AgNWs surface showed a highly homogeneous distribution of the enzyme on the surface of the metallic nanowires. The width of the AgNWs-Tyr nanostructure increased uniformly (width ~600 nm). This can be explained by the high affinity between AgNWs and tyrosinase, which maximized the contact surface, reducing the insulating effects while facilitating the electron transfer-as demonstrated in the next section. AgNWs were also characterized using AFM. As observed in Figure 4, AgNWs deposited onto the ITO glass showed a length of~10 µm and a width of~400 nm. The AFM image of Tyr deposited on the ITO-AgNWs surface showed a highly homogeneous distribution of the enzyme on the surface of the metallic nanowires. The width of the AgNWs-Tyr nanostructure increased uniformly (width~600 nm). This can be explained by the high affinity between AgNWs and tyrosinase, which maximized the contact surface, reducing the insulating effects while facilitating the electron transfer-as demonstrated in the next section.

Electrochemical Characterization of the AgNWs-Tyr Biosensor
Cyclic voltammetry (CV) was used to study the electrochemical response of the AgNWs-Tyr biosensor. Experiments were carried out in catechol 10 −4 M in PBS solution 0.01 M, pH 7.0, at a scan rate of 100 mV/s. The responses of bare ITO and ITO covered by AgNWs to catechol were registered for comparison purposes and are shown in Figure 5. The response of ITO to catechol was characterized by a cathodic peak at −150 mV, pro-

Electrochemical Characterization of the AgNWs-Tyr Biosensor
Cyclic voltammetry (CV) was used to study the electrochemical response of the AgNWs-Tyr biosensor. Experiments were carried out in catechol 10 −4 M in PBS solution 0.01 M, pH 7.0, at a scan rate of 100 mV/s. The responses of bare ITO and ITO covered by AgNWs to catechol were registered for comparison purposes and are shown in Figure 5. The response of ITO to catechol was characterized by a cathodic peak at −150 mV, produced by the reduction of the o-quinone (obtained by the oxidation of catechol [17,[40][41][42]. The intensity of the peak increased when the ITO was covered with AgNWs (−13 µA for bare ITO and −20 µA for ITO-AgNWs). In addition, a redox pair corresponding to the Ag + /Ag process was also observed at 268 mV (cathodic wave) and 595 mV (anodic wave).

Electrochemical Characterization of the AgNWs-Tyr Biosensor
Cyclic voltammetry (CV) was used to study the electrochemical response of the AgNWs-Tyr biosensor. Experiments were carried out in catechol 10 −4 M in PBS solution 0.01 M, pH 7.0, at a scan rate of 100 mV/s. The responses of bare ITO and ITO covered by AgNWs to catechol were registered for comparison purposes and are shown in Figure 5. The response of ITO to catechol was characterized by a cathodic peak at −150 mV, produced by the reduction of the o-quinone (obtained by the oxidation of catechol [17,[40][41][42]. The intensity of the peak increased when the ITO was covered with AgNWs (−13 μA for bare ITO and −20 μA for ITO-AgNWs). In addition, a redox pair corresponding to the Ag + /Ag process was also observed at 268 mV (cathodic wave) and 595 mV (anodic wave). When Tyr was deposited onto the ITO electrode, the intensity of the peak corresponding to the reduction of o-quinone was clearly higher than in the absence of the enzyme (−13 μA in bare ITO and −77 μA for ITO-Tyr). The intensity of the cathodic peak increased in the presence of spherical silver nanoparticles (−93 μA for AgNPs-Tyr). This intensity increased even further in the presence of nanowires (−128 μA for AgNWs-Tyr) ( Figure 6). This result evidenced the influence of the shape of the silver nanomaterial and When Tyr was deposited onto the ITO electrode, the intensity of the peak corresponding to the reduction of o-quinone was clearly higher than in the absence of the enzyme (−13 µA in bare ITO and −77 µA for ITO-Tyr). The intensity of the cathodic peak increased in the presence of spherical silver nanoparticles (−93 µA for AgNPs-Tyr). This intensity increased even further in the presence of nanowires (−128 µA for AgNWs-Tyr) ( Figure 6). This result evidenced the influence of the shape of the silver nanomaterial and confirmed that AgNWs, having a higher aspect ratio than AgNPs, improved the electron transfer between the enzyme and the electrode.
Differential pulse voltammetry (DPV) was used to evaluate the response of the AgNWs-Tyr biosensor to increasing concentrations of catechol in PBS solution 0.01 M, pH 7.0, at a scan rate of 100 mV/s. As observed in Figure 7, the current intensity increased linearly with the catechol concentration from 24.9 to 172 µM, following the equation y = (−197.9 ± 8.61) × (−139.98 ± 0.95)(R 2 = 0.9759). The limit of detection (LOD), calculated following the 3·σ/m criterion, was 2.7·10 −6 M for AgNWs-Tyr, whereas the LOD obtained using an AgNPs-Tyr biosensor (prepared under the same conditions) was 3.2 × 10 −5 M. In addition, the sensitivity calculated for the AgNW-Tyr biosensor was 197.9 µA·mM −1 which is one to two orders of magnitude higher than the sensitivity shown by other sensors combining tyrosinase and silver nanoparticles reported in recent papers [16,43]. Some results obtained with biosensors containing AgNPs or AuNPs are collected in Table 1. confirmed that AgNWs, having a higher aspect ratio than AgNPs, improved the electron transfer between the enzyme and the electrode. Differential pulse voltammetry (DPV) was used to evaluate the response of the AgNWs-Tyr biosensor to increasing concentrations of catechol in PBS solution 0.01 M, pH 7.0, at a scan rate of 100 mV/s. As observed in Figure 7, the current intensity increased linearly with the catechol concentration from 24.9 to 172 μM, following the equation y = (−197.9 ± 8.61) × (−139.98 ± 0.95)(R 2 = 0.9759). The limit of detection (LOD), calculated following the 3·σ/m criterion, was 2.7·10 −6 M for AgNWs-Tyr, whereas the LOD obtained using an AgNPs-Tyr biosensor (prepared under the same conditions) was 3.2 × 10 −5 M. In addition, the sensitivity calculated for the AgNW-Tyr biosensor was 197.9 μA·mM −1 which is one to two orders of magnitude higher than the sensitivity shown by other sensors combining tyrosinase and silver nanoparticles reported in recent papers [16,43]. Some results obtained with biosensors containing AgNPs or AuNPs are collected in Table 1.   Differential pulse voltammetry (DPV) was used to evaluate the response of the AgNWs-Tyr biosensor to increasing concentrations of catechol in PBS solution 0.01 M, pH 7.0, at a scan rate of 100 mV/s. As observed in Figure 7, the current intensity increased linearly with the catechol concentration from 24.9 to 172 μM, following the equation y = (−197.9 ± 8.61) × (−139.98 ± 0.95)(R 2 = 0.9759). The limit of detection (LOD), calculated following the 3·σ/m criterion, was 2.7·10 −6 M for AgNWs-Tyr, whereas the LOD obtained using an AgNPs-Tyr biosensor (prepared under the same conditions) was 3.2 × 10 −5 M. In addition, the sensitivity calculated for the AgNW-Tyr biosensor was 197.9 μA·mM −1 which is one to two orders of magnitude higher than the sensitivity shown by other sensors combining tyrosinase and silver nanoparticles reported in recent papers [16,43]. Some results obtained with biosensors containing AgNPs or AuNPs are collected in Table 1.   Finally, the apparent Michaelis-Menten (K app M ) constant was calculated following the Lineweaver-Burk approach by representing 1/I lim vs. 1/[S ox ], where I lim is the intensity of the maximum and Sox is the substrate concentration (catechol in this case) [46]. These results confirm the excellent performance of AgNWs as electron mediators, probably due to the excellent affinity between the AgNWs and the enzyme.
The selectivity of the AgNWs-Tyr biosensor was evaluated by analyzing the response to catechol (a diphenol) in the presence of other phenols usually found in foods including vanillin (a monophenol) and pyrogallol (a triphenol). Two flavonoids containing a resorcinol moiety linked to a catechol moiety (quercitin and catechin) were also included in the study. Experiments were carried out using a 2 × 10 −4 M solution of catechol that was diluted 1:1 (v:v) with a 2 × 10 −4 M solution of the corresponding interferent. The intensities of the cathodic peaks at~−400 mV were compared with the response of 10 −4 M solutions of the pure components. As shown in Figure 8, the AgNWs-Tyr biosensor did not respond to other phenols usually present in foods, confirming the high degree of selectivity toward this phenol. This result can be explained taking into account that tyrosinase has high affinity toward o-diphenols, whereas it has a limited affinity toward triphenols and polyphenols. This justifies the small response observed toward solutions of vanillic acid, pyrogallol, quercitin or catechin. The decrease in the responses toward mixtures of catechol:pyrogallol, catechol:quercitin, or catechol:catechin can be explained by assuming that pyrogallol, catechin and quercitine can block some active sites, reducing the response of the biosensor to catechol.
Finally, the apparent Michaelis-Menten ( app M K ) constant was calculated following the Lineweaver-Burk approach by representing 1/Ilim vs. 1/[Sox], where Ilim is the intensity of the maximum and Sox is the substrate concentration (catechol in this case) [46]. These results confirm the excellent performance of AgNWs as electron mediators, probably due to the excellent affinity between the AgNWs and the enzyme.
The selectivity of the AgNWs-Tyr biosensor was evaluated by analyzing the response to catechol (a diphenol) in the presence of other phenols usually found in foods including vanillin (a monophenol) and pyrogallol (a triphenol). Two flavonoids containing a resorcinol moiety linked to a catechol moiety (quercitin and catechin) were also included in the study. Experiments were carried out using a 2 × 10 −4 M solution of catechol that was diluted 1:1 (v:v) with a 2 × 10 −4 M solution of the corresponding interferent. The intensities of the cathodic peaks at ~−400 mV were compared with the response of 10 −4 M solutions of the pure components. As shown in Figure 8, the AgNWs-Tyr biosensor did not respond to other phenols usually present in foods, confirming the high degree of selectivity toward this phenol. This result can be explained taking into account that tyrosinase has high affinity toward o-diphenols, whereas it has a limited affinity toward triphenols and polyphenols. This justifies the small response observed toward solutions of vanillic acid, pyrogallol, quercitin or catechin. The decrease in the responses toward mixtures of catechol:pyrogallol, catechol:quercitin, or catechol:catechin can be explained by assuming that pyrogallol, catechin and quercitine can block some active sites, reducing the response of the biosensor to catechol.

In Situ EC-AFM Studies. Reproducibility and Repeatability
In recent years, the simultaneous use of electrochemistry and AFM has emerged as a useful technique to study the electrode surfaces and surface phenomena occurring during the electrochemical process. The repeatability of the reported AgNWs-Tyr biosensors was evaluated by measuring 15 consecutive cycles in catechol 10 −4 M in PBS solution 0.01 M, pH 7.0, at a scan rate of 100 mV/s from −600 to 1200 mV. The calculated variation coefficient was 3.2%, demonstrating the effective immobilization of the enzyme with a minimal loss of enzymatic activity and minimal fouling upon successive cycling.
Surface changes occurring during cycling were further analyzed by AFM. Figure 9 shows the topography of ITO-AgNWs (left) and ITO-AgNWs-Tyr (right) before and after voltammetry.
formly covered the surface of the AgNWs, filling the holes and spaces left by the enzy during its deposition. These precipitates could easily be removed by washing with wa indicating that the precipitates were produced by crystallites of the saline buffer, or polyphenol weakly bound to the surface. Moreover, after washing, the original size w restored and the initial voltammograms could be reproduced with only minor differen with respect to the original one. According to these results, it can be concluded that presence of AgNWs can prevent the nonspecific adsorption of fouling agents.  AFM images registered after 15 consecutive cycles demonstrated the formation of precipitates that were mainly concentrated on the surface of the metallic structure. In the case of the ITO-AgNWs surface, precipitates were large and homogeneously distributed along the AgNWs surface, whereas in the AgNWs-Tyr biosensor, the precipitates uniformly covered the surface of the AgNWs, filling the holes and spaces left by the enzyme during its deposition. These precipitates could easily be removed by washing with water, indicating that the precipitates were produced by crystallites of the saline buffer, or by polyphenol weakly bound to the surface. Moreover, after washing, the original size was restored and the initial voltammograms could be reproduced with only minor differences with respect to the original one. According to these results, it can be concluded that the presence of AgNWs can prevent the nonspecific adsorption of fouling agents.
Finally, in order to evaluate the reproducibility of the AgNWs-based device, the voltammetric responses of three devices, prepared under the same conditions, were observed; these showed a relative standard deviation (RDS) of 1.65%. The excellent reproducibility could be attributed, at least in part, to the size homogeneity of the AgNWs obtained using our improved synthetic method that can help to minimize differences from one device to another ( Figure 10).
Finally, in order to evaluate the reproducibility of the AgNWs-based device, the voltammetric responses of three devices, prepared under the same conditions, were observed; these showed a relative standard deviation (RDS) of 1.65%. The excellent reproducibility could be attributed, at least in part, to the size homogeneity of the AgNWs obtained using our improved synthetic method that can help to minimize differences from one device to another ( Figure 10). Finally, the developed nanowired biosensor AgNWs-Tyr was used to assess the catechol concentration in a commercial red wine using the standard addition method (addition of 10 −5 , 5 × 10 −5 , 10 −4 , 2.5 × 10 −4 , 5 × 10 −4 , 7.5 × 10 −4 , 10 −3 , 5 × 10 −3 , 10 −2 M catechol in wine diluted 10% in PBS 0.01 M). The values obtained corresponded to the mean of three determinations and their subsequent associated standard deviations. The obtained voltammograms and the calibration curve are represented in Figure 11, [y = (26.64 ± 2.09)·log(x) + (39.56 ± 1.08)] (R 2 = 95.8115). The calculated concentration of catechol in the diluted wine sample was 32 μM, which is in good agreement with the results obtained in previous works based on electrochemical sensors for catechol detection in red wines [19].  Finally, the developed nanowired biosensor AgNWs-Tyr was used to assess the catechol concentration in a commercial red wine using the standard addition method (addition of 10 −5 , 5 × 10 −5 , 10 −4 , 2.5 × 10 −4 , 5 × 10 −4 , 7.5 × 10 −4 , 10 −3 , 5 × 10 −3 , 10 −2 M catechol in wine diluted 10% in PBS 0.01 M). The values obtained corresponded to the mean of three determinations and their subsequent associated standard deviations. The obtained voltammograms and the calibration curve are represented in Figure 11, [y = (26.64 ± 2.09)·log(x) + (39.56 ± 1.08)] (R 2 = 95.8115). The calculated concentration of catechol in the diluted wine sample was 32 µM, which is in good agreement with the results obtained in previous works based on electrochemical sensors for catechol detection in red wines [19].
Finally, in order to evaluate the reproducibility of the AgNWs-based device, the voltammetric responses of three devices, prepared under the same conditions, were observed; these showed a relative standard deviation (RDS) of 1.65%. The excellent reproducibility could be attributed, at least in part, to the size homogeneity of the AgNWs obtained using our improved synthetic method that can help to minimize differences from one device to another ( Figure 10). Finally, the developed nanowired biosensor AgNWs-Tyr was used to assess the catechol concentration in a commercial red wine using the standard addition method (addition of 10 −5 , 5 × 10 −5 , 10 −4 , 2.5 × 10 −4 , 5 × 10 −4 , 7.5 × 10 −4 , 10 −3 , 5 × 10 −3 , 10 −2 M catechol in wine diluted 10% in PBS 0.01 M). The values obtained corresponded to the mean of three determinations and their subsequent associated standard deviations. The obtained voltammograms and the calibration curve are represented in Figure 11, [y = (26.64 ± 2.09)·log(x) + (39.56 ± 1.08)] (R 2 = 95.8115). The calculated concentration of catechol in the diluted wine sample was 32 μM, which is in good agreement with the results obtained in previous works based on electrochemical sensors for catechol detection in red wines [19].

Conclusions
Silver nanowires were used as a platform for the immobilization of tyrosinase, leading to a catechol biosensor with excellent performance. The nanowires showed a high affinity toward the enzyme, and the high aspect ratio facilitated the electron transfer. The AgNWs-Tyr biosensor presented a linear range from 24.9 to 172 µM, and a detection limit of 2.7 × 10 −6 M. It showed a high sensitivity and low KM app values, related to the good accessibility of catechol molecules to the active site of the enzyme. The excellent reproducibility and repeatability obtained could be attributed to the homogeneity of the AgNWs and their ability to prevent fouling effects. The implementation of the biosensor in a wine complex matrix demonstrated the feasibility of incorporating the developed biosensor in field applications.

Data Availability Statement:
The data presented in this study are available on request from the corresponding author.

Conflicts of Interest:
The authors declare no conflict of interest.