Development of a Chemically Modified Sensor Based on a Pentapeptide and Its Application for Sensitive Detection of Verbascoside in Extra Virgin Olive Oil

In addition to their antioxidant and antimicrobial action in functional foods, beverages, and in some dermato-cosmetic products, olive phenolic compounds are also recognized for their role in the prevention of diabetes and inflammation, treatment of heart disease and, consequently, of the numerous chronic diseases mediated by the free radicals. In recent years, attention has increased, in particular, regarding one of the most important compound in extra virgin olive oil (EVOO) having glycosidic structure, namely verbocoside, due to the existence in the literature of numerous studies demonstrating its remarkable contribution to the prophylaxis and treatment of various disorders of the human body. The purpose of this study was the qualitative and quantitative determination of verbascoside in commercial EVOOs from different regions by means of a newly developed sensor based on a screen-printed carbon electrode (SPCE) modified with graphene oxide (GPHOX), on the surface of which a pentapeptide was immobilized by means of glutaraldehyde as cross-linking agent. The modified electrode surface was investigated using both Fourier-transform infrared spectroscopy (FTIR) and scanning electron microscopy (SEM) methods. This newly developed sensor has shown a high sensibility compared to the unmodified electrode, a low detection limit (LOD) of up to 9.38 × 10−8 M, and a wide linearity range between 0.1 µM and 10.55 µM. The applicability of the modified sensor was confirmed by detecting verbascoside in ten different EVOOs samples using the cyclic voltammetry (CV) method, with very good results. The validation of the electroanalytical method was performed by using the standard addition method with very good recoveries in the range of 97.48–103.77%.


Introduction
In recent years, the study of GPHOX-based materials has been expanded especially in terms of electrochemical applications. Underlying the importance of GPHOX-based materials in electrochemistry are their unique characteristics, such as very good chemical stability, high hydrophilicity, high dispersity in various solvents and ease of functionalization [1]. Typically, GPHOX is obtained by a simple wet oxidative exfoliation process of graphite using strong oxidants [2] followed by the manufacture of flexible GPHOX thin films by simple liquid phase processes such as the casting technique [3,4], vacuum filtration [5] and spray coating [6]. The physical and chemical properties of GPHOX offer promising applications for chemical sensors [7], mechanical [8] and electronic [9], energy storage [10], optoelectronic devices [11], nanoelectronics [12] and biotechnology [13]. A series of characteristics of GPHOX, including the high dispersibility in water, but also in organic solvents, the very good surface-to-volume ratio, the existence of a wide range of surface-bound reactive functional groups (mainly epoxy and hydroxyl groups) [14], gives fascinating In the specialized literature, there are numerous studies on the development and evaluation of methods for the analysis, isolation and identification of phenolic compounds from olive oils [36,37]. Among the analytical methods for the quantification of polyphenols in olive oil is the Folin-Ciocalteau spectrophotometric assay for the evaluation of phenolic compounds content [38,39] and also high-performance liquid chromatography (HPLC) [40,41] for the identification and quantification of individual phenolic compounds.
Recently, due to their high sensitivity, low cost, and the possibility of miniaturization and automation, a number of sensors and biosensors have been described in the literature, their simplicity favoring their use for olive oil analysis over classical methods [42,43]. Among electrochemical methods, voltammetric techniques have been successfully used to detect phenolic compounds in aqueous solutions and in different food matrices, for example, tea, wine, beer, and, not least, olive oil [44].
In this study, a direct electrochemical method capable of assessing and quantifying the verbascoside content in different EVOO samples is described with minimal sample preparation using an SPCE modified with a GPHOX composite film on the surface of which a pentapeptide (NH2-FESNF-CO-NH2, sequence where F-phenylalanine, Eglutamic acid, S-serine, and N-asparagine), the sequence of which is found in the primary structure of the lysozyme in egg white, has been immobilized ( Figure 2). The proposed method is simple, fast and offers a promising alternative to more complex analytical techniques. To the best of our knowledge, until now, there is no study related to the detection of verbascoside with a GPHOX-peptide modified sensor, and in the future, research should be conducted on the use of this device for the detection of other types of samples, such as pharmaceuticals in different dosage forms, dietary supplements or biological samples such as human serum. In this study, a direct electrochemical method capable of assessing and quantifying the verbascoside content in different EVOO samples is described with minimal sample preparation using an SPCE modified with a GPHOX composite film on the surface of which a pentapeptide (NH 2 -FESNF-CO-NH 2, sequence where F-phenylalanine, E-glutamic acid, S-serine, and N-asparagine), the sequence of which is found in the primary structure of the lysozyme in egg white, has been immobilized ( Figure 2). The proposed method is simple, fast and offers a promising alternative to more complex analytical techniques. To the best of our knowledge, until now, there is no study related to the detection of verbascoside with a GPHOX-peptide modified sensor, and in the future, research should be conducted on the use of this device for the detection of other types of samples, such as pharmaceuticals in different dosage forms, dietary supplements or biological samples such as human serum.

Electrode Characterisation
The construction of a robust sensor by modifying its surface with pentapeptide solution and fixing it by cross-linking involves obtaining analytically important parameters, including sensitivity, lifetime, detection, and quantification limits [45].
To observe the changes using pentapeptide in the commercial SPCE/GPHOX, its active surface was analyzed by means of two techniques, the FTIR spectrometric method and SEM, respectively.
2.1.1. FTIR Spectrometric Method FTIR spectra ( Figure 3) were recorded for both the single electrode, SPCE/GPHOX, and the modified electrode, SPCE/GPHOX-Pentapeptide, to confirm peptide film formation on the sensor surface and also to define the characteristic peaks of GPHOXrelated functional groups containing oxygen, such as epoxy, carbonyl and hydroxyl.

Electrode Characterisation
The construction of a robust sensor by modifying its surface with pentapeptide solution and fixing it by cross-linking involves obtaining analytically important parameters, including sensitivity, lifetime, detection, and quantification limits [45].
To observe the changes using pentapeptide in the commercial SPCE/GPHOX, its active surface was analyzed by means of two techniques, the FTIR spectrometric method and SEM, respectively.

FTIR Spectrometric Method
FTIR spectra ( Figure 3) were recorded for both the single electrode, SPCE/GPHOX, and the modified electrode, SPCE/GPHOX-Pentapeptide, to confirm peptide film formation on the sensor surface and also to define the characteristic peaks of GPHOX-related functional groups containing oxygen, such as epoxy, carbonyl and hydroxyl. solution and fixing it by cross-linking involves obtaining analytically important parameters, including sensitivity, lifetime, detection, and quantification limits [45].
To observe the changes using pentapeptide in the commercial SPCE/GPHOX, its active surface was analyzed by means of two techniques, the FTIR spectrometric method and SEM, respectively.

FTIR Spectrometric Method
FTIR spectra ( Figure 3) were recorded for both the single electrode, SPCE/GPHOX, and the modified electrode, SPCE/GPHOX-Pentapeptide, to confirm peptide film formation on the sensor surface and also to define the characteristic peaks of GPHOXrelated functional groups containing oxygen, such as epoxy, carbonyl and hydroxyl.  The different oxygen-containing functional groups in the GPHOX structure have been identified by the characteristic vibrational modes of the epoxy (C-O-C) group (1230-1320 cm −1 ) [43], a stretch band corresponding to the C=O group in the carboxyl group at 1721 cm −1 [46,47], a deformation vibration band related to O-H at 1404 cm −1 [48], respectively a stretching vibration 1087 cm −1 belonging to the alkoxy group C-O [49]. Moreover, in the FTIR spectrum of GPHOX, due to extensive oxidation, GPHOX has a strong and broad O-H stretching vibration band at 3410 cm −1 [50], characteristic of GPHOX due to the existence of many hydroxyl groups in its structure [51].
The FTIR method is one of the few techniques that can also be applied to the structural characterization of peptides in different media. Thus, the type I amide bands found in the range 1600-1700 cm −1 were the most intense absorption bands, indicating that a β-sheet conformation is present in the structure of the peptide film formed at the sensor surface [52]. Along with these, the band at 1595 cm −1 can be attributed to additional interactions occurring in the fibrils, such as protofilament packing [53,54]. The existence of peaks corresponding to the amide supports the presence of the pentapeptide on the whole electrode surface. These peaks are not present in the case of the unmodified electrode, i.e., SPCE/GPHOX [55]. The band observed at 1762 cm −1 can be attributed to the C=O stretching mode of the carboxyl group of either asparagine or glutamate residue or C=O from the amide group located on the C-terminal amino acid [56]. Moreover, the stretching vibration corresponding to free or non-hydrogen bonding -NH 2 groups occurs at 3445 cm −1 [57] and at 3281 cm −1 [58,59], respectively, and the absorption corresponding to wave number 3000 cm −1 corresponds to the O-H stretching vibration of the hydroxyl group in the serine structure [60].

Morphological Characterisation Using SEM
The SEM image ( Figure 4) shows the morphology of the active sensor surface. Threedimensional arrangements consisting of continuous fibers with good homogeneity are observed, showing a homogeneity of the composite nanofilm surface. On the other hand, in Figure 4, the presence of GPHOX sheets is not clearly evident. They are covered by fibers, and this is evidence of GPHOX incorporation in the peptide fibrillary network [59]. electrode, i.e., SPCE/GPHOX [55]. The band observed at 1762 cm −1 can be attributed to the C=O stretching mode of the carboxyl group of either asparagine or glutamate residue or C=O from the amide group located on the C-terminal amino acid [56]. Moreover, the stretching vibration corresponding to free or non-hydrogen bonding -NH2 groups occurs at 3445 cm −1 [57] and at 3281 cm −1 [58,59], respectively, and the absorption corresponding to wave number 3000 cm −1 corresponds to the O-H stretching vibration of the hydroxyl group in the serine structure [60].

Morphological Characterisation Using SEM
The SEM image ( Figure 4) shows the morphology of the active sensor surface. Threedimensional arrangements consisting of continuous fibers with good homogeneity are observed, showing a homogeneity of the composite nanofilm surface. On the other hand, in Figure 4, the presence of GPHOX sheets is not clearly evident. They are covered by fibers, and this is evidence of GPHOX incorporation in the peptide fibrillary network [59].

Preliminary Studies for the Characterisation of SPCE/GPHOX-Pentapeptide
Taking into account our previous work [23], where it was demonstrated that the optimal pH value for electrochemical determinations at which a stable signal was obtained was 6.5, the determinations in this study were performed in solutions obtained by dissolving in 0.1 M PBS at pH 6.5. Moreover, in the same study, the optimal amount of peptide required for sensor modification was determined using different volumes of the peptide solution, demonstrating that the anodic peak was more intense and better defined when using a volume of 20 µL [23]. Therefore, the same volume of 20 µL of the pentapeptide solution was also used in the present study to obtain the modified sensor.
Further preliminary analyses consisted of evaluating the electrochemical behavior of SPCE/GPHOX and SPCE/GPHOX-Pentapeptide in 0.1 M PBS at pH = 6.5. According to previous studies [23,60,61], the potential range in which a stable signal was obtained was from −0.4 to 1.0 V, proving to be optimal for the new peptide-based sensor described in the present study. The signal was stable after three cycles without interference or background noise, suggesting that there was no contamination of the active surface during the modification steps. Hence, the same potential range was used in the study of the kinetics of the electrochemical reaction of verbascoside. At the same time, the lack of contamination of the active surface of the electrode can also be demonstrated by the fact that when immersing SPCE/GPHOX in 10 −1 M PBS solution, the CVs do not show oxidation or reduction peaks in the studied potential range (Figure 5a).
Upon immersion of the SPCE/GPHOX-Pentapeptide sensor in 0.1 M PBS solution (Figure 5b), the CVs recorded show the electrode signal changing during the first cycle, with the signal stabilizing after three cycles. In the case of the modified electrode, the oxidation peak appears more intense at the first scan at a potential value of 0.64 V (Ipa = 32.74 µA).
At the second and third scan cycles, the oxidation peaks are evident but are not as intense as that present at the first scan. Moreover, in all three successive scans, the reduction peaks can be seen at the same potential and having approximately the same intensity (Epc = −0.02 V, Ipc = -9.58 µA).
the kinetics of the electrochemical reaction of verbascoside. At the same time, the lack of contamination of the active surface of the electrode can also be demonstrated by the fact that when immersing SPCE/GPHOX in 10 −1 M PBS solution, the CVs do not show oxidation or reduction peaks in the studied potential range (Figure 5a).
Upon immersion of the SPCE/GPHOX-Pentapeptide sensor in 0.1 M PBS solution (Figure 5b), the CVs recorded show the electrode signal changing during the first cycle, with the signal stabilizing after three cycles. In the case of the modified electrode, the oxidation peak appears more intense at the first scan at a potential value of 0.64 V (Ipa = 32.74 µA). At the second and third scan cycles, the oxidation peaks are evident but are not as intense as that present at the first scan. Moreover, in all three successive scans, the reduction peaks can be seen at the same potential and having approximately the same intensity (Epc = −0.02 V, Ipc = -9.58 µA).

Electrochemical Properties of SPCE/GPHOX-Pentapeptide in K4[Fe(CN)6]/K3[Fe(CN)6] Solution
K4[Fe(CN)6] exhibits redox activity, and this can be evidenced by CV, when an anodic and a cathodic peak is obtained due to the reversible oxidation of the ferrocyanide ion to ferricyanide, occurring at the electrode surface [62].    6 ] exhibits redox activity, and this can be evidenced by CV, when an anodic and a cathodic peak is obtained due to the reversible oxidation of the ferrocyanide ion to ferricyanide, occurring at the electrode surface [62].
The voltammetric responses of the sensors immersed in a solution containing 10 −3 M K 4 [Fe(CN) 6 ]/K 3 [Fe(CN) 6 ] dissolved in 0.1 M PBS, pH = 6.5 recorded in the same potential range were well defined and reproducible at a scan rate of 0.05 V·s −1 ( Figure 6). It can be seen that a pair of redox peaks, one anodic and one cathodic due to the ferrocyanide/ferricyanide redox process, is present in both sensors but of different intensities and potentials.  (Figure 6). It can be seen that a pair of redox peaks, one anodic and one cathodic due to the ferrocyanide/ferricyanide redox process, is present in both sensors but of different intensities and potentials.  Table 1.   Table 1. As can be seen, the peptide-modified electrode showed the highest degree of reversibility, with the separation between anodic and cathodic peaks being smaller than for the unmodified electrode and the Ipc/Ipa ratio greater than 1 (1.13). This demonstrates that the reaction process for the modified electrode is quasi-reversible. Both electrodes show similar electrochemical behavior, with almost double sensitivity in the case of the pentapeptide-modified sensor, according to the obtained parameters, and can be successfully used in further determinations.
Another important parameter in the electrochemical determination of redox processes using CV is the scan rate. Thus, the influence of scan rate on the voltammetric responses of the sensors was obtained by recording the CVs of the two electrodes in 10 In Figure 7a,c, it can be seen that as the applied scan rate increases, the anodic and cathodic peak currents also increase linearly, indicating a quasi-reversible oxidation reaction [63]. The measured values for the anodic peak currents were used to determine the linear dependence equation of Ipa on the square root of the scan rate, v 1/2 , which indicated a typical diffusion-controlled reaction [64] obtained for the unmodified sensor In Figure 7a,c, it can be seen that as the applied scan rate increases, the anodic and cathodic peak currents also increase linearly, indicating a quasi-reversible oxidation reaction [63]. The measured values for the anodic peak currents were used to determine the linear dependence equation of Ipa on the square root of the scan rate, v 1/2 , which indicated a typical diffusion-controlled reaction [64] obtained for the unmodified sensor ( Figure 7b). The dependence between Ipa and scan rate, v, using the same experimental conditions, was a linear dependence in the case of the modified peptide-based sensor (Figure 7d), which is typical for the process controlled by the adsorption of the electroactive species [65]. Therefore, the modification of the pentapeptide sensor had a favorable effect on the reaction kinetics at the electrode surface, also creating an optimal environment for an analyte-sensitive layer interaction and, therefore, a fast electron transfer, resulting in a change of the rate-determining factor of the oxido-reduction process in the case of SPCE/GPHOX-Pentapeptide [66].

Electrochemical Sensor Responses in Verbascoside Solution
The next step of the present study was to analyze the behavior of both the modified and the unmodified sensor in verbascoside solution using CV.  In the case of the unmodified sensor, the cyclic voltammogram shows an anodic oxidation peak at the potential value of Epa = 0.38 V and a cathodic reduction peak at the potential value of Epc = 0.21 V.
Regarding the behavior of the peptide-modified sensor upon immersion in verbascoside solution, it is different from the unmodified sensor, which is evident in the corresponding voltammogram (red line) in Figure 8. Thus, the oxidation of verbascoside occurs in two steps, showing two anodic and two cathodic peaks of different intensities and potentials. The first oxidation peak occurs at the potential value of Epa1 = 0.08 V, followed by the second, well-defined oxidation peak at the potential value of Epa2 = 0.35 V. When scanning in the negative direction, the first reduction peak appears at a potential value of Epc2 = 0.22 V, and the second at a potential value of Epc1 = 0.01 V. The differences between the anodic and cathodic peak potentials, Epa1 -Epc1 = 0.07 V and Epa2 -Epc2 = 0.13 V, are in agreement with the corresponding value for a reversible reaction involving two electrons at each stage [67].
The electrochemical oxidation of verbascoside follows a mechanism of compounds with catecholic structure, i.e., a two-electron and two-proton transfer process, since, in the In the case of the unmodified sensor, the cyclic voltammogram shows an anodic oxidation peak at the potential value of Epa = 0.38 V and a cathodic reduction peak at the potential value of Epc = 0.21 V.
Regarding the behavior of the peptide-modified sensor upon immersion in verbascoside solution, it is different from the unmodified sensor, which is evident in the corresponding voltammogram (red line) in Figure 8. Thus, the oxidation of verbascoside occurs in two steps, showing two anodic and two cathodic peaks of different intensities and potentials. The first oxidation peak occurs at the potential value of Epa1 = 0.08 V, followed by the second, well-defined oxidation peak at the potential value of Epa2 = 0.35 V. When scanning in the negative direction, the first reduction peak appears at a potential value of Epc2 = 0.22 V, and the second at a potential value of Epc1 = 0.01 V. The differences between the anodic and cathodic peak potentials, Epa1 -Epc1 = 0.07 V and Epa2 -Epc2 = 0.13 V, are in agreement with the corresponding value for a reversible reaction involving two electrons at each stage [67].
The electrochemical oxidation of verbascoside follows a mechanism of compounds with catecholic structure, i.e., a two-electron and two-proton transfer process, since, in the chemical structure of this compound, there are two catecholic moieties corresponding to caffeic acid and hydroxytyrosol [68] (Figure 9). The two catechol fragments have different electron densities due to the functional groups to which they are bound. Since the carboxyl group is not directly linked to the aromatic ring, the electron-attracting inductive effect will be supported by the double bond. Moreover, the positive inductive effect of the alkyl chain increases the electron density on the aromatic ring, lowering the electrochemical oxidation potential. Therefore, we can deduce that the first pair of redox peaks is due to the caffeic acid residue, and the second pair of peaks appear due to the oxidation of hydroxytyrosol. The electrochemical parameters achieved from the CVs of the two sensors immersed in 10 −4 M verbascoside solution are presented in Table 2. Table 2. The values of the parameters obtained from the CVs of the two sensors immersed in 10 −4 M verbascoside-0.1 M PBS solution of pH 6.5.

Sensor
Epa1 The reduced value of the first anodic peak potential in the case of SPCE/GPHOX-Pentapeptide indicates a lower activation energy of the oxidation process, being influenced by the adsorption of verbascoside on pentapeptide predominantly through hydrogen bonds [67] and through π-π stacking interactions between pentapeptide and verbascoside aromatic rings [69]. Therefore, in the case of this sensor, the electron transfer occurring at the active surface is faster compared to the unmodified sensor [70].
From the determinations made so far, it can be deduced that the higher anode peak intensity in the modified sensor indicates a higher sensitivity of the sensor [71] and the lower potentials at which peaks related to the oxidation-reduction process of verbascoside occur in the case of the same modified sensor may suggest a better selectivity of the sensor for this analyte [72].
The next step was the study of the influence of scan rate on the voltammetric responses of the two sensors in a solution of 10 −4 M verbascoside-0.1 M PBS, pH = 6.5, The electrochemical parameters achieved from the CVs of the two sensors immersed in 10 −4 M verbascoside solution are presented in Table 2. The reduced value of the first anodic peak potential in the case of SPCE/GPHOX-Pentapeptide indicates a lower activation energy of the oxidation process, being influenced by the adsorption of verbascoside on pentapeptide predominantly through hydrogen bonds [67] and through π-π stacking interactions between pentapeptide and verbascoside aromatic rings [69]. Therefore, in the case of this sensor, the electron transfer occurring at the active surface is faster compared to the unmodified sensor [70].
From the determinations made so far, it can be deduced that the higher anode peak intensity in the modified sensor indicates a higher sensitivity of the sensor [71] and the lower potentials at which peaks related to the oxidation-reduction process of verbascoside occur in the case of the same modified sensor may suggest a better selectivity of the sensor for this analyte [72].
The next step was the study of the influence of scan rate on the voltammetric responses of the two sensors in a solution of 10 −4 M verbascoside-0.1 M PBS, pH = 6.5, applying scan rates in the range 0.05-0.5 V·s −1 (Figure 10). Since the anodic peaks are higher and better defined compared to the catodic ones, the dependence of Ipa on the scan rate or square root of the scan rate will be studied. By increasing the scan rate, it was obvious that the maximum oxidation potentials for both sensors (Figure 10a,c) were shifted toward more positive values and the reduction potentials toward more negative values. However, in the case of SPCE/GPHOX-Pentapeptide the Epa1-Epc1 and Epa2-Epc2 differences were always close to the theoretical value of 0.13 V corresponding to a reversible two-electron, two-proton mechanism, even for high scan rates. In the case of SPCE/GPHOX, as the scan rate increases, the intensity of the oxidation peak current increases linearly with the square root of the scan rate, consistent with oxidation limited by the diffusion of an active species in solution [73]. On the other hand, in the case of SPCE/GPHOX-Pentapeptide, as the scan rate increases, the intensity of the oxidation peak current also increases linearly with the scan rate, consistent with the electron-transfer-controlled oxidation process [74]. Moreover, the anodic peak intensities were higher in the modified sensor compared to those obtained with the unmodified sensor, the presence of peptide on the sensor surface significantly improving its sensitivity. The explanation can be related to the ability of some compounds to interact simultaneously or competitively with mixtures of proteins or peptides, the nature and mechanisms of the interaction depending on various environmental factors such as ionic strength, pH or concentration of the compounds on the one hand, and on the other hand on the structural characteristics, i.e., the polarity of By increasing the scan rate, it was obvious that the maximum oxidation potentials for both sensors (Figure 10a,c) were shifted toward more positive values and the reduction potentials toward more negative values. However, in the case of SPCE/GPHOX-Pentapeptide the Epa1-Epc1 and Epa2-Epc2 differences were always close to the theoretical value of 0.13 V corresponding to a reversible two-electron, two-proton mechanism, even for high scan rates. In the case of SPCE/GPHOX, as the scan rate increases, the intensity of the oxidation peak current increases linearly with the square root of the scan rate, consistent with oxidation limited by the diffusion of an active species in solution [73]. On the other hand, in the case of SPCE/GPHOX-Pentapeptide, as the scan rate increases, the intensity of the oxidation peak current also increases linearly with the scan rate, consistent with the electron-transfer-controlled oxidation process [74]. Moreover, the anodic peak intensities were higher in the modified sensor compared to those obtained with the unmodified sensor, the presence of peptide on the sensor surface significantly improving its sensitivity. The explanation can be related to the ability of some compounds to interact simultaneously or competitively with mixtures of proteins or peptides, the nature and mechanisms of the interaction depending on various environmental factors such as ionic strength, pH or concentration of the compounds on the one hand, and on the other hand on the structural characteristics, i.e., the polarity of the molecules, steric conformation and size of the molecules [75]. Compounds with a higher molecular weight but also with a higher number of hydroxyl groups, such as verbascoside, have a higher number of sites for interaction with peptides [76]. These interactions can occur via non-covalent bonds, i.e., hydrogen bonds or van der Waals forces [77] with the formation of complex structures, the assembly process often requiring a combination of multiple interaction sites [78]. Furthermore, the presence of interactions between the supramolecular structure, i.e., the peptide and the compound of interest, can influence the kinetics of the oxidation-reduction process, increasing the rate of electron transfer to the surface of the modified sensor [79] and also the result of a synergistic potentiation interaction [80].
Taking into account all the above, it is found that the same electrochemical behavior is maintained for both sensors as in the previous determinations in electroactive 10 −3 M K 4 [Fe(CN) 6 ]/K 3 [Fe(CN) 6 ]-PBS 0.1 M solution, demonstrating, once again, the role and influence of the modification of the sensor with the pentapeptide on the kinetics of the reaction at the electrode, respectively, on the process of the electron transfer.

Development of Calibration Curve
The concentration of the solutions to be analyzed is of major importance in the response of a voltammetric sensor. The dependence between anodic peak currents and the concentration of the compound of interest, i.e., verbascoside, allows current intensity measurement to be used for quantitative applications.
Therefore verbascoside, have a higher number of sites for interaction with peptides [76]. These interactions can occur via non-covalent bonds, i.e., hydrogen bonds or van der Waals forces [77] with the formation of complex structures, the assembly process often requiring a combination of multiple interaction sites [78]. Furthermore, the presence of interactions between the supramolecular structure, i.e., the peptide and the compound of interest, can influence the kinetics of the oxidation-reduction process, increasing the rate of electron transfer to the surface of the modified sensor [79] and also the result of a synergistic potentiation interaction [80]. Taking into account all the above, it is found that the same electrochemical behavior is maintained for both sensors as in the previous determinations in electroactive 10

Development of Calibration Curve
The concentration of the solutions to be analyzed is of major importance in the response of a voltammetric sensor. The dependence between anodic peak currents and the concentration of the compound of interest, i.e., verbascoside, allows current intensity measurement to be used for quantitative applications.
Therefore   As shown in Figure 11a, a linear relationship was obtained between peak current and verbascoside concentration in the range of 0.1-10.55 µM. Exceeding this concentration, the disappearance of linearity was observed, following a plateau phase in which, although the concentration is still increasing, the intensity of the anodic peak current remains constant in value. This means that all active centers on the sensor surface are involved in the electrochemical reaction [81].
The equation of the calibration curve can help us calculate the limit of detection (LOD) and the limit of quantification (LOQ) of the pentapeptide-modified sensor [82]. These were calculated using the equations LOD = 3σ/m; LOQ = 10σ/m, where σ is the standard deviation (SD) of the current recorded in the control sample (in this case, the PBS solution 0.1 M), and m is the slope of the calibration curve [83].
The values achieved for SPCE/GPHOX-Pentapeptide are: LOD = 9.38 × 10 −8 M and LOQ = 3.12 × 10 −7 M. Considering that these values obtained are similar or lower than those reported in the literature [84,85], this sensor was used for the quantitative determination of verbascoside in real samples, i.e., samples of different EVOO.

Accuracy of the Method
The accuracy of the method was evaluated by the standard addition method on samples of known concentration [86]. The percentage recovery of verbascoside added to the sample was evaluated by the relationship between the experimentally determined concentration and the corresponding theoretical concentration [87]. The closer the recovery percentage is to 100%, the more accurate the technique will be for subsequent determination of verbascoside in commercial EVOO samples. [88]. The results obtained, presented in Table 3, show this closeness, confirming that the method is suitable for the objective proposed. As shown in Figure 11a, a linear relationship was obtained between peak current and verbascoside concentration in the range of 0.1-10.55 µM. Exceeding this concentration, the disappearance of linearity was observed, following a plateau phase in which, although the concentration is still increasing, the intensity of the anodic peak current remains constant in value. This means that all active centers on the sensor surface are involved in the electrochemical reaction [81].
The equation of the calibration curve can help us calculate the limit of detection (LOD) and the limit of quantification (LOQ) of the pentapeptide-modified sensor [82]. These were calculated using the equations LOD = 3σ/m; LOQ = 10σ/m, where σ is the standard deviation (SD) of the current recorded in the control sample (in this case, the PBS solution 0.1 M), and m is the slope of the calibration curve [83].
The values achieved for SPCE/GPHOX-Pentapeptide are: LOD = 9.38 × 10 −8 M and LOQ = 3.12 × 10 −7 M. Considering that these values obtained are similar or lower than those reported in the literature [84,85], this sensor was used for the quantitative determination of verbascoside in real samples, i.e., samples of different EVOO.

Accuracy of the Method
The accuracy of the method was evaluated by the standard addition method on samples of known concentration [86]. The percentage recovery of verbascoside added to the sample was evaluated by the relationship between the experimentally determined concentration and the corresponding theoretical concentration [87]. The closer the recovery percentage is to 100%, the more accurate the technique will be for subsequent determination of verbascoside in commercial EVOO samples. [88]. The results obtained, presented in Table 3, show this closeness, confirming that the method is suitable for the objective proposed.

Stability, Reproducibility, Repeatability and Interference Studies
The stability of the SPCE/GPHOX-Pentapeptide was evaluated by performing 30 measurements at regular intervals (one day) for one month using a 10 −4 M verbascoside solution, using the CV method. Between determinations, the sensor was stored in the refrigerator at 4 • C in a hermetically sealed box. The results showed no significant differences between the anodic currents recorded on different days, with coefficients of variation less than 5%, confirming that the sensor is stable and can be used in electroanalysis.
The reproducibility of the developed method was investigated by preparing five different sensors, using a 10 −4 M verbascoside solution for performing the test. The calculated RSD value for the Ipa depicted for all five sensors was 3.5%, demonstrating good reproducibility of sensor development.
Tests for repeatability study were performed in a 50 µM verbascoside-0.1 M PBS solution. The value of the coefficient of variation for the anodic peak determined in five consecutive measurements in the same solution did not exceed 2.5%. Between measurements, the SPCE/GPHOX-Pentapeptide was rinsed with 0.1 M PBS solution, pH = 6.5. Therefore, the sensor can be used repeatedly for the determination of verbascoside.
Under optimal experimental conditions, the effect of possible interferents in the form of organic compounds predominantly found in EVOO, such as tyrosol, hydroxytyrosol and oleuropein, on the quantification of verbascoside was evaluated using CV as a detection method. The limit of tolerance was defined as the maximum concentration of interfering compounds that results in a relative error of ±5% for the quantitative determination of verbascoside. The achieved values, presented in Table 4, show that the peaks related to the presence of our compound of interest do not change significantly upon the addition of interferents. Tolerance limits were calculated with the following results: for oleuropein 5 × 10 −5 M, and for tyrosol and hydroxytyrosol it was 2 × 10 −5 M.
Taking into account all the above, it can be deduced that the method described is capable of detecting and quantifying the substance of interest, namely verbascoside, in the presence of chemically related substances, thus being considered specific.

Quantitative Determination of Verbascoside in EVOO
To validate the SPCE/GPHOX-Pentapeptide sensor in the analysis of verbascoside from real samples, ten samples of different EVOOs were selected and subjected to analysis using the CV method for determination. Measurements were performed in the potential range between −0.4 V and 1.0 V, applying a scan rate of 0.05 V·s −1 . Figure 12 presents, as an example, the CVs of the pentapeptide-modified sensor immersed in solutions containing extracts from four of the ten EVOOs selected for determination.
using the CV method for determination. Measurements were performed in the potential range between −0.4 V and 1.0 V, applying a scan rate of 0.05 V·s −1 . Figure 12 presents, as an example, the CVs of the pentapeptide-modified sensor immersed in solutions containing extracts from four of the ten EVOOs selected for determination. In all four voltammograms shown in Figure 12, there is an anodic peak related to the presence of verbascoside in the matrices, on the basis of which this compound was quantified in the respective EVOO samples. Thus, for the calculation of the amount of verbascoside in each sample, the slope of the calibration line obtained by the voltammetric method was used, and the obtained values are shown in Table 5. All analyses were performed in triplicate. In all four voltammograms shown in Figure 12, there is an anodic peak related to the presence of verbascoside in the matrices, on the basis of which this compound was quantified in the respective EVOO samples. Thus, for the calculation of the amount of verbascoside in each sample, the slope of the calibration line obtained by the voltammetric method was used, and the obtained values are shown in Table 5. All analyses were performed in triplicate. The values obtained for the 10 samples of EVOO on the concentration of verbascoside are close, the highest value being obtained for Costa D'Oro L'extra (Italy).
The precision of the method expressed as relative standard deviation (RSD) was near ±2%, indicating the accuracy of this method.

Determination of the Antioxidant Activity of Verbascoside by DPPH Method; Correlation between Sensor Response and Spectrophotometric Measurements to Determine Antioxidant Activity
This assay provides information on the antioxidants' capacity to donate electrons or hydrogen atoms scavenging the DPPH free radical [89]. The scavenging capacity of antioxidants toward DPPH free radicals is quantified by the decrease in absorbance at 517 nm [90].
In the first step of the determination, 0.1 mM DPPH stock solution was freshly prepared by dissolving the DPPH reagent in 96% ethanol and kept at room temperature in the dark. Subsequently, 0.5 mL volumes were measured from each sample solution (the same solutions used in the electrochemical measurements) and added to 3 mL of DPPH solution. These were kept at room temperature for 20 min, and the absorbances were then measured at 517 nm against ethanol.
For all 10 samples, the percentage reduction capacity of DPPH radicals was calculated according to the following equation [91]: where AD is the absorbance of the control solutions (in the absence of antioxidant agent), and AE is the absorbance of the test solutions.
The results obtained are presented as the average of the percentage inhibition values for all sample solutions tested and are included in Table 6.  Table 6 shows that the highest percentage inhibition value was obtained for Costa D'Oro L'extra (Italy), this result being in agreement with the value obtained in the quantitative determination of verbascoside in EVOO samples, where the highest amount of verbascoside contained per kilogram EVOO was also achieved for this matrix.
The next step of the study was to establish correlations between the electrochemical signals provided by the modified sensor and the data obtained from the spectrophotometric determination of antioxidant activity by the DPPH method.
Partial Least Squares regression (PLS) was used as a prediction technique to correlate the voltammetric data obtained by the pentapeptide-modified sensor with the antioxidant activity quantified as the capacity of free radical scavenging. The independent variables (X matrix) were the entire CVs obtained with the modified sensor, and the dependent variable was the percentage of DPPH inhibition. The normalization of the data was performed by the Standard Deviation method.
The results of PLS1 regression models were presented in the form of the dependences between predicted antioxidant activity results towards measured antioxidant activity results. Figure 13 shows the dependence plot between the percentage inhibition value of DPPH free radical predicted from voltammetric data and the values measured by the spectrophotometric method. signals provided by the modified sensor and the data obtained from the spectrophotometric determination of antioxidant activity by the DPPH method.
Partial Least Squares regression (PLS) was used as a prediction technique to correlate the voltammetric data obtained by the pentapeptide-modified sensor with the antioxidant activity quantified as the capacity of free radical scavenging. The independent variables (X matrix) were the entire CVs obtained with the modified sensor, and the dependent variable was the percentage of DPPH inhibition. The normalization of the data was performed by the Standard Deviation method.
The results of PLS1 regression models were presented in the form of the dependences between predicted antioxidant activity results towards measured antioxidant activity results. Figure 13 shows the dependence plot between the percentage inhibition value of DPPH free radical predicted from voltammetric data and the values measured by the spectrophotometric method. Figure 13. Plot of predicted inhibition from the voltammetric data vs. measured inhibition obtained from DPPH spectrophotometric method. Figure 13. Plot of predicted inhibition from the voltammetric data vs. measured inhibition obtained from DPPH spectrophotometric method. Table 7 shows the quantitative data obtained from the CV-DPPH regression model. It is noted that both calibration and validation values indicate good model performance (correlation coefficient close to 1). In addition, low values of the root mean square error at calibration (RMSEC) and root mean square error at prediction (RMSEP) were obtained for the spectrophotometric method. It can be appreciated that from the data obtained with the pentapeptide-modified sensor by means of CV, it is possible to estimate accurately the antioxidant activity of the oils expressed as the inhibition capacity of the free radical DPPH.

Chemicals and Solutions
Monosodium phosphate (NaH 2 PO 4 ) and disodium phosphate (Na 2 HPO 4 ), reagents purchased from Sigma-Aldrich (St. Louis, MO, USA), were used to prepare 0.1 M phosphate buffer solution (PBS), the supporting electrolyte in the electrochemical measurements performed All solutions were prepared with ultra-pure water, MilliQ water (resistivity 18.2 MΩ·cm) obtained from a water purification system (Milli-Q Simplicity ® , Bedford, MA, USA). pH adjustment to 6.5 was performed by adding 0.1 M H 3 PO 4 or 0.1 M NaOH. PH measurement is performed using a pH meter from WTW instruments, Weilheim, Germany.
Verbascoside powder of analytical purity was purchased from Sigma-Aldrich, St Louis, MO, USA. A stock solution of verbascoside of concentration 10 −4 M, prepared by completely dissolving the powder in 0.1 M PBS, was used for electrochemical analysis.
Pentapeptide, of >95% purity (sequence NH 2 -FESNF-CO-NH 2 , where F is phenylalanine, E is glutamic acid, S is serine, and N is asparagine) was purchased from ProteoGenix, Schiltigheim, France. Peptide purity and retention time were confirmed by RP-HPLC chromatography on a Dionex UltiMate 3000 UHPLC system (Thermo Scientific, Waltham, MA, USA).
Analytically pure compounds structurally similar to verbascoside (oleuropein, tyrosol and hydroxytyrosol) used for the interference studies were also purchased from Sigma-Aldrich (St. Louis, MO, USA).
To obtain the modified sensor (SPCE/GPHOX-Pentapeptide), the SPCE modified with a GPHOX film (SPCE/GPHOX) (purchased from Metrohm DropSens, Oviedo, Spain) was used, onto the surface of which the pentapeptide solution was added and cross-linked with glutaraldehyde. The pentapeptide solution used had a concentration of 10 mg/mL. The 0.1 mM DPPH (2,2-diphenyl-1-picrylhydrazyl) stock solution was prepared by weighing 0.0036 g DPPH reagent (purchased from Sigma-Aldrich) and dissolving in 100 mL 96% ethanol (Sigma-Aldrich). The resulting solution was kept at room temperature and in the dark until use.

Electrodes and Equipment
A Biologic SP 150 potentiostat/galvanostat (Bio-Logic Science Instruments SAS France) coupled with EC-Lab Express software operating in Windows was used to record, characterize and optimize the electrode signals. Three electrodes were connected to this device: the Ag/AgCl/KCl 3M reference electrode, the platinum (Pt) wire auxiliary electrode, and the SPCE working electrode, simultaneously introduced into the electrochemical cell.
The Partner AS 220/C/2 analytical balance was used for weighing the substances, and the most efficient way to dissolve the substances and homogenize the suspensions was by means of an Elmasonic ultrasonic bath (Carl Roth GmbH, Karlsruhe, Germany). The pH meter used for pH measurement was Inolab pH 7310, WTW instruments, Weilheim, Germany.
FTIR spectra were acquired with a Bruker ALPHA FT-IR spectrometer (BrukerOptik GmbH, Ettlingen, Germany) using OPUS software (BrukerOptik GmbH, Ettlingen, Germany) in the wavenumber range between 4000 and 500 cm −1 , using the attenuated total reflectance (ATR) method as sample exposure mode. The ZnSe crystal was carefully cleaned with ultrapure water and isopropanol between measurements. The background was the spectrum obtained in the air.
The surface morphology of the samples was examined with a Verios G4 UC scanning electron microscope (Thermo Scientific, Waltham, MA, USA) equipped with an energydispersive X-ray spectroscopy analyzer (Octane Elect Super SDD detector (AMETEK, Tokyo, Japan). The samples were coated with 10 nm platinum using a Leica EM ACE 200 coating system (Leica Microsystems, Vienna, Austria) to provide electrical conductivity. Then, the samples were fixed with double adhesive tape on the cylindrical conductor supports. SEM investigations were performed in High Vacuum mode using a secondary electron detector (Everhart-Thornley detector, ETD) at an accelerating voltage of 5 kV.
For the spectrophotometric method based on the reaction of antioxidant compounds with the free radical DPPH, sample absorbances were measured using a Rayleigh UV2601 UV/Vis Double Beam Spectrophotometer (Beijing Beifen-Ruili Analytical Instrument, Beijing, China).

Obtaining the Chemically Modified Sensor
For the preparation of the SPCE/GPHOX-Pentapeptide sensor, several steps were carried out. Initially, a 20 µL peptide solution (5 µL in each step) was added to the SPCE/GPHOX surface by the drop-and-dry method sequentially, in four steps, with drying pauses. Drying was carried out at room temperature for 30 min. The addition of the pentapeptide solution was performed using an Eppendorf micropipette. The next step was to hold the sensor over a container of 5 mL 2% glutaraldehyde for 1 min, the glutaraldehyde vapor ensuring the immobilization of the peptide on the electrode surface, a process known as cross-linking. The resulting sensor was stored at 4 • C until use, for a maximum of 72 h [92]. Figure 14 shows the steps in the preparation of the chemically modified sensor.

Methods of Analysis
In the present study, in order to highlight the oxidation-reduction processes taking place on the electrode surface and also to validate the results obtained, the CV method was performed as the electroanalytical technique. This is one of the most widely used methods to characterize electrochemical systems, providing both qualitative and quantitative information about the system studied. CV can provide information on the mechanism of a chemical reaction and the kinetics of the process, and it is also possible to determine the antioxidant properties of active compounds, which depend on their chemical structure and redox properties [93].

Real Samples and Preparation of the Solutions to Be Analysed
To study the sensitive properties of the sensor, 10 extracts from different EVOOs obtained by liquid-liquid extraction were analyzed [94]. An amount of 5 g of each oil was mixed with 10 mL methanol-water solution (40:10, v/v). The hydromethanolic extracts were separated using a separation funnel, after which 5 mL methanolic extract was added to 45 mL 0.1 M PBS of pH 6.5, which was the sample analyzed with the sensor made in this study. Table 8 shows all the commercial samples of olive oils studied.

Methods of Analysis
In the present study, in order to highlight the oxidation-reduction processes taking place on the electrode surface and also to validate the results obtained, the CV method was performed as the electroanalytical technique. This is one of the most widely used methods to characterize electrochemical systems, providing both qualitative and quantitative information about the system studied. CV can provide information on the mechanism of a chemical reaction and the kinetics of the process, and it is also possible to determine the antioxidant properties of active compounds, which depend on their chemical structure and redox properties [93].

Real Samples and Preparation of the Solutions to Be Analysed
To study the sensitive properties of the sensor, 10 extracts from different EVOOs obtained by liquid-liquid extraction were analyzed [94]. An amount of 5 g of each oil was mixed with 10 mL methanol-water solution (40:10, v/v). The hydromethanolic extracts were separated using a separation funnel, after which 5 mL methanolic extract was added to 45 mL 0.1 M PBS of pH 6.5, which was the sample analyzed with the sensor made in this study. Table 8 shows all the commercial samples of olive oils studied. The study of antioxidant activity using this spectrophotometric assay is based on the decolorization of the stable DPPH radical, strongly colored purple-red, by an antioxidant substance. The absorbance measurement is made at 517 nm, the decolorization being an indicator of antioxidant efficacy [95]. Applying this method provides an easy and rapid way to determine antioxidants by spectrophotometry, and various chemical compounds or natural products with antioxidant activity can be evaluated.

Data Analysis
PLS is a method that reduces the variables used to predict to a smaller set of predictors, then is used to perform a regression [96]. PLS1 corresponds to the case where there is only one dependent variable. The PLS1 regression model was used to establish correlations between electrochemical sensor responses and antioxidant activity determined by the DPPH spectrophotometric method (% inhibition degree was the dependent variable).
Multivariate data analysis was performed using Matlab, Excel, and Unscrambler.

Conclusions
Verbascoside is a pharmacologically active compound with much recent evidence supporting its biological and safety activities [97], having a special structure with many reactive sites [98], and the determination and quantification of this compound in EVOO samples is of real use, considering the health benefits that the consumption of olive oil as a food product can provide.
Therefore, this study reported, for the first time, the fabrication of an electrochemical sensor by modifying a GPHOX-based SPCE with a pentapeptide containing the NH 2 -FESNF-CO-NH 2 sequence, used for the accurate determination of verbascoside from real matrices with complex composition, i.e., samples of different EVOOs. Applying CV as a detection technique has demonstrated very good analytical performance with applicability in laboratory practice. Kinetic studies of electrochemical processes confirmed the superiority of the modified SPCE/GPHOX-Pentapeptide sensor over the unmodified SPCE/GPHOX sensor, obtaining with SPCE/GPHOX-Pentapeptide a wide range of linearity (0.1-10.55 µM) and a low detection limit (9.38 × 10 −8 M), respectively. In addition, the influence of other related compounds on the voltammetric response was reduced due to the favorable specificity of the newly developed sensor. Moreover, the antioxidant activity of the compound of interest was achieved by DPPH spectrophotometric assay, thus asserting the antioxidant activity of verbascoside in EVOO samples. Additionally, a good correlation between the results of the DPPH assay and CV was obtained.
The method developed and described in this work has a number of advantages, including good precision, sensitivity, time and low cost of analysis. The development of this peptide-based sensor may also prove effective in quality control of other types of