Development of Sonogel-Carbon based biosensors using sinusoidal voltages and currents methods

Accepted Manuscript Title: Development of Sonogel-Carbon Based Biosensors Using Sinusoidal Voltages and Currents Methods Authors: Juan Jos´e Garc´ıa Guzm´an, Laura Cubillana Aguilera, Dolores Bellido Milla, Ignacio Naranjo Rodr´ıguez, Cecilia Lete, Jose Mar´ıa Palacios Santander, Stelian Lupu PII: DOI: Reference:

S0925-4005(17)31593-9 http://dx.doi.org/10.1016/j.snb.2017.08.161 SNB 23028

To appear in:

Sensors and Actuators B

Received date: Revised date: Accepted date:

16-5-2017 1-8-2017 20-8-2017

Please cite this article as: Juan Jos´e Garc´ıa Guzm´an, Laura Cubillana Aguilera, Dolores Bellido Milla, Ignacio Naranjo Rodr´ıguez, Cecilia Lete, Jose Mar´ıa Palacios Santander, Stelian Lupu, Development of Sonogel-Carbon Based Biosensors Using Sinusoidal Voltages and Currents Methods, Sensors and Actuators B: Chemicalhttp://dx.doi.org/10.1016/j.snb.2017.08.161 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Development of Sonogel-Carbon Based Biosensors Using Sinusoidal Voltages and Currents Methods

Juan José García Guzmána, Laura Cubillana Aguileraa, Dolores Bellido Millaa, Ignacio Naranjo Rodrígueza, Cecilia Leteb,*, Jose María Palacios Santandera,*, Stelian Lupuc,*

a

Institute of Research on Electron Microscopy and Materials (IMEYMAT), Department of

Analytical Chemistry, Faculty of Sciences, Campus de Excelencia Internacional del Mar (CEIMAR), University of Cadiz, República Saharaui, S/N. 11510 Puerto Real, Cadiz-Spain. b

Institute of Physical Chemistry “Ilie Murgulescu” of the Romanian Academy, 202 Splaiul

Independentei, 060021 Bucharest, Romania. c

Department of Analytical Chemistry and Environmental Engineering, Faculty of Applied

Chemistry and Materials Science, University Politehnica of Bucharest, 1-7 Gh. Polizu Street, 011061 Bucharest, Romania.

*

Corresponding authors. Phone: +40 21 3163101; E-mail: [email protected] (C. Lete). Phone: +34 956 016357;

E-mail: [email protected] (J. Palacios). Phone: +40 21 4023886; E-mail: [email protected] (S. Lupu).

Highlights  Use of low cost Sonogel-Carbon electrode materials for biosensors  Novel sinusoidal currents preparation procedure of bio-composite materials  The voltammetric biosensors were applied in the dopamine, hydroquinone and catechol detection

1

 Application

of

prepared

biosensors

in

electroanalysis

of

pharmaceuticals

ABSTRACT In this work, biosensors based on Sonogel-Carbon electrodes modified with poly(3,4ethylenedioxythiophene) - tyrosinase biocomposite materials have been developed using sinusoidal voltages

and currents

methods. The

sinusoidal voltages method uses a

sinusoidal voltage signal of a 50 mHz fixed frequency and high amplitude superimposed on a direct current potential to modify the Sonogel-Carbon electrodes surface with the poly(3,4-ethylenedioxythiophene) - tyrosinase layer. A new preparation method involving the application of a sinusoidal current signal with fixed frequency and amplitude over a direct current has been developed as well. The experimental parameters of these methods have been optimized and selected in such a way to highlight the contribution of the sinusoidal component in the biosensors preparation. The advantages of the new preparation methods in terms of electrocatalytic and analytical efficiencies are presented. The analytical performances of the proposed biosensors towards benchmark analytes like dopamine, hydroquinone, and catechol have been investigated. The lowest limit of detection value of 7.10 μM and a high sensitivity value of 0.0037 µA/µM for dopamine determination were obtained with a biosensor prepared via the new sinusoidal currents method. The biosensors were successfully applied in the quantification of dopamine in pharmaceutical products.

Keywords: sinusoidal voltage and current methods, electrodeposition, biosensor, SonogelCarbon electrodes, dopamine, hydroquinone.

1. Introduction 2

Currently, there is a growing demand for continuous, fast, selective and sensitive monitoring of compounds of clinical, agrifood and/or environmental interest. Nowadays, the development of the technology is giving place to a new and complete generation of smart and composite materials that arise impressive potential for the development of new (bio)sensors. Amperometric (bio)sensors based on electrochemical transducers are very promising tools in this context [1–3]. Compared to laboratory-based methodology like gas/liquid chromatography,

electrochemical enzymatic biosensors

are usually

characterized by simple instrumentation, no sample treatment, high specificity, low-cost, rapid response, sensitivity, relatively compact size and easiness of implementation to detect biomolecules [4-6]. The successful enzyme immobilization onto the transducer surface strongly influences the analytical performance of biosensors by addressing key problems like loss of enzyme, decreased enzymatic activity, and long-time operational stability. The enzyme immobilization can be achieved by several approaches, such as adsorption, microencapsulation, covalent bonding, and cross-linking [7]. Recently, it has been demonstrated that organic conducting polymers (CPs), like poly(thiophene) and its derivatives, such as poly(3,4-ethylenedioxythiophene) (PEDOT), can provide a suitable microenvironment for enzymes immobilization, retaining the enzymatic activity [8–10]. PEDOT has attracted particular interest in this sense and is widely used in electroanalysis due to its electrochemical properties and ability to incorporate inorganic and bio/organic components [11–15], metal particles [16] and organic molecules [17,18]. In most of these reported applications , the electrodeposition of CPs onto the electrode surface has been performed by potentiostatic (at 0.95 V vs. Ag/AgCl reference electrode), potentiodynamic and galvanostatic methods.

4

In general,

the thickness of the enzyme-entrapped polymeric film can be controlled by the duration of

3

the potentiostatic deposition procedure [12,18,19]. The main advantage of CPs use in biosensors relies on the incorporation of the biological receptor (enzyme, nucleic acids or antibodies), mediators or even synthetic organic molecules (e.g. cyclodextrines, calixarenes,

phenylboronic

acid),

within

the

polymeric

matrix

during

the

electropolymerization of the corresponding monomers [20–22]. These electrochemical approaches for biosensors preparation have been extensively used by many researchers because the co-immobilization of enzymes/biological receptors within CPs matrices competes and even overcomes the traditional immobilization methods [7] used in this field. Thus, they represent the state-of-the-art methods in electrochemical biosensing technology.

However, in the past few years, our Group has developed a new electrochemical immobilization methodology : the in situ electrochemical deposition of both the CP component and the biological element by using sinusoidal voltages (SV) procedure [10,23– 27]. This novel electrodeposition method is based on the superimposition of a sinusoidal voltage on a direct current (d.c.) fixed potential in order to reach the potential value at which 3,4-ethylenedioxythiophene (EDOT) monomer polymerizes. By means of the SV a finer control of the enzyme immobilization and the porosity of the biocomposite coatings are achieved. The keystone lies on the proper choice of the frequencies and amplitudes of the sinusoidal voltages, what may lead to an improved enzymatic activity of the immobilized enzyme. Moreover, the increased porosity of the biocomposite material provided better sensitivity of the analytical measurements. Biosensors built by means of SV methodology on Au microelectrode arrays have been successfully applied to the determination of dopamine and catechol with very good performance.

4

In the present paper, a new methodology based on sinusoidal currents (SC) for the in situ electrodeposition of PEDOT and tyrosinase enzyme onto the surface of Sonogel-Carbon (SNGC) electrodes in order to allow higher porosity coatings is reported. Sonogel-Carbon material was employed as transducer due to their important advantages with respect to metal-based electrodes (Au or Pt): low cost, ease of modification (versatility), renewable surface (mechanically or electrochemically) or even disposability, good reproducibility, sensitivity and very high biocompatibility [28–30]. The main characteristic of SC method lies on the application of a sinusoidal current of fixed frequency and amplitude over a carefully selected d.c. current. Our hypothesis relies in the assumption that the use of sinusoidal currents amplitude may affect the porosity and morphology of the deposited CPs, increasing at the same time the stability of the resulting bio-composite material, preventing the enzyme leakage, and maintaining or even enhancing the enzyme biocatalytic performance.

A complete study regarding the optimization of the main

parameters influencing the SC method is described. The tuning of the frequency and amplitude of the applied sinusoidal current ensures a finer control of the porosity, morphology and even of the electrochemical polymerization process itself. Actually, the biosensors prepared by the novel SC approach proved better analytical performance than those obtained by SV methods and comparable to those previously published. Up to the extent of our knowledge, this is the first time that SC in situ electropolymerization approach is reported, being a novelty in the field of CPs electrodeposition and biosensing. The analytical performance of SC and SV prepared biosensors was tested for several analytes such as dopamine, hydroquinone, catechol and their mixtures, in terms of quality analytical parameters. Finally, the determination of dopamine in pharmaceutical products was also successfully accomplished.

5

2. Material and methods 2.1. Reagents Tyrosinase (E.C. 1.14.18.1, from mushroom, 3610 units/mg solid, Sigma, St. Louis, MO, USA), dopamine hydrochloride (Fluka), hydroquinone (Sigma-Aldrich), catechol (SigmaAldrich), and 3,4-ethylenedioxythiophene (EDOT, Aldrich), were of analytical reagent grade. Dopamine hydrochloride (5 mg/mL) concentrate for solution for intravenous infusion was from Zentiva (Romania). All other chemicals were of analytical reagent grade. Double distilled water was used for the preparation of all aqueous solutions.

2.2. Electrochemical measurements The

electrochemical

measurements

were

carried

out

using

an

Autolab

potentiostat/galvanostat 302N (Ecochemie, The Netherlands) with bipotentiostat module coupled to a PC running the GPES software, in a three-electrode configuration: SonogelCarbon electrodes (SNGC) having an inner diameter of 1.15 mm and obtained according to the procedure referred in literature [28, 29] were used as working electrodes; Ag/AgCl/KCl (3M) electrode (Metrohm) and a glassy carbon rod (Metrohm) were used as reference and counter electrode or auxiliary electrode, respectively. Before the use, the working SNGC electrodes were polarized by using an electrochemical procedure in 0.1 M H2SO4 aqueous solution as follows: the working electrode potential was poised at -0.7 V for 10 s, and then stepped at +1.8 V for 10 s, the procedure being repeated 8 times. The cyclic voltammetry measurements were performed with the bipot module of the potentiostat by simultaneous polarization of two SNGC electrodes. In order to ensure a proper activity of the immobilized Tyr, the analytical determinations of the target analytes have been performed in air-saturated buffered aqueous solutions.

6

2.3. Electrodeposition procedures of PEDOT-enzyme layers The electrodeposition of PEDOT-Tyr layers has been performed from an aqueous solution containing the optimum concentrations of 0.01 M EDOT, 2 mg/mL of Tyr, and 0.05 M phosphate buffer solution (PBS) at pH 7 by using the following procedures: (A) Sinusoidal voltages (SV) of a 50 mHz fixed frequency value, with excitation amplitude (Eac) of ±350 mV, were applied at fixed d.c. potential (Edc) value of 0.60 V to one of the SNGC electrode. The investigated deposition times were of 300 and 600 s, assuming 100% polymerization efficiency. The selected deposition time values control the thickness of the electrodeposited PEDOT-Tyr layer. The obtained modified electrodes were denoted as SNGC/PEDOT-Tyr-SV. In order to highlight the contribution of the enzyme, the electrodeposition of only PEDOT layers onto another SNGC electrode was also achieved by using the SV procedure described above in a solution containing 0.01 M EDOT and 0.05 M PBS at pH 7, and the modified electrode was denoted as SNGC/PEDOT-SV. The SV preparation procedure was implemented by using the FRA manual control feature of the potentiostat. (B) Sinusoidal currents (SC) of fixed frequency value of 100 mHz and amplitude of 1.0 μA were superimposed onto a d.c. current value of 4.0 μA, for electrodeposition times of 300 and 600 s, these being the optimum electrochemical parameters. Actually, the investigated electrochemical parameters were as follows: frequencies of the SC signal of 50 and 100 mHz, amplitude values of the SC signal of 0.5, 1, and 1.5 μA, and d.c. current values of 2, 3, 3.5, and 4 μA, respectively. The obtained modified electrode was referred to as SNGC/PEDOT-Tyr-SC. The PEDOT layer was also electrodeposited onto SNGC electrode using the SC procedure described above in a solution containing 0.01 M EDOT and 0.05 M PBS at pH 7, and the modified electrode was denoted as SNGC/PEDOT-SC. For comparison, the PEDOT-Tyr layer has been also electrodeposited by using a constant

7

current of 4.0 μA (i.e. galvanostatic mode) for the same electrodeposition times (the modified electrode was referred to as SNGC/PEDOT-Tyr-Galv). After the preparation, the modified electrodes were rinsed with doubly distilled water and used further in electrochemical characterization and analytical applications. When not in use, the modified electrodes were stored in a refrigerator for operational-stability and shelflife studies.

2.4. Surface characterization 2.4.1. SEM microscopy Scanning electron microscopy (SEM) studies were carried out on a QUANTA 200 (FEI Company, Hillsboro, Oregon, USA) operating at 25 kV and equipped with a Microanalyzer (EDAX) to perform energy dispersive X-ray spectroscopy (EDS).

2.4.2. AFM microscopy Surface topological studies were performed using an atomic force microscope (AFM) Veeco Nanoscope IIIa, in tapping mode. Phosphorus (n) doped silicon cantilevers, with spring constants in the range 20–80 N·m−1, were used. Calibration of the microscope was achieved by imaging calibration gratings supplied by the manufacturer. AFM images were examined for artifacts, and reproducibility was checked in the usual way, i.e. by changing the AFM cantilever and by either moving (during the experiment) the sample in the X- or Y-directions or by varying the scanning angle and the frequency.

2.5. Analytical measurements The target analytes: dopamine, hydroquinone and catechol, were quantified analytically in air-saturated aqueous buffer solutions (pH 7) at T = 294 ± 1 K by using cyclic voltammetry

8

(CV) under the following analytical protocol: the response of the biosensor, i.e. the cyclic voltammetric response of one SNGC/PEDOT-Tyr electrode, was recorded after the addition of analyte aliquots. For bipotentiostatic analytical measurements, the cyclic voltammetric signals were recorded simultaneously for both SNGC/PEDOT-Tyr and SNGC/PEDOT modified electrodes after addition of analyte aliquots. Standard addition method was used in the analytical determination of dopamine from Zentiva pharmaceutical formulations according to this procedure: a volume of 20 mL PBS was spiked with a known volume of dopamine chloride concentrate solution from a Zentiva vial in order to reach 50 µM dopamine concentration into the cell, and then the voltammetric response of the biosensor was recorded. Then, three aliquots of dopamine standard solution were added and the voltammetric response was registered after the addition of each aliquot. All the measurements were carried out in triplicates.

3. Results and discussion 3.1. Electrochemical preparation and characterization of the PEDOT and PEDOT-Tyr materials Two preparation methods were used in the development of biosensors based on PEDOTTyr, namely the sinusoidal voltage (SV) and current (SC) procedures. The aim of this study is to compare the performances of these methods in terms of efficiency and reliability for the electrodeposition of the biocomposite material onto SNGC electrodes. The SV procedure is based on the use of a SV signal with fixed frequency and amplitude superimposed on a d.c. potential (Edc) situated in a potential region where no electrochemical polymerization reaction takes place, i.e. Edc = 0.60 V. The approach based on the use of a SV signal superimposed on a d.c potential value of 0.60 V ensures the separation of the sinusoidal and d.c. components contribution to the electrodeposition of

9

the PEDOT-Tyr material. The SC method is based on the application of a sinusoidal current of fixed frequency and amplitude over a d.c. current. The d.c. current value was selected upon investigation of the electrodeposition of PEDOT only in galvanostatic mode in order to ensure a resulting potential value located in a region where only the SC component induces the electropolymerization of the monomer. The thickness of the PEDOT-Tyr and PEDOT materials was controlled by the electrodeposition time assuming 100% electropolymerization efficiency. The immobilization of Tyr enzyme within the PEDOT matrix is achieved during the electropolymerization process thanks to electrostatic interactions between the negatively charged groups of the enzyme and the positive charges originating from the polymer backbone. The applied SC and/or SV signal as well as the resulting potential/current responses were recorded simultaneously during the electrodeposition process. In Fig. 1A and 1B the resulting responses of the system after the application of the SC and SV signals are shown. From figure 1A, one can observe that the system's response is reaching potential values around 0.90 V where the electropolymerization of the EDOT monomer takes place. This response is obtained when a SC signal of 100 mHz frequency and ±1 µA amplitude was superimposed on a d.c current of 4 µA. It should be noticed the sinusoidal shape of the potential response in Fig. 1A and this provides an interesting feature of this method: the potential value is changing accordingly to the applied SC signal and the electropolymerization rate is increased during the anodic cycle of the SC signal and diminished or the process is even interrupted on the cathodic cycle of the SC signal. This behavior ensures the increase of the porosity of the electrodeposited polymer, compared with potentiostatic and galvanostatic deposition methods, and consequently an enhanced amount of immobilized enzyme is achieved. Obviously, the electropolymerization of the monomer does not occur at potential values lower than 0.80 V. On the other hand, the

10

application of a d.c. current of 4 µA provides a potential response value of ca. 0.95 V ensuring the electropolymerization of the EDOT monomer and, consequently, the electrodeposition of the PEDOT-Tyr coating (see Figure S1 from supplementary material). In the case of the SC method, the contribution of the applied SC signal increases the resulting potential to a region where the electropolymerization reaction occurs, i.e. potential values higher than 0.85 V, and ensures a sinusoidal variation of the potential response which provides a higher porosity of the electrodeposited polymer and an enhanced amount of immobilized enzyme. From Fig. 1B it can be observed a similar shape of the current response as a result of the application of a SV signal. The same sinusoidal shape of the current response is ensuring the successful deposition of the PEDOT-Tyr material with increased porosity and enhanced enzyme retention capability. The obtained modified electrodes were further investigated by means of cyclic voltammetry in the presence of a redox soluble probe, i.e. the potassium ferricyanide. Figure 1C reports the cyclic voltammograms recorded at a SNGC electrode before and after modification with PEDOT-Tyr material using the SC preparation method. A decrease of the cathodic peak current by 10% and an increase of the peak potentials separation from 90 to 260 mV were observed for the modified electrode compared with the unmodified electrode, demonstrating the successful modification of the electrode surface. The SC-modified electrode was also investigated by using electrochemical impedance spectroscopy (EIS) in the presence of both forms of the ferri/ferrocyanide redox couple. The EIS spectrum of the SC-based biosensor shows that the overall impedance is dominated by the charge transfer process for almost all the frequency range, compared with that of the unmodified electrode where a small semicircle appears at high frequency followed by a straight line at low frequency, as expected for bare electrodes (see Figure S2 from supplementary material). This finding confirms the successful modification of the electrode surface with a coating

11

characterized by a large charge transfer resistance, mainly due to the presence of the immobilized enzyme. In the case of the galvanostatic deposition of the PEDOT-Tyr material using a constant current of 4 µA, for the same electrodeposition time of 300 s, a similar shape of the EIS spectrum of the modified electrode can be observed (see Figure S3 from supplementary material). In this case, the overall impedance is higher than that related to the biosensor prepared via SC method because the applied constant current is ensuring a continuous electrodeposition of the PEDOT-Tyr coating for all the period of time, i.e. 300 s. Due to the sinusoidal nature of both applied signal and system response, the SC method ensures the preparation of PEDOT-Tyr coating characterized by a smaller thickness and a higher porosity. Figure 1D shows the cyclic voltammograms for a SNGC electrode modified by SV method and that of an unmodified electrode. In this case a decrease of the peak currents is observed together with an increase in the peak potential separation, this behavior attesting the electrodeposition of a PEDOT-Tyr coating with a higher thickness compared to that obtained by SC method. This result is also confirmed by the SEM data (see section 3.2.1). Comparing the CVs of SC- and SV-based biosensors, the decrease of the cathodic peak current suggests that a higher amount of tyrosinase was immobilized in the case of the biosensor prepared via SC method (see Figure S4 from supplementary material).

Figure 1 near here

3.2. Morphological characterization of the coatings 3.2.1. SEM Characterization In Figure 2, SEM images at different magnifications (160×, 800× and 25,000×) corresponding to PEDOT-Tyr coatings deposited onto SNGC electrodes by SV and SC

12

methods are shown. Furthermore, an example of EDS spectrum corresponding to a SNGC/PEDOT-Tyr-SC biosensor is also included. As it can be seen in Figure 2, the electrodeposition method based on SV procedure leads to a thicker film of PEDOT-Tyr coating on the SNGC electrode surface (see Figures 2A and 2D) than SC method. However, when increasing the magnification, a more compact and homogeneous surface can be noticed for SV-based polymer/enzymatic coatings (see Figures 2B, 2C, 2E, and 2F). Hence, it seems that SC method drives to a rougher more porous electrodeposited layer, what may be better for the enzyme immobilization and its biocatalytic performance. Moreover, it was found out a better analytical performance of the SNGC/PEDOT-Tyr-SC due to the smaller thickness of the coating (see Section 3.3). On the other hand and due to the presence of the Kα peak for S, EDS spectrum demonstrates the presence of the polymer on the electrode surface (see Figure 2G). The other peaks correspond to the typical EDS signals for an unmodified Sonogel-Carbon electrode [29]. P peak is due to the use of the electrode for previous studies in PBS.

Figure 2 near here

3.2.2. AFM characterization Figures 3 and 4 collect information about surface characterization of the SNGC/PEDOTTyr-SV and SNGC/PEDOT-Tyr-SC electrodes, respectively, by AFM. In all cases, several statistical parameters, such as average roughness coefficient (Ra), surface skewness (Ssk) and surface kurtosis (Sku) were calculated. Ra coefficient gives an idea about the roughness of material surface composing the electrode; Ssk value represents the degree of bias of the roughness shape (if Ssk > 0, peaks are predominant on the surface; on the contrary, if S sk < 0, valleys, holes or pores are more abundant); and Sku value is a measure of the sharpness

13

of the roughness profile (if Sku > 3, surface is spiked and if Sku < 3, surface is much softer). Looking at the different 3D and 2D plots obtained at several surface expositions (10 and 3 μm2), it can be seen that SV-based electrodeposited polymer/enzymatic films show a surface composed of more or less big like-sheet structures, meanwhile SC-based films seem to be more granular. In other words, PEDOT-Tyr-SV seems to be more compact and less porous than PEDOT-Tyr-SC layers. This fact corroborates the conclusions obtained from SEM characterization: the surface corresponding to the SNGC/PEDOT-Tyr-SC electrode shows higher porosity and seems rougher than the corresponding SV-based biosensor. The calculated statistical parameters also drive to a similar conclusion. The roughness parameters for SNGC/PEDOT-Tyr-SV are: Ra = 123.95 nm; Ssk = -0.821 nm; and Sku = 6.64 nm, and for SNGC/PEDOT-Tyr-SC are Ra = 197.22 nm; Ssk = -0.337 nm; and Sku = 3.05 nm. As it can be observed, Ra value for SC-based films is higher than for SV-based films, indicating a rougher and more porous surface. Concerning the S sk and Sku parameters, both surfaces can be characterized for showing more holes or valleys than peaks and a very high degree of sharpness (porous with conical shapes).

Figure 3 near here

Figure 4 near here

3.3. Analytical performance 3.3.1. Linear response range, sensitivity, limits of detection and quantification, selectivity The SNGC electrodes modified with PEDOT-Tyr material using both SV and SC methods were applied in the electroanalysis of dopamine, hydroquinone, and catechol. Figure 5A

14

reports the cyclic voltammograms recorded at SNGC electrode modified via SV method for dopamine electroanalysis in aqueous buffered solution. The quantification of dopamine was done using the cathodic peak current of the enzymatically produced quinone derivative at the corresponding peak potential of ca. 0.14 V. There was a linear increase of the cathodic peak current with dopamine concentration over the range from 40 to 300 μM, with a sensitivity of 0.0022 μA/μM, computed as the slope of the calibration plot (see Fig. 5B), and a correlation coefficient of 0.9946. The limits of detection (LD) and quantification (LQ) were assessed using the criteria: 3 s/m for LD, and respectively 10 s/m, for LQ, where s is the standard error of the regression line intercept, and m is the slope of the regression line. The obtained LD and LQ values were of 7.50 μM DA and respectively 25.01 μM DA.

Figure 5 near here

The capability of the SNGC/PEDOT-Tyr-SV based biosensor to measure the hydroquinone in the presence of a high amount of dopamine of 300 μM was also investigated. Fig. 5C shows the CVs recorded in an aqueous buffered solution containing 300 μM dopamine and increasing hydroquinone concentrations in the range 40 - 600 μM. It can be observed that a second cathodic peak appears at ca. 0.024 V that is ascribed to the reduction of the quinone derivative corresponding to the added hydroquinone. It should be noted that there is a peak potential separation of ca. 116 mV between the cathodic peaks potential of the two analytes, dopamine and hydroquinone, attesting the capability of the biosensor to measure one analyte in the presence of the another one. For hydroquinone quantification there was also a linear increase of the cathodic peak current with analyte concentration in the range 80 - 600 μM hydroquinone, with sensitivity of 0.0011 μA/μM and correlation coefficient

15

of 0.9971 respectively (see Fig. 5D). The LD and LQ values for hydroquinone determination in the presence of dopamine were of 12.93 and 43.10, respectively. The analytical performance of the biosensor prepared by using the new SC method has been also investigated. Figure 6A reports the cyclic voltammograms recorded at SNGC electrode modified via SC method for dopamine electroanalysis in aqueous buffered solution. The quantification of dopamine was done using the cathodic peak current of the enzymatically produced quinone derivative at the corresponding peak potential of ca. 0.14 V. There was a linear increase of the cathodic peak current with dopamine concentration over the range from 20 to 300 μM, with a sensitivity of 0.0037 μA/μM, computed as the slope of the calibration plot (see Fig. 6B), and a correlation coefficient of 0.9988. It should be noted the increase of the sensitivity by ca. 70% for the SC-based biosensor compared to that obtained by SV method. The obtained LD and LQ values were of 7.10 μM DA and, respectively, 23.65 μM DA.

Figure 6 near here

The capability of the SNGC/PEDOT-Tyr-SC based biosensor to measure the hydroquinone in the presence of a high amount of dopamine of 300 μM was also investigated. Fig. 6C shows the CVs recorded in an aqueous buffered solution containing 300 μM of dopamine and increasing hydroquinone concentrations in the range 20 - 600 μM. It can be observed that a second cathodic peak appears at ca. 0.004 V that is ascribed to the reduction of the quinone derivative corresponding to the added hydroquinone. It should be noted that there is a peak potential separation of ca. 136 mV between the cathodic peaks potential of the two analytes, dopamine and hydroquinone, attesting the capability of the biosensor to measure one analyte in the presence of the another one. For hydroquinone quantification

16

there was also a linear increase of the cathodic peak current with analyte concentration in the range 80 - 600 μM hydroquinone, with sensitivity of 0.001 μA/μM and correlation coefficient of 0.9988, respectively (see Fig. 6D). The LD and LQ values for hydroquinone determination in the presence of dopamine were of 15.88 μM HQ and 52.91 μM HQ, respectively. The SNGC/PEDOT-Tyr-SC based biosensor was also applied in the voltammetric determination of catechol. A linear response range from 20 to 300 μM catechol, with a correlation coefficient of 0.9975, and a LD of 12.9 μM catechol were obtained. The determination of catechol in the presence of a high amount of hydroquinone of 600 μM using this biosensor provided the following analytical parameters: a linear response range from 20 to 300 μM catechol, a correlation coefficient of 0.9971, and a LD of 24.51 μM catechol. These results demonstrate the capability of the SC-based biosensor to quantify a wide range of analytes, including their mixtures.

3.3.2. Determination of the Michaelis-Menten constant The kinetic parameters, that are the Michaelis-Menten constant (KM) and the maximum velocity, expressed as maximum current (Imax), of the enzymatic reaction have been determined using the Lineweaver-Burk equation (1): (1) where I is the current which is related to the initial velocity of the enzymatic reaction, KM is the Michaelis-Menten constant, Imax is the maximum current that can be related to the maximum velocity measured under saturated substrate conditions, and C is the concentration of the substrate (the target analyte). A smaller Michaelis-Menten constant value indicates a greater affinity of the enzyme towards the substrate, i.e. the target analyte. The KM and Imax kinetic parameters were assessed using the 1/I vs. 1/C plot for dopamine 17

electroanalysis at the SNGC/PEDOT-Tyr-SV based biosensor, and the following values were obtained: KM = 126.44 μM and, respectively, Imax = 2.06 μA. The same procedure was performed to calculate kinetics parameters of different analytes such as hydroquinone and catechol. Resulting values are: KM = 96.10 μM and Imax = 1.11 μA for hydroquinone; KM = 69.50 μM and Imax = 1.04 μA for catechol, respectively. The obtained low KM values indicates a high affinity of immobilized enzyme Tyr toward these analytes. Additionally KM and Imax kinetic parameters were also calculated using the same procedure for dopamine electroanalysis at the SNGC/PEDOT-Tyr-SC based biosensor. The obtained values were: KM = 69.05 μM and Imax = 1.85 μA. This assay was also repeated to determine KM and Imax for other analytes such as hydroquinone and catechol. The calculated values are KM = 134.86 μM and Imax = 1.24 μA for hydroquinone, and KM = 93.28 μM, Imax = 0.72 μA for catechol, respectively.

3.3.3. Repeatability, reproducibility, and stability The repeatability of the obtained SNGC/PEDOT-Tyr-SV biosensor was determined by measuring for 3 times the analytes concentration, i.e. dopamine, hydroquinone, catechol, each at 100 μM level and it was expressed as the relative standard deviation (RSD%). Values of 3.12 %, 3.71 % and 4.18 % have been obtained, respectively. The repeatability of the SNGC/PEDOT-Tyr-SC biosensor for these analytes (dopamine, hydroquinone, catechol) has been also investigated and it was also expressed as the relative standard deviation (RSD%). Values of 3.35 %, 1.09 % and, respectively, 3.46 % have been obtained. The reproducibility (RSD%) between two SNGC/PEDOT-Tyr-SV biosensors prepared under the same experimental conditions for measuring a 100 µM dopamine was of 8.42%.

18

Reproducibility for three SNGC/PEDOT-Tyr-SC biosensors was also determined and a value of 4.49% has been obtained. These values attest the good repeatability and reproducibility of the prepared biosensors. The stability of the prepared SNGC/PEDOT-Tyr-SV and SNGC/PEDOT-Tyr-SC biosensors was also investigated by measuring 100 µM dopamine in different days over a 1 month period of time. Between measurements, the biosensors were kept in a refrigerator at 4 ºC. The stability was assessed over a month measuring 100 µM of dopamine and comparing the peak current values, in order to check the decrease of the signal. However an increase was detected over the time, between the first day and the last one; this behavior was found in both biosensors SNGC/PEDOT-Tyr-SV and SNGC/PEDOT-Tyr-SC. The signals were 5.8 and 1.1 times higher for SNGC/PEDOT-Tyr-SV and SNGC/PEDOT-TyrSC biosensors, respectively. This finding reveals that the biosensors posses a very good stability over a large period of time. The SNGC/PEDOT-Tyr-SC biosensor displayed overall better analytical performance compared with the biosensor prepared via SV method and this demonstrates the usefulness of the proposed new SC preparation method. Furthermore, the figures of merit of the analytical performance of the SNGC/PEDOT-TyrSC biosensor are comparable with those of other biosensors/sensors published in the literature (see Table 1).

Table 1. Comparison of analytical performance of SC-based biosensor with other biosensors/sensors. Biosensor / sensor type

Linear range (M)

Detection

Ref.

limit (M) Dopamine MEA/PEDOT-tyrosinase

(SV, 1×10−5 – 1.5×10−4

frequency range 100 kHz to 0.1 Hz)

19

0.99×10−6

[10]

(SV, 1×10−5 – 3×10−4

Au-disk/PEDOT-tyrosinase

4.18×10−6

[23]

6×10−7

[24]

2.4×10−7

[26]

frequency range 100 kHz to 0.1 Hz) IDE/PEDOT-tyrosinase-glutaraldehyde

5×10−5 – 2.5×10−4

(SV, fixed frequencies of 1 kHz and 0.05 Hz, d.c. potential value of 1.05 V) MEA/PEDOT-tyrosinase

fixed 2×10−5 – 3×10−4

(SV,

frequency of 0.05 Hz, d.c. potential value of 0.60 V, amplitude of ±0.35 V) Au-QCM/PEDOT-tyrosinase

2×10−5 – 6×10−4

3.9×10−6

[27]

Carbon paste/tyrosinase

1.5×10−5 – 2.5×10−4

1.5×10−5

[31]

Tyrosinase entrapped in polyacrylamide 1.2×10−4 – 3.6×10−4

3.96×10−5

[32]

5.2×10−7

[33]

5×10−6

[34]

microgels Tyrosinase/Multi-walled

carbon 5×10−6 – 2.3×10−5

nanotube/Nafion/GCE carbon 5×10−6 – 5×10−5

Single-walled

nanotubes/Polypyrrole/tyrosinase 2×10−3 – 8×10−3

Carbon paste/tyrosinase from crude

7.5×10−4

[35]

extract of cara root SNGC/PEDOT-Tyrosinase (SC, fixed 2×10−5 – 3×10−4

7.1×10−6

frequency of 0.1 Hz, d.c. current of 4

This work

µA, amplitude of 1 µA) Hydroquinone PEDOT/nitrogen-doped graphene

1×10−6 – 1×10−5

1.8×10−7

[36]

Pt/ZrO2-RGO/GCE

1×10−6 – 1×10−3

4×10−7

[37]

Au/CNT/Polypyrrole/HRP

1.6×10−5 – 2.4×10−4

6.42×10−6

[38]

3.8×10-5

[39]

1.59×10−5

This

Graphite-epoxy

resin-sweet

potato 2.5×10-4 – 4.0×10-3

tissue (source of polyphenol oxidase) SNGC/PEDOT-Tyrosinase (SC, fixed 8×10−5 – 6×10−4 frequency of 0.1 Hz, d.c. current of 4

work

µA, amplitude of 1 µA) Catechol MEA/PEDOT-tyrosinase

(SV,

1×10−5 – 6×10−5

frequency range 100 kHz to 0.1 Hz) 20

5.58×10−6

[10]

Tyrosinase entrapped in polyacrylamide 5×10−7 – 2.4×10−5

3×10−7

[32]

2.2×10−7

[33]

microgels Tyrosinase/Multi-walled

carbon 1×10−6 – 2.3×10−5

nanotube/Nafion/GCE PEDOT/nitrogen-doped graphene

1×10−6 – 1×10−5

2.6×10−7

[36]

Pt/ZrO2-RGO/GCE

1×10−6 – 4×10−4

4×10−7

[37]

Carbon paste/Tyrosinase/DNA

1×10−6 – 5×10−5

1×10−6

[40]

Tyrosinase-MWCNT-MNP/SPE

1×10−5 – 8×10−5

7.61×10−6

[41]

4.9×10−6 – 1.1×10 −3

5.8×10−7

[42]

1.29×10−5

This

Tyrosinase-IL-MWCNT-DHP/GCE

SNGC/PEDOT-Tyrosinase (SC, fixed 2×10−5 – 3×10−4 frequency of 0.1 Hz, d.c. current of 4

work

µA, amplitude of 1 µA) SV: sinusoidal voltages; SC: sinusoidal currents; SNGC: Sonogel-Carbon electrode; MEA: gold disk microelectrodes array; IDE: interdigitated gold band microelectrodes array; Au-QCM: gold coated quartz crystal; RGO: reduced graphene oxide; GCE: glassy carbon electrode; CNT: carbon nanotubes; DNA: deoxyribonucleic acid; HRP: horseradish peroxidase; MWCNT: multi-walled carbon nanotubes; MNP: magnetic nanoparticles; SPE: screen-printed electrode; IL: 1-butyl-3-methylimidazolium chloride (ionic liquid); DHP: dihexadecylphosphate.

3.3.4. Interference study The interference study has been performed by investigating several compounds like ascorbic acid (AA), uric acid (UA), and epinephrine (EP), at the SV and SC-based biosensors. These compounds are usually contemporary present in real samples related to dopamine electroanalysis. Ascorbic acid is the major interfering specie, while epinephrine is a structurally related catecholamine. Uric acid could also influence the electroanalysis of dopamine. In the design of the interference study, the concentrations of the interfering species were as follows: ascorbic acid (1 mM), uric acid (50 µM), and epinephrine (500 µM). These concentration values are similar to those usually encountered in real sample analysis. The cyclic voltammograms recorded at the SC-based biosensor in the presence of

21

1 mM ascorbic acid and various dopamine concentrations ranging from 20 to 300 µM revealed that there was no interference from the ascorbic acid (see Figure S5 from supplementary material). It was observed that a second anodic peak appears at ca. +0.3 V for dopamine concentrations higher than 100 µM, but there is no influence on the cathodic peak located at ca. +0.14 V that is used for dopamine quantification. Actually, a relative error of less than +5% of the peak current related to the lowest dopamine concentration of 20 µM from the linear response range was observed. This behavior may be due to the fact that dopamine is positively charged at the working pH of 7, while ascorbic acid is negatively charged and repelled out from the electrode surface by the PEDOT matrix. A similar behavior was observed in the presence of uric acid. In addition, the presence of epinephrine resulted in a second cathodic peak located at ca. -0.2 V, but with no influence on the cathodic peak used for dopamine measurement (see Figure S6 from supplementary material). Therefore, the dopamine electroanalysis can be performed by measuring the current of the cathodic peak situated at +0.14 V, which was not affected by the presence of the interfering species. Similar results were obtained for the SV-based biosensor, excepting the case for lower concentration of interfering specie (500 µM ascorbic acid) that produced a +10.9% relative error of the peak current corresponding to the lowest dopamine concentration of 20 µM. Consequently, the SC-based biosensor displayed the best antiinterference capability which could be useful for dopamine electroanalysis in real samples.

3.4. Analytical applications 3.4.1. Determination of dopamine in pharmaceutical products The SNGC/PEDOT-Tyr-SV biosensor was applied in the quantification of dopamine in pharmaceutical products (Zentiva, solution for intravenous infusion, labeled concentration 5 mg/mL) using the standard addition method as follows: to a known volume of 0.1 M

22

PBS of pH 7 placed in the electrochemical cell, a known volume of Zentiva solution was added in order to reach a final concentration in the electrochemical cell of 50 µM dopamine. The measurements were done in triplicate. The obtained dopamine concentration value was of 60.9 (± 12.6) µM. The recovery of 121.8% is good taking into account that the Zentiva solution contains a large number of species, like sodium metabisulfite, maleic acid, ethanol, propylene glycol, and sodium chloride. These results demonstrate the good analytical performance of the SNG/PEDOT-Tyr-SV biosensor. The same assay was performed with PEDOT-Tyr-SC biosensor. The measurements were also done in triplicate. The obtained dopamine concentration value was of 55.3 (± 6.5) µM. In this case the recovery of 110.6 % shows an improved analytical performance of this biosensor compared to the previous one.

4. Conclusions The preparation and the analytical applications of tyrosinase based biosensors obtained via SC and SV methods have been presented. The electrochemical and morphological characterization of the proposed biosensors demonstrated that the SC method ensured the preparation of PEDOT-Tyr coatings having a smaller thickness and a porous structure. The analytical performance, in terms of limits of detection and quantification, linear response range, reproducibility, stability, Michaelis-Menten constant, precision and accuracy of the biosensor obtained via SC method was superior. The analytical application of both types of biosensors in the electroanalysis of pharmaceutical products revealed better accuracy and precision for the biosensor obtained by SC method. These results demonstrate the usefulness of the new SC preparation method in the development of electrochemical biosensors based on low cost electrode materials.

23

Acknowledgements: This work was supported by a grant from the Romanian National Authority for Scientific Research, CNCS–UEFISCDI, Project number PN-II-ID-PCE2011-3-0271. J.J. Garcia-Guzmán greatly acknowledges the PhD scholarship from EDUCA for supporting a research stage at the University Politehnica of Bucharest.

24

REFERENCES

[1]

E. T. S. G. da Silva, D. E. P. Souto, J. T. C. Barragan, J.de F. Giarola, A. C. M. de Moraes, L. T. Kubota, Electrochemical biosensors in point-of-care devices: Recent advances

and

future

trends,

Chemelectrochem,

4

(2017)

1–18.

http://dx.doi.org/10.1002/celc.201600758 [2]

L. Rotariu, F. Lagarde, N. Jaffrezic-Renault, C. Bala, Electrochemical biosensors for fast detection of food contaminants – trends and perspective, Trends in Analytical Chemistry, 79 (2016) 80–87. http://dx.doi.org/10.1016/j.trac.2015.12.017

[3]

P. Yáñez-Sedeño, S. Campuzano, J. M. Pingarrón. Electrochemical sensors based on magnetic molecularly imprinted polymers: A review, Anal. Chim. Acta 960 (2017) 1–17. http://dx.doi.org/10.1016/j.aca.2017.01.003

[4]

E. B. Bahadır, M. K. Sezgintürk. Applications of commercial biosensors in clinical, food, environmental, and biothreat/biowarfare analyses, Anal. Biochem. 478(1) (2015) 107–120. http://dx.doi.org/10.1016/j.ab.2015.03.011

[5]

M. Pohanka, P. Skládal, Electrochemical biosensors – principles and applications, J. Appl. Biomed. 6 (2008) 57–64.

[6]

A. Amine, F. Arduini, D. Moscone, G. Palleschi, Recent advances in biosensors based on enzyme inhibition, Biosens. Bioelectron.

76 (2016)

180–194.

http://dx.doi.org/10.1016/j.bios.2015.07.010 [7]

A. Amine, H. Mohammadi, I. Bourais, G. Palleschi, Enzyme inhibition-based biosensors for food safety and environmental monitoring, Biosens. Bioelectron. 21 (2006) 1405–1423. http://dx.doi.org/10.1016/j.bios.2005.07.012

[8]

B. Lakard, G. Herlem, S. Lakard, A. Antoniou, B. Fahys, Urea potentiometric biosensor based on modified electrodes with urease immobilized polyethylenimine

25

films,

Biosens.

Bioelectron.

19

(2004)

1641–1647.

http://dx.doi.org/10.1016/j.bios.2003.12.035 [9]

M. Singh, P. K. Kathuroju, N. Jampana, Polypyrrole based amperometric glucose biosensors,

Sens.

Actuators

B

Chem.

143

(2009)

430–443.

http://dx.doi.org/10.1016/j.snb.2009.09.005 [10] S. Lupu, C. Lete, P. C. Balaure, F. J. del Campo, F. X. Muñoz, B. Lakard, J-Y. Hihn, In situ electrodeposition of biocomposite materials by sinusoidal voltages on microelectrodes array for tyrosinase based amperometric biosensor development, Sens.

Actuators

B

Chem.

181

(2013)

136–143.

http://dx.doi.org/10.1016/j.snb.2013.01.060 [11] S. Lupu, C. Lete, M. Marin, N. Totir, P. C. Balaure, Electrochemical sensors based on platinum electrodes modified with hybrid inorganic–organic coatings for determination of 4-nitrophenol and dopamine, Electrochim. Acta 54 (2009) 1932– 1938. http://dx.doi.org/10.1016/j.electacta.2008.07.051 [12] S. Lupu, In situ electrochemical preparation and characterization of PEDOT-Prussian blue

composite

materials,

Synt.

Met.

161

(2011)

384–390.

http://dx.doi.org/10.1016/j.synthmet.2010.12.015 [13] S. Lupu, F. J. del Campo, F. X. Muñoz, Development of microelectrode arrays modified with inorganic–organic composite materials for dopamine electroanalysis, J.

Electroanal.

Chem.

639

(2010)

147–153.

http://dx.doi.org/10.1016/j.jelechem.2009.12.003 [14] V. Tsakova, R. Seeber, Conducting polymers in electrochemical sensing: factors influencing the electroanalytical signal, Anal. Bioanal. Chem. 408(26) (2016) 7231– 7241. http://dx.doi.org/10.1007/s00216-016-9774-7

26

[15] L. Pigani, C. Rioli, G. Foca, A. Ulrici, R. Seeber, F. Terzi, C. Zanardi, Determination of polyphenol content and colour index in wines through PEDOT-modified electrodes,

Anal.

Bioanal.

Chem.

408(26)

(2016)

7329–7338.

http://dx.doi.org/10.1007/s00216-016-9643-4 [16] S. Lupu, B. Lakard, J-Y. Hihn, J. Dejeu, Novel in situ electrochemical deposition of platinum nanoparticles by sinusoidal voltages on conducting polymer films, Synt. Met. 162 (2012) 193–198. http://dx.doi.org/10.1016/j.synthmet.2011.11.031 [17] H. Yamato, M. Ohwa, W. Wernet, Stability of polypyrrole and poly(3,4ethylenedioxythiophene) for biosensor application, J. Electroanal. Chem. 397 (1995) 163–170. http://dx.doi.org/10.1016/0022-0728(95)04156-8 [18] S. Lupu, F. Parenti, L. Pigani, R. Seeber, C. Zanardi, Differential pulse techniques on modified conventional-size and microelectrodes. Electroactivity of poly[4,4’bis(butylsulfanyl)-2,2’-bithiophene] coating towards dopamine and ascorbic acid oxidation,

Electroanalysis

15

(2003)

715–725.

http://dx.doi.org/10.1002/elan.200390090 [19] R. Seeber, F. Terzi, C. Zanardi, Functional Materials in Amperometric Sensing. Polymeric, Inorganic, and Nanocomposite Materials for Modified Electrodes, First Edition, Springer, 2014. [20] U. Lange, N. V. Roznyatovskaya, V. M. Mirsky, Conducting polymers in chemical sensors

and

arrays,

Anal.

Chim.

Acta

614

(2008)

1–26.

http://dx.doi.org/10.1016/j.aca.2008.02.068 [21] S. Zhang, G. Wright, Y. Yang, Materials and techniques for electrochemical biosensor design and construction, Biosens. Bioelectron. 15(5-6) (2000) 273–282. http://dx.doi.org/10.1016/S0956-5663(00)00076-2

27

[22] M. Gerard, A. Chaubey, B.D. Malhotra, Application of conducting polymers to biosensors,

Biosens.

Bioelectron.

17(5)

(2002)

345–359.

http://dx.doi.org/10.1016/S0956-5663(01)00312-8 [23] S. Lupu, C. Lete, P. C. Balaure, D. I. Caval, C. Mihailciuc, B. Lakard, J.-Y. Hihn, F. Javier del Campo, Development of amperometric biosensors based on nanostructured tyrosinase-conducting polymer composite electrodes, Sensors (Basel) 13(5) (2013) 6759–6774. http://dx.doi.org/10.3390/s130506759 [24] S. Lupu, C. Lete, F. J. del Campo, Dopamine electroanalysis using electrochemical biosensors prepared by a sinusoidal voltages method, Electroanalysis 27 (2015) 1649–1659. http://dx.doi.org/10.1002/elan.201400680 [25] S. Lupu, F. J. del Campo, F. X. Muñoz, Sinusoidal voltage electrodeposition and characterization of conducting polymers on gold microelectrode arrays, J. Electroanal.

Chem.

687

(2012)

71–78.

http://dx.doi.org/10.1016/j.jelechem.2012.09.035 [26] C. Lete, B. Lakard, J.-Y. Hihn, F. J. del Campo, S. Lupu, Use of sinusoidal voltages with fixed frequency in the preparation of tyrosinase based electrochemical biosensors for dopamine electroanalysis, Sens. Actuators B Chem. 240 (2017) 801– 809. http://dx.doi.org/10.1016/j.snb.2016.09.045 [27] C. Lete, D. Berger, C. Matei, S. Lupu, Electrochemical and microgravimetric studies of

poly[3,4-ethylenedioxythiophene]-tyrosinase

biocomposite

material

electrodeposited onto gold electrodes by a sinusoidal voltages method, J Solid State Electrochem. 20(11) (2016) 3043–3051. http://dx.doi.org/10.1007/s10008-016-3270z

28

[28] M. del M. Cordero-Rando, J. L. Hidalgo-Hidalgo de Cisneros, E. Blanco, I. NaranjoRodríguez, The sonogel-carbon electrode as a sol−gel graphite-based electrode, Anal. Chem. 74 (2002) 2423–2427. http://dx.doi.org/10.1021/ac010782u [29] L. M. Cubillana-Aguilera, J. M. Palacios-Santander, I. Naranjo-Rodríguez, J. L. Hidalgo-Hidalgo-de-Cisneros, Study of the influence of the graphite powder particle size on the structure of the Sonogel-Carbon materials, J Sol-Gel Sci. Techn. 40 (2006) 55–64. http://dx.doi.org/10.1007/s10971-006-9151-7 [30] D. Bellido-Milla, L. M. Cubillana-Aguilera, M. El Kaoutit, M. P. Hernández-Artiga, J. L. Hidalgo-Hidalgo De Cisneros, I. Naranjo-Rodríguez, J. M. Palacios-Santander, Recent advances in graphite powder-based electrodes, Anal. Bioanal. Chem. 405 (2013) 3525–3539. http://dx.doi.org/10.1007/s00216-013-6816-2 [31] E.S. Forzani, G.A. Rivas, V.M. Solis, Amperometric determination of dopamine on an enzymatically modified carbon paste electrode, J. Electroanal. Chem. 382 (1995) 33–40. http://dx.doi.org/10.1016/0022-0728(94)03640-O [32] J.P. Hervás Pérez, M. Sánchez-Paniagua López, E. López-Cabarcos, B. López-Ruiz, Amperometric tyrosinase biosensor based on polyacrylamide microgels, Biosens. Bioelectron. 22 (2006) 429–439. http://dx.doi.org/10.1016/j.bios.2006.05.015 [33] Y.-C. Tsai, C.-C. Chiu, Amperometric biosensors based on multiwalled carbon nanotube-Nafion-tyrosinase nanobiocomposites for the determination of phenolic compounds,

Sens.

Actuators

B

Chem.

125

(2007)

10–16.

http://dx.doi.org/10.1016/j.snb.2007.01.032 [34] K. Min, Y.J. Yoo, Amperometric detection of dopamine based on tyrosinase– SWNTs–Ppy

composite

electrode,

Talanta

http://dx.doi.org/10.1016/j.talanta.2009.08.032

29

80

(2009)

1007–1011.

[35] C. Sulzbacher Caruso, I. da Cruz Vieira, O. Fatibello Filho, Determination of epinephrine and dopamine in pharmaceutical formulations using a biosensor based on carbon paste modified with crude extract of cara root (Dioscorea bulbifera), Anal. Lett. 32 (1999) 39–50. http://dx.doi.org/10.1080/00032719908542597 [36] W.M. Si, W. Lei, Z. Han, Q.L. Hao, Y.H. Zhang, M.Z. Xia, Selective sensing of catechol and hydroquinone based on poly(3,4-ethylenedioxythiophene)/nitrogendoped graphene composites, Sens. Actuators B Chem. 199 (2014) 154–160. http://doi.org/10.1016/j.snb.2014.03.096 [37] A.T.E. Vilian, S.M. Chen, L.H. Huang, M.A. Ali, F.M.A. Al-Hemaid, Simultaneous determination of catechol and hydroquinone using a Pt/ZrO2–RGO/GCE composite modified glassy carbon electrode, Electrochim. Acta 125 (2014) 503–509. http://doi.org/10.1016/j.electacta.2014.01.092 [38] S. Korkut, B. Keskinler, E. Erhan, An amperometric biosensor based on multiwalled carbon nanotube-poly(pyrrole)-horseradish peroxidase nanobiocomposite film for determination

of

phenol

derivatives,

Talanta

76

(2008)

1147–1152.

http://doi.org/10.1016/j.talanta.2008.05.016 [39] K.O. Lupetti, G. Zanotto-Neto, O. Fatibello-Filho, Sweet potato tissue-epoxy resin composite biosensor for hydroquinone determination in photographic process wastewater,

J.

Braz.

Chem.

Soc.

17

(2006)

1329–1333.

http://dx.doi.org/10.1590/S0103-50532006000700020 [40] P. Dantoni, S.H.P. Serrano, A.M. Oliveira Brett, I.G.R. Gutz, Flow-injection determination of catechol with a new tyrosinase/DNA biosensor, Anal. Chim. Acta 366 (1998) 137–145. http://doi.org/10.1016/S0003-2670(98)00102-0

30

[41] B. Perez-Lopez, A. Merkoci, Magnetic nanoparticles modified with carbon nanotubes for electrocatalytic magnetoswitchable biosensing applications, Adv. Funct. Mater. 21 (2011) 255–260. http://doi.org/10.1002/adfm.201001306 [42] F.C. Vicentini, B.C. Janegitz, C.M.A. Brett, O. Fatibello-Filho, Tyrosinase biosensor based on a glassy carbon electrode modified with multi-walled carbon nanotubes and 1-butyl-3-methylimidazolium chloride within a dihexadecylphosphate film, Sens. Actuators B Chem. 188 (2013) 1101–1108. http://doi.org/10.1016/j.snb.2013.07.109

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Authors’ Biographies

Juan José García Guzmán is a PhD student in his last year. He has worked in the Department of Analytical Chemistry at University of Cadiz (Cadiz, Spain) for seven years. His main research interests include nanomaterials, (bio)sensors, conducting polymers modified electrodes, and electroanalysis of agri-food/environmental samples.

Laura Cubillana-Aguilera is at present an assistant professor in the Department of Analytical Chemistry at University of Cadiz (Cadiz, Spain). She got the PhD degree in 2007 at the same University. Her main research interests include composite electrodes, nanomaterials

(nanoparticles,

carbon

nanotubes,

etc.),

electroanalysis,

materials

characterization (SEM, TEM, DRX, EDS, AFM, etc.) and chemometrics.

Dolores Bellido Milla is associate professor of the Department of Analytical Chemistry at University of Cadiz (Cadiz, Spain) since 2004. Her field of research is related to the determination of analytes of interest in real samples (wine, beer, oils, etc.) by spectrophotometry,

microwave-digestion/atomic

absorption

spectroscopy

and

electrochemistry (biosensors).

Ignacio Naranjo-Rodríguez is full professor of the Department of Analytical Chemistry at University of Cadiz (Cadiz, Spain) since 2012. His main research interests include composite electrodes, nanomaterials, electrochemistry, conducting polymers and biosensors.

Cecilia Lete received her Ph. D. degree in 2001 at Bucharest University, Romania. Currently she is senior scientist at Romanian Academy, Institute of Physical Chemistry ‘‘Ilie Murgulescu’’, Electrochemistry Department. Her research interests include electrochemical sensors and biosensors based on new nanostructured (bio)materials.

José María Palacios-Santander received his PhD degree in 2003 at University of Cadiz (Cadiz, Spain). In 2009 he got a postdoc fellowship with Prof. Renato Seeber, at University of Modena and Reggio Emilia (Modena, Italy). Nowadays, he is Lecturer in the Department of Analytical Chemistry at University of Cadiz and the Secretary of Department of Analytical Chemistry at the Faculty of Science. His main research interests 32

include composite electrodes, nanomaterials (nanoparticles, carbon nanotubes, etc.), electroanalysis, chemometrics and scanning electrochemical microscopy.

Stelian Lupu is full professor at the Department of Analytical Chemistry and Environmental Engineering of the University Politehnica of Bucharest. He has obtained a Ph.D. degree in 2001 from the University of Bucharest, Romania, and the Habilitation à Diriger des Recherches in 2012 from the University of Franche-Comté, France. His general research interests include chemical sensors and biosensors, microelectrodes and their arrays, conducting polymers modified electrodes, and electroanalysis of biologically active compounds and organic pollutants.

33

Captions to figures

Figure 1. (A) The voltage response recorded upon the application of a SC signal with 100 mHz frequency and ±1 μA amplitude over a d.c. current of 4 µA. (B) The current response recorded upon the application of a SV signal with 50 mHz frequency and ±350 mV amplitude over a d.c. potential of 0.60 V. (C) Cyclic voltammograms recorded at both unmodified SNGC and SNGC/PEDOT-Tyr-SC modified electrodes in aqueous solution containing 5 mM K3Fe(CN)6, and 1 M KCl at potential scan rate of 50 mV/s. (D) Cyclic voltammograms recorded at both unmodified SNGC and SNGC/PEDOT-Tyr-SV modified electrodes in aqueous solution containing 5 mM K3Fe(CN)6, and 1 M KCl at potential scan rate of 50 mV/s.

Figure 2. SEM images of PEDOT-Tyr coatings deposited onto SNGC electrodes by SV and SC methods at different magnifications: (A) PEDOT-Tyr-SV 160×, (B) PEDOT-TyrSV 800×, (C) PEDOT-Tyr-SV 25000×, (D) PEDOT-Tyr-SC 160×, (E) PEDOT-Tyr-SC 800× and (F) PEDOT-Tyr-SC 25000×, and example of EDS spectrum (G) for the SNGC/PEDOT-Tyr-SC modified electrode.

Figure 3. 3D and 2D AFM images of SNGC/PEDOT-Tyr-SV electrode at surface expositions: (A) and (B) 10 μm2, and (C) and (D) 3 μm2.

Figure 4. 3D and 2D AFM images of SNGC/PEDOT-Tyr-SC electrode at surface expositions: (A) and (B) 10 μm2, and (C) and (D) 3 μm2.

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Figure 5. (A) Cyclic voltammograms recorded at SNGC/PEDOT-Tyr-SV modified electrode in 0.1 M PBS of pH 7 with different dopamine concentrations ranging from 40 to 300 μM and (B) the corresponding calibration plot. (C) CVs recorded at SNGC/PEDOTTyr-SV modified electrode in 0.1 M PBS of pH 7 containing 300 μM dopamine, and with increasing hydroquinone concentrations ranging from 40 to 600 μM and (D) the corresponding calibration plot. Potential scan rate: 50 mV/s.

Figure 6. (A) Cyclic voltammograms recorded at SNGC/PEDOT-Tyr-SC modified electrode in 0.1 M PBS of pH 7 with different dopamine concentrations ranging from 20 to 300 μM and (B) the corresponding calibration plot. (C) CVs recorded at SNGC/PEDOTTyr-SC modified electrode in 0.1 M PBS of pH 7 containing 300 μM dopamine, and with increasing hydroquinone concentrations ranging from 20 to 600 μM and (D) the corresponding calibration plot. Potential scan rate: 50 mV/s.

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