Development and application of graphite-SiO2/Al2O3/Nb2O5-methylene blue (GRP-SiAlNb-MB) composite for electrochemical determination of dopamine

Juliana de Fátima Giarola; Keyller Bastos Borges; César Ricardo Teixeira Tarley; Fernanda Midori de Oliveira; Emerson Schwingel Ribeiro; Arnaldo César Pereira

doi:10.1016/j.arabjc.2015.03.006

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Original article

10 (

); 430-438

doi:

10.1016/j.arabjc.2015.03.006

Development and application of graphite-SiO₂/Al₂O₃/Nb₂O₅-methylene blue (GRP-SiAlNb-MB) composite for electrochemical determination of dopamine

Juliana de Fátima Giarola^{^a}, Keyller Bastos Borges^{^a,⁎}, César Ricardo Teixeira Tarley^{^b}, Fernanda Midori de Oliveira^{^b}, Emerson Schwingel Ribeiro^{^c}, Arnaldo César Pereira^{^a}

a Departamento de Ciências Naturais, Universidade Federal de São João del-Rei (UFSJ), Campus Dom Bosco, Praça Dom Helvécio 74, Fábricas, 36301-160 São João del-Rei, MG, Brazil

b Departamento de Química, Universidade Estadual de Londrina (UEL), Centro de Ciências Exatas, Rodovia Celso Garcia Cid, PR 445, Km 380, 86050-482 Londrina, PR, Brazil

c Departamento de Química Inorgânica, Instituto de Química, Universidade Federal do Rio de Janeiro (UFRJ), 21941-909 Rio de Janeiro, RJ, Brazil

⁎Corresponding author at: Departamento de Ciências Naturais, Universidade Federal de São João del-Rei, Campus Dom Bosco, Praça Dom Helvécio 74, Fábricas, 36301-160 São João del-Rei, Minas Gerais, Brazil. Tel./fax: +55 32 3379 2483. keyller@ufsj.edu.br (Keyller Bastos Borges)

Received: 2014-9-22, Accepted: 2015-3-8, Published: 2015-03-01

Disclaimer:
This article was originally published by Elsevier and was migrated to Scientific Scholar after the change of Publisher.

Peer review under responsibility of King Saud University.

Abstract

In the present paper an amperometric sensor based on graphite-SiO₂/Al₂O₃/Nb₂O₅-methylene blue (GRP-SiAlNb-MB) composite has been successfully prepared for dopamine (DA) determination in real samples. The electrochemical behavior of DA at the GRP-SiAlNb-MB has been evaluated by employing cyclic voltammetry. The best ratio (m/m) of GRP-SiAlNb-MB composite was found to be 1:0.54. Under optimized conditions (pH 7.5 in 0.15 mol L⁻¹ phosphate buffer) the amperometry method responds linearly to DA from 5.0 up to 500.0 μmol L⁻¹ (r = 0.995) with limits of detection and quantification of 1.49 and 4.97 μmol L⁻¹, respectively. The developed method was successfully applied for DA determination in real samples of pharmaceutical formulations and can be used for routine quality control analysis of pharmaceutical formulations containing DA. The use of inorganic matrix SiAlNb was found to be very useful to adsorb MB in the composite material with further improvement of the anodic peak current of DA.

Keywords

Methylene blue

Carbon paste

Inorganic material

Amperometry

Pharmaceutical formulation

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1 Introduction

The development of highly selective and stable new sensors aiming at the determination of phenolic compounds in medicinal, pharmaceutical and biotechnological samples is of paramount importance for quality control. Chemical modifications of solid electrodes have been extensively studied for this purpose since it helps to obtain various sensor configurations (Trojanowicz, 2011; Lavecchia et al., 2010; Alonso-Lomillo et al., 2010 ). Nonetheless, the choice of electrodic material as a substrate for the immobilization of electroactive species plays an important role in the preparation of chemically modified electrodes (CME). In this sense, graphite paste electrode has received growing attention during years owing to its properties including good adsorption properties toward the chemical modifiers, renewable surface and presence of porous surface (Kalcher et al., 2006). Methylene blue (MB) has been widely employed for electrode modification as chemical modifier playing an important electrocatalytic role (Arvand et al., 2003). However, a brief overview of literature demonstrates that the majority of electrochemical methods that makes use of MB are focused on biosensor preparation using horseradish peroxidase (HRP) as enzyme for hydrogen peroxide determination. In this case, the enzyme and MB are usually deposited onto glassy carbon or gold electrode surface (Liu et al., 2000; Lei et al., 1996; Xu et al., 2003, 2004; Qian et al., 1998; Gu and Hasebe, 2004 ), which makes very weak this physical immobilization, thus resulting in a loss of MB during sensor use. This shortcoming can be overcome by using inorganic matrix in the sensor preparation, such as nano-SiO₂ (Xian et al., 2006), nano-TiO₂ (Xiao et al., 2008) and SiO₂/M_xO_y mixed oxide (Zaitseva et al., 2002; Ribeiro et al., 2003), which has been very useful to strongly adsorb MB aiming at sensor preparation.

Recently, our research group has reported the outstanding features of ternary mixed oxide (SiO₂/Al₂O₃/Nb₂O₅) to strongly adsorb DNA in the MWCNT/SiO₂/Al₂O₃/Nb₂O₅/DNA composite for electrochemical determination of promethazine and amitriptyline as well as to preconcentrate metal ions (Marco et al., 2013a,b; Costa et al., 2011; Tarley et al., 2010 ).

Dopamine (DA), 3,4-dihydroxyphenylethylamine, is an important phenolic compound which occurs naturally as neurotransmitter in the mammalian central nervous system. The deficiency of DA can cause some serious diseases such as Parkinson, epilepsy and senile dementia in humans (Ge et al., 2009; Smith and Devlin, 1992). Nowadays, catecholamine-based pharmaceuticals are widely used in medicine. So it is extremely important to determine DA quickly, precisely and accurately for quality control.

DA can be determined by electrochemical techniques, but some difficulties still remains, since its oxidation needs a high over potential at bare electrodes and the products are often adsorbed on the electrode surface, resulting in electrode fouling and unstable analytical signal. Different electroanalytical approaches have been dedicated to the development of new modified electrodes for DA monitoring. In general the materials commonly used are based on functionalized polymers or electrochemically modified polymers such as: poly(acrylic acid) multi-walled carbon nanotubes (MWCNTs) (Liu et al., 2007), deoxyribonucleic acid (DNA)/poly(p-aminobenzensulfonic acid) bilayer (Lin et al., 2007), RNA film (Kang and Lin, 2006), functionalized single-walled carbon nanotube (SWCNT) (Zhang et al., 2007), poly(eriochrome black T) (Yao et al., 2007), functionalized thiadiazole (Kalimuthu and John, 2009), poly 3-(5-chloro-2-hydroxyphenylazo)-4,5-dihydroxynaphthalene-2,7-disulfonic acid (CDDA) film (Ensafi and Khayamian, 2009), poly(acid chrome blue K) (Zhang et al., 2009), mesoporous carbon/Nafion (Zheng et al., 2009), and poly(vinyl alcohol) (Li and Lin, 2006). Such approach for sensor preparation shows slight drawbacks including time-consuming to be prepared and, in some cases, low reproducibility, thus resulting in a loss of stability. Other reports concerning the DA determination by ECL (electrochemiluminescence) can be cited (Zhang et al., 2013). ECL is a sensitive technique that has been widely used for sensor preparation; however, it requires complex modification procedures on the electrode and, thus, an accuracy optimization of several variables is crucial for its success since electrochemical and chemiluminescence measurements are integrated, which requires high skills for the analyst. In this sense, graphite paste electrodes prepared from composites based on mixed oxide and chemical modifier methylene blue (MB) are considered as a suitable alternative in comparison with the film preparation aiming the development of a reliable and feasible method for routine analysis of DA in pharmaceutical samples.

Therefore, the main objective of this paper was to prepare a new graphite-SiO₂/Al₂O₃/Nb₂O₅-methylene blue (GRP-SiAlNb-MB) composite and employ it as an electrochemical sensor for DA determination in pharmaceutical formulation. The quickly and ease to prepare, the high stability of the MB adsorbed onto SiAlNb as well as the satisfactory sensitivity are the highlights of proposed sensor for DA determination.

2 Experimental

2.1

2.1 Chemical and reagents

Dopamine (DA) was obtained from Sigma Aldrich (St. Louis, MO, USA). Dopamine injectable samples (Dopamine Hydrochloride 5 mg mL⁻¹, Teuto®), which contain sodium chloride and sodium metabisulfite in aqueous medium, were obtained from a local hospital. A pure graphite powder (GRP), anhydrous monobasic sodium phosphate, dibasic sodium phosphate and methylene blue (MB) were purchased from Synth® (Diadema, SP, Brazil). Tris[hydroxymethyl]aminomethane (Trizma), 4-[2-hydroxyethyl] piperazine-1-ethanesulfonic acid (HEPES), piperazine-1,4-bis[2-ethanesulfonic acid] (PIPES), sodium hydroxide, nitric acid, acetic acid and mineral oil were acquired from Sigma–Aldrich® (St. Louis, MO, USA). The material SiO₂/Al₂O₃/Nb₂O₅ (designated as SiAlNb), obtained by sol–gel process, was prepared according to previously described procedure (Costa et al., 2011; Tarley et al., 2010). All other reagents were of analytical grade, and their solutions were prepared with distilled and deionized water (resistivity: >18 MΩ cm⁻¹, 25 °C; Millipore® Milli-Q® purification system, Bedford, MA, USA).

2.2

2.2 Instrumentation and optimization of the sensor

The electrochemical measurements were performed using an Autolab potentiostat/galvanostat (PGSTAT 12) interfaced with a personal computer for data acquisition and potential control. All the experimental conditions were controlled with General Purpose Electrochemical System (GPES) software. A conventional three electrode cell was used at 25 ± 1 °C. An Ag/AgCl/KCl (3.0 mol L⁻¹) electrode, a platinum wire, and the GRP-SiAlNb-MB were used as the reference, auxiliary and working electrodes, respectively. Scanning electron microscopy (SEM) analyses of materials were performed in a Model JSM 6360-LV JEOL scanning electron microscope. Prior to the analyses, the samples were coated with a thin layer of gold/palladium alloy, using a Bal-Tec MED 020 equipment, in order to minimize charging under the incident electron beam. A Shimadzu FTIR-8300 spectrometer operating in transmission mode in the range of 4000–400 cm⁻¹ at 4 cm⁻¹ resolution and using the conventional KBr pellet technique was employed in order to elucidate the functional groups present in the materials. HPLC experiments were performed on a Shimadzu (Kyoto, Japan) HPLC system consisting of two LC 10AD solvents pumps, an SCL 10 A_vp system controller, a CTO 10 AS column oven and a 7125 model Rheodyne injector (Cotati, USA) with a 20 μL loop. Separations were obtained at 40 °C on a Shimadzu CLC-ODS (250 × 4.6 mm id, 5 μm particle size, Shimadzu, Kyoto, Japan). The analytical column was protected with a RP-18 end capped guard column (Merck, Darmstadt, Germany). Brief, the mobile phase for the analysis consisted of 0.2 mol L⁻¹ phosphate buffer pH 3: methanol (3: 7, v/v) at a flow rate of 1.0 mL min⁻¹ (Chen et al., 2003).

2.3

2.3 Preparation of the sensor

Twenty five milliliters of 0.1 mmol L⁻¹ methylene blue (MB) solution, equivalent to 0.799 mg, was added to 200 mg of SiAlNb. This mixture was maintained at rest for 15 min. After this step, the mixture was mechanically stirred for 2 h to allow the electroactive specie to adsorb onto the silica matrix. Shortly thereafter, the modified material (SiAlNb-MB) was filtered and then dried at room temperature. After this step, the material was mixed (19.5 mg) with the graphite powder (designed as GRP: 10.5 mg) dispersed in 7 μL of mineral oil until a complete homogeneous carbon paste was obtained. The paste was introduced into the bottom cavity of a glass tube in order to construct an electrode (designed as GRP-SiAlNb-MB) for DA determination. It worth emphasize that the cationic form of the dye, MB⁺Cl⁻, Scheme 1, was adsorbed on the SiAlNb material surface by an ion exchange reaction:

where NbOH stands for niobium hydroxide (V) dispersed on SiAlNb surface.

The methylene blue dye immobilized is strongly adhered on the surface and in the pores of the SiAlNb material, thus allowing obtain an electrode (GRP-SiAlNb-MB) with high stability to several redox cycles (Zaitseva et al., 2002; Ribeiro et al., 2003).

2.4

2.4 Preparation of reference solutions

Stock standard solutions of DA were prepared by dissolution of the analyte in water to obtain a final concentration of 0.01 mol L⁻¹. This standard solution was diluted to give the following concentrations of active pharmaceutical ingredient: 5, 10, 20, 50, 100, 150, 200, 250, 300, 350, 400, 450 and 500 μmol L⁻¹. All of these solutions were stored at −20 °C in the absence of light. Working standard solutions were prepared daily by diluting the stock solutions to an appropriate concentration with 0.15 mol L⁻¹ phosphate buffer pH 7.5.

2.5

2.5 Preparation of pharmaceutical formulation

DA injectable formulation was prepared by dissolution of 10 μL of drug in 10 mL of 0.15 mol L⁻¹ phosphate buffer pH 7.5. This final solution (32.64 μmol L⁻¹) was analyzed by the electrochemical system.

2.6

2.6 Method optimization

Cyclic voltammograms were obtained with the potential ranging from −0.3 V to 0.3 V by varying the solution concentration of MB adsorbed on silica matrix. For this task, the amount of SiAlNb-MB in the graphite-SiAlNb-methylene blue composite was set at 60% (m/m). Then, after the final optimization of %MB in the inorganic material, the amount of SiAlNb-MB in the composite was studied with 50%, 60%, 65% and 70% (m/m).

The dependence of sensor response in the presence of DA was evaluated by amperometry. Different buffer solutions (pH 7.0) at 0.1 mol L⁻¹ concentration were studied: phosphate, HEPES, Tris and Pipes. Next, the variation of pH between 5.0 and 8.0 using a phosphate buffer solution at 0.1 mol L⁻¹ concentration was studied. Finally, the influence of different concentrations of buffer solution (0.10, 0.15, 0.20 and 0.25 mol L⁻¹) was evaluated.

3 Results and discussions

3.1

3.1 FT-IR spectra and scanning electron microscopy (SEM) images of materials

The FT-IR spectra of materials are depicted in Fig. 1. A band at 3444 cm⁻¹ observed for pure graphite powder, SiAlNb, SiAlNb-MB and GRP-SiAlNb-MB is attributed to the stretch of O–H groups. At 1686 cm⁻¹ the signal is referred to the O–H and H₂O deformation vibration of water molecules adsorbed onto materials. Higher intense peaks in the region of 1067–1158 cm⁻¹, were observed, as expected for the material SiAlNb, which corresponds to the Si–O stretches in the SiAlNB matrix (Tarley et al., 2010). The band at 964 cm⁻¹ is attributed to either the Si–OH and Si–Nb stretches, while the signals at 790, 577 and 434 cm⁻¹ are assigned, respectively to stretching modes of Si–O–Si, Nb–O–Nb and deformation of Si–O–Si (Marco et al., 2013a,b). It is worth emphasizing the great difference of peak intensity at 2919, 2858 and 1459 cm⁻¹ observed for the GRP-SiAlNb-MB material when compared to the other materials.

FT-IR spectra of pure graphite powder, SiAlNb, SiAlNb-MB and GRP-SiAlNb-MB materials.

Fig. 2 shows the scanning electron micrographs of pure graphite powder, SiAlNb, SiAlNb-MB and GRP-SiAlNb-MB materials. The micrographs of SiAlNb and SiAlNb-MB are very similar, which indicates that the morphology of mixed oxide is not affected by the presence of MB. The pure graphite powder presents typical morphological features of carbonaceous materials with particles sizes very irregular. GRP-SiAlNb-MB material, in contrast, presented morphological features very different from those ones achieved for pure graphite powder, while SiAlNb and SiAlNb-MB presented particles that are highly aggregated and able to form a robust and dense network structure.

Sem images of (a) pure graphite powder, (b) SiAlNb, (c) SiAlNb-MB and (d) GRP-SiAlNb-MB with magnification 1200 times.

3.2

3.2 Optimization of GRP-SiAlNb-MB sensor in the absence of DA

The dye MB can form molecular associations, normally as dimers and sometimes as higher order aggregates, even in the solution phase or immobilized (Ribeiro et al., 2003). Therefore the influence of MB on the GRP-SiAlNb-MB composite was evaluated under different concentrations. Fig. 3 shows cyclic voltammograms for the GRP-SiAlNb-MB sensor at pH 7.0 in the presence of 0.1 mol L⁻¹ KCl. It can be seen that peak current and peak separation depend upon MB concentration. The best electrochemical performance was achieved by using MB concentration at 10⁻⁴ mol L⁻¹. At lower concentration of MB (10⁻⁵ mol L⁻¹), a decrease of current was observed, which is due to a lower concentration of MB immobilized on the surface of the SiAlNb material. However, the use of higher concentration (10⁻³ mol L⁻¹) leads to the formation of a higher ratio of dimer MB on the surface of the SiAlNb material which reduces the analytical current. Thus, the best current was achieved by MB concentration in solution of 10⁻⁴ mol L⁻¹, being this value adopted for further studies.

Cyclic voltammograms obtained in 0.1 mol L−1 KCl at pH 7.0 for the GRP-SiAlNb-MB prepared with different concentrations of methylene blue at ν = 20 mV s−1.

The influence of SiAlNb-MB amount (m/m) in the carbon paste (graphite, GRP) is illustrated in Fig. 4b. As observed in Fig. 4a a high anodic peak current, I_ap, was achieved by increasing MB concentration. However, SiAlNb-MB amount higher than 65% (m/m) provides a decrease on the anodic peak current due to the increased resistivity of the carbon paste, as well as a poor consistency of paste and weak adhesion force into the cavity of electrode. Fig. 4b shows the influence of SiAlNb-MB amount as a function of peak separation (ΔE_p). It was observed that there are slight differences on the response using 70% and 65% (m/m) of SiAlNb-MB. Therefore, taking into account that the highest value of anodic peak current has been achieved using 65% (m/m) of SiAlNb-MB, this amount was chosen for further experiments. It is worth emphasizing that other electrodes, prepared with higher percentages did not show good consistency and also showed ratio between anodic peak current and cathodic peak current (Ipa/Ipc) below than that found for the sensor prepared with 65% (m/m) of SiAlNb-MB.

(a) Variation in Iap/μA depending on the percentage of SiAlNb-MB (m/m) in carbon paste. (b) Variation in Ep/V as a function of the percentage of SiAlNb-MB (m/m) in carbon paste. Conditions: KCl0.1 mol L−1, pH 7.0 at ν = 20 mV s−1.

After optimizing the concentration of MB immobilized on the modified silica, the influence of the scan rate on the peak current of MB was investigated to assess whether the electron transfer process is diffusion or adsorption controlled. It was observed a good linear relationship between the anodic peak current and the square root of the scan rate, ν^1/2 (r = 0.99987), thus indicating that the oxidation process is diffusion controlled, i.e., the mass transport is the limiting factor of the process (Andrieux and Savéant, 1978).

3.3

3.3 Optimization of GRP-SiAlNb-MB sensor in the presence of DA

3.3.1

3.3.1 Amperometric optimization

For amperometric measurements, the potential applied to the system is of paramount importance because its magnitude is directly related to the sensitivity of the analytical system. The dependence of applied potential (E_ap) on the sensor response was investigated at 0.1 mmol L⁻¹ DA in 0.1 mol L⁻¹ phosphate buffer pH 7.0. According to the data shown in Table 1, a better analytical signal was achieved at 250 mV vs. Ag/AgCl. Nevertheless, in order to improve the selectivity of sensor, a lower potential (250 mV) was set for further experiments. Additionally, at higher potential values, the DA oxidation can takes place at the graphite surface, which is between 500 and 700 mV (Njagi et al., 2010).

Table 1 Influence of the applied potential (E_ap) on the amperometric response of the sensor proposed in the presence of dopamine in phosphate buffer 0.1 mol L⁻¹ pH 7.5.

E_ap. (mV)	0.0	50	100	150	200	250	300	350
ΔI (μA)	0.06	0.33	0.08	0.11	1.69	5.07	5.27	5.41

3.4

3.4 Optimization of experimental and operational parameters for GRP-SiAlNb-MB sensor

3.4.1

3.4.1 Influence of buffer solution type

The influence of different buffers solutions on the sensor response was investigated by using phosphate, HEPES, Tris and Pipes for a 0.1 mol L⁻¹ concentration at pH 7.0. As observed from Table 2, the greatest variation anodic current (ΔI) was obtained in phosphate buffer. This result may be related to the increased mobility of phosphate ions caused by the smaller size of these ions, enabling ease of charge transfer between GRP-SiAlNb-MB and DA solution, resulting in a higher ΔI.

Table 2 Influence of different buffer solutions 0.1 mol L⁻¹ pH 7.5 on the current variation (ΔI) solution of 0.1 mmol L⁻¹ dopamine.

Buffer solution	ΔI (μA)
Phosphate	5.06
Hepes	4.10
Pipes	2.51
Tris	3.83

3.4.2

3.4.2 Influence of pH on the sensor performance

The effect of pH on the sensor response was performed in 0.1 mol L⁻¹ phosphate buffer by varying the pH values from 5.0 to 8.0. The results show that the change in current (ΔI) was higher with the increasing of pH of buffer solution until 7.5 (Fig. 5), being this value chosen for further experiments. The higher electrode reaction for DA in more alkaline medium allowed us to conclude that analyte oxidation involves the loss of proton to the medium.

Influence of the pH on the response of the sensor in DA 1 mmol L−1. Conditions: ν = 20 mV s−1 and 0.1 mol L−1phosphate buffer.

3.4.3

3.4.3 Phosphate solution concentration

Phosphate solutions (pH 7.5) at different concentrations (0.10, 0.15, 0.20 and 0.25 mol L⁻¹) were analyzed in order to study the ionic strength effect of the electrolyte on the proposed sensor. The best values were found to be when electrolyte concentration varying from 0.15 to 0.25 mol L⁻¹ were employed, thus indicating that the ions migrated at a constant velocity in this concentration range (Table 3). Thus, in order to reduce the buffer consumption the concentration of 0.15 mol L⁻¹ was used for further studies.

Table 3 Influence of the concentration of phosphate buffer pH 7.5 on the current variation (ΔI) solution 0.1 mmol L⁻¹ dopamine.

[Buffer phosphate] (mol L⁻¹)	ΔI (μA)
0.005	6.45
0.100	9.58
0.150	11.64
0.200	12.14
0.250	12.24

3.5

3.5 Final analytical conditions

After final optimization of the proposed method i.e., concentration of 10⁻⁴ mol L⁻¹ MB, 65% (m/m) of SiAlNb-MB in the composite, E_ap = 250 mV, pH 7.5 in 0.15 mol L⁻¹ phosphate buffer. Differential pulse voltammetry and square wave voltammetry also were evaluated as electrochemical techniques for DA determination under the best operational parameters (data not shown). Nevertheless, we found that the proposed sensor exhibited the best analytical response when measurements were performed by amperometry.

Fig. 6a shows the Amperogram obtained by successive additions of DA in the electrochemical cell solution containing 0.15 mol L⁻¹ phosphate buffer pH 7.5. As seen in Fig. 6b, the additions of the analyte are sufficient to generate ΔI linearly proportional to concentration of DA. The calibration curve obtained for determination of DA showed sensitivity equal to 0.008 A L mol⁻¹. In order to check the benefits of MB and SiAlNb in amperometric measurements, experiments under optimized conditions, using different electrodes configuration including, GRP, GRP-SiAlNb and GRP-SiAlNb-MB were carried out (Table 4). As observed, the sensitivity of GRP-SiAlNb-MB was higher than other electrodes, thus indicating that MB plays an electrocatalytic role on the eletrooxidation of DA as has been previously reported (Argüello et al., 2008). This supposition can also be confirmed from the low obtained active area of GRP-SiAlNb-MB. The active area of electrodes were 0.081 for Graphite, 0.067 for GRP-SiAlNb and 0.053 cm² GRP-SiAlNb-MB, respectively, in which were determined from the slope of plot Ip vs. ν^1/2, according to Randles–Sevcik equation (Eq. (1)), using 1.0 mmol L⁻¹ K₄Fe(CN)₆ as probe in 1.0 mol L⁻¹ KCl as supporting electrolyte.

(1)

Ip = 2.69 \times 10^{5} n^{3 / 2} {AD}^{1 / 2} ν^{1 / 2} C

where n = 1 and D is the diffusion coefficient of K₄Fe(CN)₆ of 7.6 × 10⁻⁶ cm² s⁻¹

(a) Amperogram for the determination of DA concentrations obtained for 5–500 mol L−1. (b) Analytical curve constructed from Amperogram (a). Measurements obtained using 65% (w/w) SiAlNb-MB by GRP. Conditions: 0.15 mol L−1phosphate buffer, pH 7.5 and Eap = 250mV vs. Ag/AgCl.

Table 4 Comparative study of sensitivity of graphite (GRP), GRP-SiAlNb and GRP-SiAlNb-MB under optimized conditions and dopamine concentration varying from 25 to 125 μmol L⁻¹.

Electrode configuration	Sensitivity (A L mol⁻¹)
Graphite	0.003
GRP-SiAlNb	0.004
GRP-SiAlNb-MB	0.008

The limits of detection and quantification of the proposed method were calculated using the method based on the analytical curve parameters (ICH, 1995). In this method, the limit of detection (LOD) can be expressed as in Eq. (2) and the limit of quantitation (LOQ) expressed in Eq. (3):

(2)

LOD = 3 \times s / a

(3)

LOQ = 10 \times s / a

where s is the estimated blank standard deviation (n = 10) (0.0402 μA) and a is the slope of the curve.

The present method provided a linear range from 5.0 to 500.0 μmol L⁻¹, described by equation: ΔI = 0.008 (±7.69E−5) (Dopamine, μmol L⁻¹) + 0.409 (±0.021) with good linear correlation coefficient (r = 0.995). The limits of detection and quantification were found to be 1.49 and 4.97 μmol L⁻¹, respectively. The limit of detection and linear range were compared with previous electroanalytical methods for dopamine determination (Table 5). As observed, the proposed sensor showed better limits of detection and quantification when compared to other proposed sensors for DA determination. Moreover, the developed sensor showed good sensitivity, wide linear response range, and quick response to the detection of DA.

Table 5 Comparative data on different electroanalytical methods for dopamine determination.

Different kind of electrodes	Linear range (μmol L⁻¹)	LOD (μmol L⁻¹)	Reference
Polyphenol oxidase biosensor modified with crude extract of root Cara	2000–8000	750	Caruso et al. (1999)
Electrode modified with peroxidase crude extract of zucchini (Cucurbita pepo)	500–3000	26	Lupetti et al. (2005)
Glassy carbon electrode with cobalt hexacyanoferrate film (CoHCFe) modified	120–500	8.9	Castro et al. (2008)
Electrode with carbon paste and Th(IV)-hexacyanoferrate (Th-HCF)	8–2000	5.6	Farhadi et al. (2008)
Graphite-SiO₂/Al₂O₃/Nb₂O₅-methylene blue (GRP-SiAlNb-MB) composite	5–500	1.49	This work

LOD = limit of detection.

This modified electrode presented an excellent repeatability, with a relative standard deviation (SD) of 2.3% for a series of six successive measurements of a 100 μM dopamine solution.

Furthermore, the sensor also showed good stability when subjected to 20 cycles by cyclic voltammetry (data not shown), showing no significant differences between the peak currents of the voltammograms. The sensor preparation also can be considered very fast and simple to be performed. The influence of AA, UA and glucose on DA detection was also evaluated. As observed from Table 6, no interference from these compounds was observed on DA detection.

Table 6 Study of the selectivity of the proposed sensor with possible interfering in biological samples (uric and ascorbic acid).

Interfering	[DA] expected (μmol L⁻¹)	[DA] found (μmol L⁻¹)	Relative response (%)
Uric acid	150	153.4 (±0.1)	102.3 (±0.7)
Ascorbic acid	150	151.5 (±0.3)	101 (±1.1)
Uric acid + ascorbic acid	150	154.6 (±0.4)	103.1 (±0.9)

Reproducibility between different electrodes (n = 3) was also estimated and the response of sensor at the same dopamine concentration (100 μM) was 2.62 ± 0.02 μA cm⁻².

3.6

3.6 Application of the method

Under optimal conditions the proposed sensor was applied to the DA determination in pharmaceutical sample (Dopamine hydrochloride, 5 mg mL⁻¹, Teuto®, equivalent to 32.64 mmol L⁻¹). Table 7 shows the comparison between the value of the label and the results obtained using the proposed sensor and High Performance Liquid Chromatography (HPLC), employed as reference method. The values found, with a 95% confidence level, are compared with those obtained from standard HPLC technique.

Table 7 Determination of dopamine in injectable pharmaceutical product using the chromatographic method and the proposed method (n = 3).

Sample	Reference method (mmol L⁻¹)	Proposed method (mmol L⁻¹)	Relative error (%)
1	29.39(±0.17)	29.82(±0.21)	1.46

4 Conclusions

This study demonstrated that the sensor based on MB immobilized on inorganic material modified with Al₂O₃ and Nb₂O₅ is a viable alternative for the determination of DA by amperometry. From characterization data it was observed that MB is better adsorbed in the GRP-SiAlNb matrix than SiAlNb. Additionally, the GRP-SiAlNb-MB material presented particles highly aggregated able to form a robust and dense network structure an interesting feature for sensor preparation. Under optimum experimental conditions, the method presented good LOD and LOQ, good sensitivity and wide linear response range. Therefore, considering the parameters presented, the sensor presented a methodology of low-cost, easy to perform, quick, sensitive and linear for determination of DA. It was successfully applied for the determination of DA in real samples of pharmaceutical formulations.

Acknowledgements

The authors are grateful to Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), INCT de Bioanalítica, Fundação Araucária do Paraná and to FAPERJ (Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro) for financial support and for granting research fellowships, and to the student of scientific initiation Felipo Doval Rojas Soares of IQ/UFRJ for the preparation of the material SiAlNb. This work is a collaboration research project of members of the Rede Mineira de Química (RQ-MG) supported by FAPEMIG.

References

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Liu B., Liu Z., Chen D., Kong J., Deng J., . An amperometric biosensor based on the coimmobilization of horseradish peroxidase and methylene blue on a β-type zeolite modified electrode. Fresenius J. Anal. Chem.. 2000;367:539-544.
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Liu A.H., Honma I., Zhou H.S., . Simultaneous voltammetric detection of dopamine and uric acid at their physiological level in the presence of ascorbic acid using poly(acrylic acid)-multiwalled carbon-nanotube composite-covered glassy-carbon electrode. Biosens. Bioelectron.. 2007;23:74-80.
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Marco J.P., Borges K.B., Tarley C.R.T., Ribeiro E.S., Pereira A.C., . Development of a simple, rapid and validated square wave voltametric method for determination of promethazine in raw material and pharmaceutical formulation using DNA modified multiwall carbon nanotube paste electrode. Sens. Actuat. B – Chem.. 2013;177:251-259.
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Marco J.P., Borges K.B., Tarley C.R.T., Ribeiro E.S., Pereira A.C., . Development and application of an electrochemical biosensor based on carbon paste and silica modified with niobium oxide, alumina and DNA (SiO2/Al2O3/Nb2O5/DNA) for amitriptyline determination. J. Electroanal. Chem.. 2013;704:159-168.
[Google Scholar]
Njagi J., Chernov M.M., Leiter J.C., Andreescu S., . Amperometric detection of dopamine in vivo with an enzyme based carbon fiber microbiosensor. Anal. Chem.. 2010;82:989-996.
[Google Scholar]
Qian Z., Shi X., Zhuang J., Kong J., Deng J., . Alkyl chain length effect on the horseradish peroxidase-catalytic reduction of hydrogen peroxide mediated by methylene blue at alkanethiol self-assembled monolayer electrodes. Bioelectrochem. Bioenerg.. 1998;46:193-198.
[Google Scholar]
Ribeiro E.S., Rosatto S.S., Gushikem Y., Kubota L.T., . Electrochemical study of Meldola’s blue, methylene blue and toluidine blue immobilized on a SiO₂/Sb₂O₃ binary oxide matrix obtained by the sol–gel processing method. J. Solid State Electrochem.. 2003;7:665-670.
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Smith T.E., Devlin T.M., . Textbook of Biochemistry with Clinical Correlations. New York: Wiley/Liss; 1992.
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Xian Y.Z., Liu F., Xian Y., Zhou Y.Y., Jin L.T., . Preparation of methylene blue-doped silica nanoparticle and its application to electroanalysis heme proteins. Electrochim. Acta. 2006;51:6527-6532.
[Google Scholar]
Xiao M.W., Wang L.S., Wu Y.D., Huang X.J., Dang Z., . Electrochemical study of methylene blue/titanate nanotubes nanocomposite and its layer-by-layer assembly multilayer films. J. Solid State Electrochem.. 2008;12:1159-1166.
[Google Scholar]
Xu J.Z., Zhu J.J., Wu Q., Hu Z., Chen H.Y., . An amperometric biosensor based on the coimmobilization of horseradish peroxidase and methylene blue on a carbon nanotubes modified electrode. Electroanalysis. 2003;15:219-224.
[Google Scholar]
Xu J.Z., Zhang Y., Li G.X., Zhu J.J., . An electrochemical biosensor constructed by nanosized silver particles doped sol–gel film. Mater. Sci. Eng. C. 2004;24:833-836.
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Yao H., Sun Y.Y., Lin X.H., Tang Y.H., Huang L.Y., . Electrochemical characterization of poly(eriochrome black T) modified glassy carbon electrode and its application to simultaneous determination of dopamine, ascorbic acid and uric acid. Electrochim. Acta. 2007;52:6165-6171.
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Zaitseva G., Gushikem Y., Ribeiro E.S., Rosatto S.S., . Electrochemical property of methylene blue redox dye immobilized on porous silica–zirconia–antimonia mixed oxide. Electrochim. Acta. 2002;47:1469-1474.
[Google Scholar]
Zhang Y.Z., Pan Y., Su S., Zhang L.P., Li S.P., Shao M.W., . A novel functionalized single-wall carbon nanotube modified electrode and its application in determination of dopamine and uric acid in the presence of high concentrations of ascorbic acid. Electroanalysis. 2007;19:1695-1701.
[Google Scholar]
Zhang R., Jin G.D., Chen D., Hu X.Y., . Simultaneous electrochemical determination of dopamine, ascorbic acid and uric acid using poly(acid chrome blue K) modified glassy carbon electrode. Sens. Actuat. B. 2009;138:174-181.
[Google Scholar]
Zhang L., Cheng Y., Lei J., Liu Y., Hao Q., Ju H., . Stepwise chemical reaction strategy for highly sensitive electrochemiluminescent detection of dopamine. Anal. Chem.. 2013;85:8001-8007.
[Google Scholar]
Zheng D., Ye J.S., Zhou L., Zhang Y., Yu C.Z., . Simultaneous determination of dopamine, ascorbic acid and uric acid on ordered mesoporous carbon/Nafion composite film. J. Electroanal. Chem.. 2009;625:82-87.
[Google Scholar]

Show Sections

[1] Alonso-Lomillo M.A., Dominguez-Renedo O., Arcos-Martinez M.J., . Electrochemical sensors in the development of selective methods for antiepileptic drugs determination. Comb. Chem. High Throughput Screen.. 2010;13:650-657.
[Google Scholar]

[2] Andrieux C.P., Savéant J.M., . Heterogeneous (chemically modified electrodes, polymer electrodes) vs. homogeneous catalysis of electrochemical reactions. J. Electroanal. J. Electroanal. Chem. Interf. Electrochem.. 1978;93:163-168.
[Google Scholar]

[3] Argüello J., Leidens V.L., Magosso H.A., Ramos R.R., Gushikem Y., . Simultaneous voltammetric determination of ascorbic acid, dopamine and uric acid by methylene blue adsorbed on a phosphorylated zirconia–silica composite electrode. Electrochem. Acta. 2008;54:560-657.
[Google Scholar]

[4] Arvand M., Sohrabnezhad S., Mousavi M.F., Shamsipur M., Zanjanchi M.A., . Electrochemical study of methylene blue incorporated into mordenite type zeolite and its application for amperometric determination of ascorbic acid in real samples. Anal. Chim. Acta. 2003;491:193-201.
[Google Scholar]

[5] Caruso C.S., Vieira I.C., Fatibello-Filho O., . 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.. 1999;32:39-50.
[Google Scholar]

[6] Castro S.L.S., Mortimer R.J., de Oliveira M.F., Stradiotto N.R., . Electrooxidation and determination of dopamine using a Nafion®-cobalt hexacyanoferrate film modified electrode. Sensors. 2008;8:1950-1959.
[Google Scholar]

[7] Chen J., Shi Y.-P., Liu J.-Y., . Determination of noradrenaline and dopamine in Chinese herbal extracts from Portulaca oleracea L. by high-performance liquid chromatography. J. Chromatogr. A. 2003;1003:127-132.
[Google Scholar]

[8] Costa L.M., Ribeiro E.S., Segatelli M.G., Nascimento D.R., Oliveira F.M., Tarley C.R.T., . Adsorption studies of Cd(II) onto Al2O3/Nb2O5 mixed oxide dispersed on silica matrix and its on-line preconcentration and determination by flame atomic absorption spectrometry. Spectrochim. Acta B. 2011;66:329-337.
[Google Scholar]

[9] Ensafi A.A., Khayamian M.T.T., . A differential pulse voltammetric method for simultaneous determination of ascorbic acid, dopamine, and uric acid using poly (3-(5-chloro-2-hydroxyphenylazo)-4,5-dihydroxynaphthalene-2,7-disulfonic acid) film modified glassy carbon electrode. J. Electroanal. Chem.. 2009;633:212-220.
[Google Scholar]

[10] Farhadi K., Kheiri F., Golzan M., . Th(IV)-hexacyanoferrate modified carbon paste electrode as a new electrocatalytic probe for simultaneous determination of ascorbic acid. J. Braz. Chem. Soc.. 2008;19:1405-1412.
[Google Scholar]

[11] Ge B., Tan Y., Xie Q., Ma M., Yao S., . Preparation of chitosan–dopamine-multiwalled carbon nanotubes nanocomposite for electrocatalytic oxidation and sensitive electroanalysis of NADH. Sen. Actuat. B. 2009;137:547-554.
[Google Scholar]

[12] Gu T., Hasebe Y., . Peroxidase and methylene blue-incorporated double stranded DNA–polyamine complex membrane for electrochemical sensing of hydrogen peroxide. Anal. Chim. Acta. 2004;525:191-198.
[Google Scholar]

[13] ICH, 1995. ICH Validation of Analytical Procedures: Methodology, Technical Requirements for the Registration of Pharmaceuticals for Human Use. In: International Conference on Harmonization. Geneva, Switzerland.

[14] Kalcher K., Švancara I., Metelka R., Vytřas K., Walcarius A., . Heterogeneous carbon electrochemical sensors. In: Grimes C.A., Dickey E.C., Pishko V.M., eds. Encyclopedia of Sensors. Stevenson Ranch: American Scientific Publishers; 2006. pp. 283–243
[Google Scholar]

[15] Kalimuthu P., John S.A., . Electropolymerized film of functionalized thiadiazole on glassy carbon electrode for the simultaneous determination of ascorbic acid, dopamine and uric acid. Bioelectrochemistry. 2009;77:13.
[Google Scholar]

[16] Kang G.F., Lin X.Q., . RNA modified electrodes for simultaneous determination of dopamine and uric acid in the presence of high amounts of ascorbic acid. Electroanalysis. 2006;18:2458-2466.
[Google Scholar]

[17] Lavecchia T., Tibuzzi A., Giardi M.T., . Biosensors for functional food safety and analysis. Adv. Exp. Med. Biol.. 2010;698:267-281.
[Google Scholar]

[18] Lei C., Zhang Z., Liu H., Kong J., Deng J., . Biosensoring of hydrogen peroxide using new methylene blue N incorporated in a montmorillonite-modified horseradish peroxidase immobilization matrix as an electron shuttle. Anal. Chim. Acta. 1996;332:73-81.
[Google Scholar]

[19] Li Y.X., Lin X.Q., . Simultaneous electroanalysis of dopamine, ascorbic acid and uric acid by poly (vinyl alcohol) covalently modified glassy carbon electrode. Sens. Actuat. B. 2006;115:134-139.
[Google Scholar]

[20] Lin X.Q., Kang G.F., Lu L.P., . DNA/Poly(p-aminobenzensulfonic acid) composite bi-layer modified glassy carbon electrode for determination of dopamine and uric acid under coexistence of ascorbic acid. Bioelectrochemistry. 2007;70:235-244.
[Google Scholar]

[21] Liu B., Liu Z., Chen D., Kong J., Deng J., . An amperometric biosensor based on the coimmobilization of horseradish peroxidase and methylene blue on a β-type zeolite modified electrode. Fresenius J. Anal. Chem.. 2000;367:539-544.
[Google Scholar]

[22] Liu A.H., Honma I., Zhou H.S., . Simultaneous voltammetric detection of dopamine and uric acid at their physiological level in the presence of ascorbic acid using poly(acrylic acid)-multiwalled carbon-nanotube composite-covered glassy-carbon electrode. Biosens. Bioelectron.. 2007;23:74-80.
[Google Scholar]

[23] Lupetti K.O., Ramos L.A., Vieira I.C., Fatibello-Filho O., . A zucchini-peroxidase biosensor applied to dopamine determination. II Farmaco. 2005;60:179-183.
[Google Scholar]

[24] Marco J.P., Borges K.B., Tarley C.R.T., Ribeiro E.S., Pereira A.C., . Development of a simple, rapid and validated square wave voltametric method for determination of promethazine in raw material and pharmaceutical formulation using DNA modified multiwall carbon nanotube paste electrode. Sens. Actuat. B – Chem.. 2013;177:251-259.
[Google Scholar]

[25] Marco J.P., Borges K.B., Tarley C.R.T., Ribeiro E.S., Pereira A.C., . Development and application of an electrochemical biosensor based on carbon paste and silica modified with niobium oxide, alumina and DNA (SiO2/Al2O3/Nb2O5/DNA) for amitriptyline determination. J. Electroanal. Chem.. 2013;704:159-168.
[Google Scholar]

[26] Njagi J., Chernov M.M., Leiter J.C., Andreescu S., . Amperometric detection of dopamine in vivo with an enzyme based carbon fiber microbiosensor. Anal. Chem.. 2010;82:989-996.
[Google Scholar]

[27] Qian Z., Shi X., Zhuang J., Kong J., Deng J., . Alkyl chain length effect on the horseradish peroxidase-catalytic reduction of hydrogen peroxide mediated by methylene blue at alkanethiol self-assembled monolayer electrodes. Bioelectrochem. Bioenerg.. 1998;46:193-198.
[Google Scholar]

[28] Ribeiro E.S., Rosatto S.S., Gushikem Y., Kubota L.T., . Electrochemical study of Meldola’s blue, methylene blue and toluidine blue immobilized on a SiO₂/Sb₂O₃ binary oxide matrix obtained by the sol–gel processing method. J. Solid State Electrochem.. 2003;7:665-670.
[Google Scholar]

[29] Smith T.E., Devlin T.M., . Textbook of Biochemistry with Clinical Correlations. New York: Wiley/Liss; 1992.

[30] Tarley C.R.T., Ávila T.C., Segatelli M.G., Lima G.F., Peregrino G.S., Scheeren C.W., Dias S.L.P., Ribeiro E.S., . Silica–Alumina–Niobia (SiO₂/Al₂O₃/Nb₂O₅) matrix obtained by the sol–gel processing method: new material for online extraction of zinc ions. J. Braz. Chem. Soc.. 2010;21:1106-1116.
[Google Scholar]

[31] Trojanowicz M., . Recent developments in electrochemical flow detections – a review: Part II. Liquid chromatography. Anal. Chim. Acta. 2011;688:8-35.
[Google Scholar]

[32] Xian Y.Z., Liu F., Xian Y., Zhou Y.Y., Jin L.T., . Preparation of methylene blue-doped silica nanoparticle and its application to electroanalysis heme proteins. Electrochim. Acta. 2006;51:6527-6532.
[Google Scholar]

[33] Xiao M.W., Wang L.S., Wu Y.D., Huang X.J., Dang Z., . Electrochemical study of methylene blue/titanate nanotubes nanocomposite and its layer-by-layer assembly multilayer films. J. Solid State Electrochem.. 2008;12:1159-1166.
[Google Scholar]

[34] Xu J.Z., Zhu J.J., Wu Q., Hu Z., Chen H.Y., . An amperometric biosensor based on the coimmobilization of horseradish peroxidase and methylene blue on a carbon nanotubes modified electrode. Electroanalysis. 2003;15:219-224.
[Google Scholar]

[35] Xu J.Z., Zhang Y., Li G.X., Zhu J.J., . An electrochemical biosensor constructed by nanosized silver particles doped sol–gel film. Mater. Sci. Eng. C. 2004;24:833-836.
[Google Scholar]

[36] Yao H., Sun Y.Y., Lin X.H., Tang Y.H., Huang L.Y., . Electrochemical characterization of poly(eriochrome black T) modified glassy carbon electrode and its application to simultaneous determination of dopamine, ascorbic acid and uric acid. Electrochim. Acta. 2007;52:6165-6171.
[Google Scholar]

[37] Zaitseva G., Gushikem Y., Ribeiro E.S., Rosatto S.S., . Electrochemical property of methylene blue redox dye immobilized on porous silica–zirconia–antimonia mixed oxide. Electrochim. Acta. 2002;47:1469-1474.
[Google Scholar]

[38] Zhang Y.Z., Pan Y., Su S., Zhang L.P., Li S.P., Shao M.W., . A novel functionalized single-wall carbon nanotube modified electrode and its application in determination of dopamine and uric acid in the presence of high concentrations of ascorbic acid. Electroanalysis. 2007;19:1695-1701.
[Google Scholar]

[39] Zhang R., Jin G.D., Chen D., Hu X.Y., . Simultaneous electrochemical determination of dopamine, ascorbic acid and uric acid using poly(acid chrome blue K) modified glassy carbon electrode. Sens. Actuat. B. 2009;138:174-181.
[Google Scholar]

[40] Zhang L., Cheng Y., Lei J., Liu Y., Hao Q., Ju H., . Stepwise chemical reaction strategy for highly sensitive electrochemiluminescent detection of dopamine. Anal. Chem.. 2013;85:8001-8007.
[Google Scholar]

[41] Zheng D., Ye J.S., Zhou L., Zhang Y., Yu C.Z., . Simultaneous determination of dopamine, ascorbic acid and uric acid on ordered mesoporous carbon/Nafion composite film. J. Electroanal. Chem.. 2009;625:82-87.
[Google Scholar]