Applied electrochemical biosensor based on covalently self assembled monolayer at gold surface for determination of epinephrine in the presence of Ascorbic acid

Moghadam Zohreh; Sayed Mehdi Ghoreishi; Mohsen Behpour; Motaghedifard Mohammadhassan

doi:10.1016/j.arabjc.2012.11.004

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Original article

10 (

1_suppl

); S657-S664

doi:

10.1016/j.arabjc.2012.11.004

Applied electrochemical biosensor based on covalently self assembled monolayer at gold surface for determination of epinephrine in the presence of Ascorbic acid

Moghadam Zohreh^{^a}, Sayed Mehdi Ghoreishi^{^a,^b,⁎}, Mohsen Behpour^{^a}, Motaghedifard Mohammadhassan^{^a}

a Department of Analytical Chemistry, Faculty of Chemistry, University of Kashan, Kashan, Islamic Republic of Iran

b Deylaman Institute of Higher Education, Lahijan, Islamic Republic of Iran

⁎Corresponding author at: Department of Analytical Chemistry, Faculty of Chemistry, University of Kashan, Kashan, Islamic Republic of Iran. Tel.: +98 3615912395. s.m.ghoreishi@kashanu.ac.ir (Sayed Mehdi Ghoreishi)

Received: 2012-7-10, Accepted: 2012-11-10, Published: 2012-02-01

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 this paper, a new electrochemical sensor for the determination of epinephrine (Epi) in the presence of ascorbic acid (AA) is described. The characterization of Au TMBH self-assembled monolayer modified electrode (TMBH SAM-ME) was investigated by cyclic voltammetry (CV) using the [Fe(CN)₆]^−3/−4 redox couple. The mediated oxidation of Epi at the modified electrode was investigated by voltammetric methods and the values of transfer coefficient (α), the ionic exchanging current density (i_o), catalytic rate constant (k_h) and diffusion coefficient (D) were calculated. By double potential step chronoamperometric experiments (DPCHA) at the modified electrode was obtained two linear segments of 1.7–24.9 μM and 24.9–91.7 μM by a detection limit (3σ) of 0.19 ± 0.01 μM for Epi. The advantages of this modified electrode were reproducibility and repeatability, stability and anti fouling effect against oxidation products of Epi at the surface of TMBH SAM-ME. Finally, the modified electrode was shown agreeable responses to recovery of Epi from real sample solutions by standard addition method.

Keywords

Self-assembled monolayer

Gold electrode

Electrochemical impedance spectroscopy

Epinephrine

Double potential step chronoamperometric calibration

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1 Introduction

Chemical modification of the electrode surface is of great importance in electrochemistry including a wide spectrum of promising applications. In particular, thin films and self-assembled mono layers (SAMs) have been used in electro analytical chemistry for the modification of the electrodes to develop sensors (Shervedani et al., 2008; Shervedani and Mozaffari, 2006) and biosensors (Shervedani et al., 2006; Shervedani and Mehrjardi, 2009). Further modification of the SAMs may allow fabrication of highly sensitive and more sophisticated sensors and biosensors for trace analysis.

Schiff bases, named after Hugo Schiff, are formed when any primary amine reacts with an aldehyde or a ketone under specific conditions. Structurally, a Schiff base (also known as imine or azomethine) is a nitrogen analog of an aldehyde or ketone in which the carbonyl group (C⚌O) has been replaced by an imine or azomethine group (Raman et al., 2009). Schiff bases are some of the most widely used organic compounds. They are used as pigments and dyes, catalysts, intermediates in organic synthesis, and as polymer stabilizers. Schiff bases have also been shown to exhibit a broad range of biologic activities, including antifungal, antibacterial, antimalarial, antiproliferative, anti-inflammatory, antiviral, and antipyretic properties (De Souza et al., 2007; Guo et al., 2007). The imine group present in such compounds has been shown to be critical to their biologic activities. The applications in electrochemistry, bioinorganic, biosensors, antimicrobial activity, fluorescence properties, catalysis, metallic deactivators and separation processes are reported for these components (Joseyphus and Nair, 2009).

The electrochemical impedance spectroscopy (EIS) is a powerful, nondestructive and informative technique, which is usually used for characterization and study of corrosion phenomena (Mansfeld and Lorenz, 1991), fuel cells and batteries (Lasia, 1999), coatings and conductive polymers (Inzelt and Lang, 1994), adsorption behavior of thin films (Benavente et al., 1996), the SAMs (Janek et al., 1997) and electron transfer kinetics (Protsailo and Fawcett, 2000). Recently, the EIS has been used in analytical chemistry to trace modification steps of chemically modified electrodes based on SAMs and to quantify the inorganic (Shervedani and Mozaffari, 2005) or biologic (Janata, 2002) species in solution.

To our knowledge, there is no report on the electrocatalytic determination of Epi in the presence of AA using SAM modified gold electrodes (SAM-Au). Thus, in the present work, 2-hydroxy-N′1-[(E)-1-(3-methyl-2-thienyl) methylidene] benzohydrazide (TMBH) as a Schiff base, which is a bio mimetic analog, was chosen to form Au-TMBH SAM modified electrode. The structural integrity and compactness of the modified surface were then investigated by characterization of the electrode in the presence of a reversible redox reaction probe. The investigations were aimed at evaluating the antifouling performance of this monolayer as a suitable sensor material for the analysis of neurotransmitters like Epinephrine (Epi). Finally, the proposed Au-TMBH SAM electrode was successfully examined for determination of Epi in a real sample.

2 Experimental

2.1

2.1 Chemicals and apparatus

2-hydroxy-N′1-[(E)-1-(3-methyl-2-thienyl) methylidene] benzohydrazide (TMBH), has been synthesized in inorganic laboratory of the University of Kashan and characterized by physical and spectroscopic data. Epinephrine and other materials used in this work were of analytical grade (Merck®) and used without further purification.

The cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) measurements were performed on Auto lab Potentiostat/Galvanostat, PGSTAT 35 (Eco chemie Utrecht, Netherlands), equipped with the General Purpose Electrochemical System (GPES 4.9,006 software) and NOVA, in a conventional three electrode glass cell containing poly crystalline gold disk (Metrohm, 2 mm diameter) as a working electrode, a Pt plate (99.99%, 5 cm²) as an auxiliary electrode, and a Ag/AgCl (3 M KCl) as the reference electrode.

2.2

2.2 Synthesis of TMBH

The thiol Schiff-base; TMBH, was synthesized in the usual manner by reaction of 3-methyl-thiophene-2-carbaldehyde with salicylhydrazone in a 1:1 M ratio in methanol as follows. 3-methyl-thiophene-2-carbaldehyde (0.01 mol, 1.26 g) and salicylhydrazone (0.01 mol, 1.52 g); were placed in 100 mL round-bottomed flask equipped with a condenser and a magnetic bar. Methanol (50 ml) was then added to the mixture and the mixture was refluxed far 3 h while stirring, and then cooled to room temperature. The solid product was filtered, and the product was recrystallized from ethanol. Analytical calculated for C₁₃H₁₂N₂O₂S: C, 59.98; H, 4.65; N, 10.76%. Found: C, 58.17; H, 4.59; N, 10.88%; IR bands (KBr, cm⁻¹), υ_C⚌N, 1594 cm⁻¹; Yield = 80%.

2.3

2.3 Electrode preparation

The polycrystalline gold disk electrodes were polished using aqueous slurries of alumina (0.3 down to 0.05 μm, Buehler®), sonicated in water/chloroform/water for 5 min. Then, the gold working electrode was kept in Piranha solution (1:3, v/v; 30% H₂O₂ and concentrated H₂SO₄ [caution: Piranha solution is extremely corrosive and must be handled carefully]) for 3.0 min and rinsed thoroughly with double-distilled water and then cleaned electrochemically by cycling the electrode potential between 0.000 and +1.500 V vs. a reference electrode, with a 100 mV s⁻¹ scan rate in 0.5 M sulfuric acid and 0.000 until −1.400 V with a 100 mV s⁻¹ scan rate in 0.5 M NaOH until reproducible voltammograms were observed. A roughness factor of 3.25 was obtained for bare Au electrodes from the ratio of the real to geometric surface area (Oesch and Janata, 1983; Carvalhal et al., 2005). Immediately prior to modification, the cleaned Au electrode was thoroughly rinsed with de ionized water, and placed into a 1.0 mM TMBH ethanol solution for 8 h to form Au-TMBH SAM electrode. Finally, after modification of gold electrode, the oxidation of Epi was investigated by electrochemical impedance spectroscopy and voltammetric methods such as cyclic voltammetry, linear sweep voltammetry, chronocoulometry and double potential step chronoamperometry at the surface of modified electrode. Finally, this electrode was tested for detection of Epi in human blood serum.

3 Results & discussion

3.1

3.1 Electrochemical characterization of the self assembled monolayer of TMBH

3.1.1

3.1.1 Characterization of TMBH in H₂SO₄ media

The CV of the bare-Au electrode and TMBH SAM-ME in 0.5 M H₂SO₄ shows that the gold oxidation/reduction currents are suppressed at the TMBH SAM-ME in comparison with the bare-Au electrode. The electrode surface coverage (θ) is a key factor, which can be used to estimate the surface state of electrode. The surface coverage can be determined through a decrease in the area (Q) associated with gold oxide reduction at 910 mV (Sabatani and Rubinstein, 1987). The results are summarized in Table 1.

Table 1 Partial surface coverage obtained for TMBH SAM on Au electrode by different methods.

Method	Parameters	Equation	θ (%)
CV in H₂SO₄	Q_bare = 1567.8 & Q_modified = 434.7 μC cm⁻²	% θ = 100 [1 − (Q_modified /Q_bare)]	52.0
CV in NaOH	Q_bare = 1567.8 & Q_modified = 146.5 μC cm⁻²	% θ = [(3 × Q_{modified in NaOH})/(Q_{bare,in H2SO4}/2)]	56.1
CV in probe Fe^+2/+3	i_p = 36.0 & i˚_p = 10.9 μA	% θ = [1 − (i_{p modified}/i˚_{p bare})] × 100	69.7
EIS in probe Fe^+2/+3	R˚_ct = 153.9 & R_ct = 6932.1 Ω	% θ = [1 − (R˚_{ct bare}/R_{ct modified})] × 100	97.8

θ: The electrode surface coverage.

Q: The charge density of surface.

I: The anode or cathode peak current of redox probe in cyclic voltammetry.

R_ct: The charge transfer resistant in electrochemical impedance spectroscopy.

3.1.2

3.1.2 Electrochemical desorption in NaOH media

The first cyclic voltammograms recorded in the potential range from 0.000 to −1.400 V in 0.5 M NaOH solution on a bare polycrystalline Au electrode and the Au-TMBH modified electrode. We attributed the large cathodic peak observed in the first potential cycle to the reduction of adsorbed TMBH molecules, and the maxima around −920 mV, observed before the main peak, to physically adsorbed molecules (Figure 1). A partial surface coverage may be obtained for TMBH using desorption peak area (see Table 1). So, the surface coverage of Au in NaOH is 56.1%.

Successive cyclic voltammograms obtained for desorption of TMBH from Au-TMBH SAM electrode in 0.5 M NaOH solution. Scan rate: 100 mV s−1. A = 0.0314 cm2.

3.1.3

3.1.3 Cyclic voltammetry of TMBH in the presence of [Fe(CN)₆]^−3/−4

Since the structure of the monolayer on the Au and the amount of the charge on the surface were expected to change by changing the pH, the cyclic voltammograms of the bare Au and Au-TMBH SAM modified electrodes were recorded in the presence of 5.0 mM [Fe(CN)₆]^−3/−4 redox probe, in pH 3.0, 6.0 and 8.0 (Figure 2), respectively. [Fe(CN)₆]^−3/−4 produced a couple of well-defined redox waves at Au electrode with an average peak-to-peak separation (ΔE_p) of 88 mV at 100 mV s⁻¹. After the self-assembly of TMBH, the shape of CV changed dramatically, small redox reaction of [Fe(CN)₆]^−3/−4 was apparently observed because of the blocking effect of TMBH on the electron transfer of [Fe(CN)₆]^−3/−4. At pH 3.0 (Figure 2b), the redox probe showed more reversible behavior at the bare Au electrode (curve a) in comparison with TMBH electrode (curve b), which the surface coverage (%θ) was calculated to be 50.0%. The ΔE_p increased from 128 to 325 mV. Also, the redox reaction current of probe in this pH was more than two other pH. This behavior is resultant of: (a) Electrostatic attraction between positively charged TMBH monolayer and negatively charged probe. (b) Electrostatic repulsion between positively charged TMBH molecules, causing an open channel in the structure of SAM that facilitates the electron transfer (Shervedani and Bagherzadeh, 2008). (c) The insulation effect of monolayer that retards the electron transfer process. At pH 8.0 (Figure 2d): the Au-TMBH SAM can expose a neutrally charged insulting monolayer at pH 8.0. The faradaic currents for the probe redox reaction were decreased (Figure 2d, compare curves b and c). Reasonably, hydrogen bonds have more chance to form between amine groups, and π–π stocking is more effectively formed in these conditions. Indeed, Au-TMBH SAM shows opening and closing behaviors in acidic and basic solutions, respectively, for/and/against electron transfer process (Jun and Beng, 2004). A value may be obtained for θ using Eq. (1) (Shervedani et al. 2007):

(1)

% θ = [1 - (i_{p modified} / i_{p bare}^{°})] \times 100

where i^˚_{p bare} and i_{p modified} are peak currents of redox probe at bare Au and Au-TMBH SAM electrodes under the same conditions. The values obtained for i˚_p and i_p were 19.7 and 39.4 μA at pH 3.0, 10.9 and 36.0 μA at pH 6.0 and 4.9 and 39.5 μA at pH 8.0, respectively. Thus, values of 69.7 and 87.6 were estimated for θ at pH 6.0 and 8.0. These behaviors were followed more precisely by the EIS studies as follows.

Cyclic voltammograms obtained on (a) Bare Au and (b) at pH 3.0 (c) at pH 6.0 and (d) at pH = 8.0, on Au-TMBH electrode in 0.1 M PBS, 5.0 mM [Fe(CN)6]3−/4−. Scan rate 100 mV s−1.

3.1.4

3.1.4 Electrochemical impedance spectroscopy of TMBH in the presence of [Fe(CN)6]^−3/−4

The complex plane plots obtained on bare Au and Au-TMBH SAM electrodes in the presence of 5.0 mM [Fe(CN)₆]^−3/−4 redox probe at pH 6.0, respectively, were approximated according to Ref. (Shervedani and Mozaffari, 2006), were satisfied using a CPE model and the kinetics were extracted (Table 1). By comparisons of results, it peer that at pH 6.0; the R_ct was increased by a factor of 45.04 from bare-Au to Au-TMBH SAM electrode and the electrode showed more irreversible behavior.

The values of θ obtained from oxidation/reduction of Au electrode in 0.5 M H₂SO₄, and those obtained using CV (Figure 2c) and EIS (at pH⚌6) measurements in the presence of [Fe(CN)₆]^−3/−4 were relatively larger than that obtained by the desorption method in NaOH. This behavior is explained as follows: some factors, like hydrogen bonding (Shervedani and Bagherzadeh, 2008), π–π interaction between aromatic rings (Shervedani et al., 2007), dipole–dipole, and/or Van Der Waals (VDW) interactions, can impose a barrier to the charge transfer between the probe and metal base of the electrode. In another word, the total insulating effect, including contributions of Au–S covalent bonds (i.e. real surface coverage) and the inter chain interactions (e.g., hydrogen bonding, π–π, dipole–dipole, and VDW interactions), are evaluated by methods of CV in H₂SO₄, CV or EIS in Fe^+2/+3 probe; while by second method (CV in NaOH), only the contribution of Au-S covalent bonds (real surface coverage) is evaluated. The difference (i.e., θ_{average of (CV in} H₂SO_{4), (CV in Fe}^+2/+3_{) and (EIS in Fe}^+2/+3₎ − θ _{(CV in NaOH)}) can give an insight about the contributions of other factors (e.g., hydrogen bonding, π–π, dipole–dipole, and VDW interactions) in the insulating effect. Thus, the surface coverage obtained by second method (56.1%) is almost the nearest value to the real θ.

3.1.5

3.1.5 Surface titration and evaluation of the surface pK_a

For determination of surface pK_a of SAM, the complex plane plots obtained at Au-TMBH SAM electrode are presented in Figure 2. A. The EIS data were approximated based on the procedure explained in previous part. The R_ct of the modified electrode was pH dependent. The pH-metric titration curve was constructed using R_ct as a function of solution pH (inset of Figure 3A). A surface pK_a of 6.5 ± 0.1 was evaluated from the midpoint of titration curve for Au-TMBH SAM electrode, respectively. Although the EIS is an efficient method for this goal, it still remains time consuming.

(A) Complex plane plots obtained on Au-TMBH SAM electrode in the presence of 0.5 mM [Fe(CN)6]3−/4− in phosphate buffer solution at pH 3.0–8.0. Dc potential, +200 mV; ac amplitude, 10 mV; frequency range, 10 kHz to 100 mHz. (B) Cyclic voltammograms obtained on Au-TMBH SAM electrode in 0.1 M PBS, 5.0 mM [Fe(CN)6]3−/4−; at pH 4.0–8.0.

Also, the surface pK_a of the Au-TMBH SAM electrode was estimated by recording the cyclic voltammograms in a wide range of pH (Figure 3B), and then, monitoring of i_p and ΔE_p as functions of pH. Value of 6.2 ± 0.1 was obtained from junction point of the titration curves for Au-TMBH SAM, respectively. This value is in good agreement with that obtained by EIS and confirms the validity of CV, as a simple method, for estimation of surface pK_a. The results support the changes, and thus, modification took place on the Au electrode, i.e. self assembled monolayer of TMBH.

3.2

3.2 Electrochemical behavior of epinephrine at Au-TMBH SAM electrode

Differential pulse voltammograms obtained for the redox reaction of Epinephrine at pH 6.0, in PBS on bare Au and the Au-TMBH SAM electrodes showed oxidation potentials of +0.228 and +0.200 V, respectively, indicating that the kinetic of Epi on the Au-TMBH SAM electrode was more rather quick than that on bare Au electrode and the presence of the TMBH at the electrode surface reduces the over potential of Epi oxidation, shifting the potential value by −28 mV, respectively. The oxidation peak current of Epi on the Au TMBH SAM-modified electrode was 1.85 times greater than that on the bare Au electrode. So two major factors are important in evaluation of the catalytic effect of electrode on the electro-oxidation process: a poor decrease in the over potential of oxidation and an increase in the current density as compared with the bare electrode. The π–π interaction between the phenyl structure of Epi and salicylic structure of TMBH on the surface of the Au electrode may accelerate the electron transfer of Epi in comparison with the bare electrode.

3.3

3.3 Study of pH effect

According to Nernst equation (Bockris and Reddy, 1970), the variations of oxidation potential of Epi in pH 2.0–8.0 have indicated a two-electron two-proton process following Eq. (2):

(2)

E_{pa} = E^{°} + (0.0591 / n) \log [{(Ox)}_{a} / {(R)}_{b}] - (0.0591 m / n) pH

In which a and b are coefficients of oxidant and reducer in the reaction equation, m is the number of transferred protons and n the number of transferred electrons. To verify this matter, we recorded the voltammograms of Epi on the modified electrode as a function of solution pH. The results indicated that Epi anodic peak potential (E_pa) was shifted to the negative direction by increasing the solution pH. A good linear relation was found between the E_pa of Epi and the solution pH in the range of 2.0–8.0 by E_pa(V) = 0.0661 (V/pH) pH – 0.5946 (V), R² = 0.9946, implying that protons took part in the electrode reaction. A slope of −0.0661 V/pH indicated that equal number of protons and electrons were accomplished in oxidation process. Assuming n as 2, a value of 2.24 was obtained for the number of transferred proton. Consequently, a two-proton – two-electron mechanism can be proposed for Epi, which is in good agreement with the previous reports. In addition, the anodic peak current increased with increasing the pH up to 6.0; beyond that the voltammograms and the peak currents merged each other. Therefore, pH = 6.0, was selected for further studies.

3.4

3.4 Study of scan rate

The influence of scan rate (ν) on the electrochemical behavior of Epi (20 μM) on the TMBH -modified electrode was investigated. According to Eq (3) for a totally irreversible diffusion controlled process (Bard and Faulkner, 1991):

(3)

I_{p} = 3.01 \times 10^{5} n {[(1 - α) n_{α}]}^{1 / 2} {AD}^{1 / 2} C ν^{1 / 2}

where D is the diffusion coefficient and C is the concentration of reagent, in the scan rate range of 25.0–105 mV s⁻¹, the oxidation peak current increases linearly with the square root of the scan rate, typical of a diffusion–controlled process of Epi on the modified electrode. Therefore the linear regression equation for Epi was found to be:

I_{pa} = 1.7336 ν^{1 / 2} - 0.0328, R^{2} = 0.9918

And considering (1 − α)n_α = 0.46 (see below), D = 3.5 × 10⁻⁶ cm² s⁻¹ and A = 0.0314 cm², it is estimated that the total number of electrons involved in the anodic oxidation of Epi is n ≈ 2.0.

3.5

3.5 Electron transfer coefficient of Epi at the surface of Au-SAM modified electrode

The linear sweep voltammograms of Au-TMBH SAM electrode in 0.1 M PBS (pH 6.0) containing different concentrations of Epi, with a sweep rate of 25 mV s⁻¹ were estimated. The rising part of the voltammogram is known as the Tafel region, which is affected by the electron transfer kinetics between Epi and modified electrode. If deprotonation of Epi is a sufficiently fast step, the number of electrons involved in the rate-determining step can be estimated from the slope of the Tafel plot. The Tafel slope (a) can be obtained from the slope of E_p vs. log I using Eq. (4) (Bard and Faulkner, 1991):

(4)

E_{p} = 2.3 RT / n_{α} α F (\log i_{o}) - 2.3 RT / n_{α} α F (\log i)

The Tafel slope was found to be 130 mV (Figure 4, inset), which indicates that a one-electron transfer process is the rate limiting step assuming a transfer coefficient (α) is about 0.54. Also, the ionic exchanging current density (i_o) by average of Tafel intercepts for Epi was found to be 0.14 μA cm⁻².

Linear sweep voltammograms of Au-TMBH SAM electrode in 0.1 M PBS, (pH = 6) containing 10.0, 20.0, 30.0 and 40.0 μM Epi at a sweep rate of 25 mV s−1; (A) Tafel plot derived from linear sweep voltammograms.

3.6

3.6 Evaluating of diffusion coefficient & catalytic rate constant

Double potential Chronocoulometric measurements of Epi at Au-TMBH SAM electrode were carried out at the working electrode potentials of 280.0 and 0.0 mV for various concentrations of Epi (Figure 5). For an electro active material (Epi in this case) with a diffusion coefficient of D, the current observed for the electrochemical reaction at the mass transport limited condition is described by the Cottrell equation (Bard and Faulkner, 1991). Experimental plots of Q vs. t^1/2 were employed, with the best fits for different concentrations of Epi. The slopes of the resulting straight lines were then plotted vs. Epi concentration. From the resulting slope and Cottrell equation the mean value of the D was found to be (3.5 ± 0.2) × 10⁻⁶ cm² s⁻¹.

Double potential chronocoulograms obtained at Au-TMBH SAM electrode in 0.1 M phosphate buffer solution (pH 6.0) for different concentrations of Epi. The a–d corresponds to 18.0, 26.0, 34.0 and 43.0 μM of Epi. (A) Plots of Q vs. t−1/2 obtained from chronocoulograms a–d. (B) Plot of the slope of the straight lines against Epi concentration.

The rate constant for the chemical reaction between Epi and the redox sites in Au-TMBH SAM electrode, k_h, can be evaluated using chronoamperometry according to the method described by Galus (Galus, 1976):

(5)

I_{C} / I_{L} = γ^{1 / 2} [π^{1 / 2} erf (γ^{1 / 2}) + (\exp (- γ) / γ^{1 / 2})]

where I_C is the catalytic current of Au-TMBH SAM electrode in the presence of Epi, I_L is the limited current in the absence of Epi, and γ = k_hC_bt (C_b is the bulk concentration of Epi) is the argument of error function. In the cases where γ exceeds 2, the error function is almost equal to 1.0 and the above equation can be reduced to:

(6)

I_{C} / I_{L} = π^{1 / 2} γ^{1 / 2} = π^{1 / 2} {(k_{h} C_{b} t)}^{1 / 2}

where k_h and t are the catalytic rate constant (mol⁻¹ L s⁻¹) and time elapsed (s), respectively. The above equation can be used to calculate the rate constant of the catalytic process, k_h. Having measured the catalytic current, i.e., I_C, it is possible to carry out the electrode process under identical conditions, but in the absence of Epi, in order to determine I_L. From the slope of I_C/I_L vs. t^1/2 plot, the value of k_h can be simply calculated for a given concentration of the substrate. The calculated value of k_h is (2.04 ± 0.03) × 10² mol⁻¹ L s⁻¹ using the slope of I_C/I_L − t^1/2 plot. This value of k_h also explains the sharp feature of the catalytic peak observed for the catalytic oxidation of Epi at the surface of Au-TMBH SAM electrode.

3.7

3.7 7. Evaluation of detection limit

DPV method was used to determine the concentration of Epi. The plot of peak current vs. Epi concentration consisted of two linear segments with slopes of 0.0205 ± 0.001 and 0.0127 ± 0.0001 μA L μmol⁻¹ in the concentration ranges of 3.3–23.3 μM and 23.3–100.0 μM, respectively. The decrease in sensitivity (slope) of the second linear segment is likely due to kinetic limitation. The detection limit (3σ) of Epi was found to be 0.29 ± 0.01 μM. So, the stability of Au-TMBH SAM electrode was investigated by recording the electrode response in 0.1 M PBS pH = 6 containing 40.0 μM Epi, once in every day. There were no significant changes in the electrode response, where the electrode was kept in 0.1 M PBS, pH 6, during the measurements for five days. The relative standard deviation of the peak currents for five time measurements, and each time (n = 3) was found to be 5.3%. The repeatability of the electrode response was estimated by making repetitive determinations of 40.0 μM Epi using one Au-TMBH SAM electrode. The relative standard deviation of the peak currents for (n = 20) was found to be 2.6%. The reproducibility of the electrode response was examined by fabrication of a set of four Au-TMBH SAM electrodes, and then, recording the electrode response in 0.1 M PBS, pH = 6, containing 40.0 μM Epi. The relative standard deviation of the peak currents for (n = 4) was found to be 3.2%. These examinations indicated high stability, repeatability, and reproducibility of the Au-TMBH SAM electrode response to Epi.

Also, Double potential step chronoamperometric experiments were recorded at the Au-TMBH electrode by polarizing the potentials to 280 and 0.0 mV. Figure 6 shows a well resolved double-step chronoamperometric evolutions obtained in the absence (buffer alone) and presence of consecutive addition of 50 μL of 1.0 mM epinephrine in phosphate buffer solution (pH 6.0) to 25 mL phosphate buffer. It shows that at the conditions employed for this work, the epinephrine electrooxidation was irreversible. Figure 6 (inset) clearly shows linear relationship between transient catalytic current (measured at 4 s) and epinephrine concentrations. Two linear segments were obtained for the low (1.7–24.9 μM) and high (24.9–91.7 μM) concentration ranges, respectively:

I_pa (μA) = (0.0155 ± 0.001) [epinephrine] (μM) + (0.2400 ± 0.004) R² = 0.9944
I_pa (μA) = (0.0088 ± 0.003) [epinephrine] (μM) + (0.4481 ± 0.002) R² = 0.9850

Typical double potential step chronoamperometric transients at Au-TMBH SAM electrode in PBS solution (pH 6.0) following addition of epinephrine. The a–t corresponds to buffer, 2.0, 3.5, 5.0, 6.5, 8.5, 11.5, 15.0, 18.0, 21.5, 25.0, 31.5, 38.0, 45.0, 51.5, 58.0, 65.0, 72.0, 78.0, 85.0 and 92.0 μM, respectively. The potential was stepped from 0.28 to 0.0 and back to 0.28 V. Other chronoamperograms obtained during same experiments are omitted here for clarity purpose. Inset is the plot of chronoamperometric current at t = 4 s vs. epinephrine concentration.

The detection limit (3σ) of Epi by this method was found to be 0.19 ± 0.003 μM. A comparison of the proposed method to the reported ones presented in Table 2 indicated that the Au-TMBH SAM electrode is superior to the existing electrodes with regard to detection limit, working concentration range and determination of Epi.

Table 2 Comparison of the detection limit and working range of Au-TMBH SAM-modified electrode with the other reported electrodes.

Electrode	Modifier	Peak potential shift (mV)	Detection limit (M)	Working concentration range (M)	Reference
Au	2-(2,3-Dihydroxy phenyl)-1,3-dithiane	155	5.1 × 10⁻⁷	7.0 × 10⁻⁷_ 5.0 × 10⁻⁴	Mazloum-Ardakani et al. (2011)
Au	Homocysteine	–	1.0 × 10⁻⁷	5.0 × 10⁻⁵_ 8.0 × 10⁻⁴	Zhang et al. (2002)
Au	Triazole	–	1.0 × 10⁻⁸	1.0 × 10⁻⁷_ 6.0 × 10⁻⁴	Sun et al. (2006)
Au	l-Cysteine	–	1.0 × 10⁻⁸	1.0 × 10⁻⁷_ 2.0 × 10⁻⁶	Wang et al. (2002)
Au	TMBH	28	1.9 × 10⁻⁷	3.3 × 10⁻⁶_ 1.0 × 10⁻⁴	Proposed method

M: as mole/liter.

3.8

3.8 The behavior of Epi and ascorbic acid mixture at the surface of Au TMBH SAM-modified electrode

The DPVs recorded for a single AA and Epi and a mixture of AA and Epi at the bare Au electrode and Au TMBH SAM-modified electrode in 0.1 M PBS (pH 6.0) show that at the bare Au electrode, the oxidation peaks of Epi and AA appear at 246 and 345 mV, respectively. The mixture of Epi and AA gives a broad peak at about 274 mV. Thus the determination of Epi in the presence of AA is a difficult task to achieve. However, at the Au TMBH SAM-modified electrode (Figure 7), the oxidation peak of Epi appeared at 214 mV but no peak was found for AA. At this electrode the current response for 2.0 × 10⁻⁵ M Epi is 0.32 μA and after addition of AA up to 1.4 × 10⁻⁴ M, the current response changes only 4.1%, which indicates that the determination error of Epi concentration was in the permission region ±5% (Zheng and Zhou, 2007), but in more concentrations of AA, this error increased. Therefore, this electrode has substantial advantage in comparison by other SAM modified electrodes (Ensafi et al., 2010a,b; Luczak, 2011 ).

DPV curves of (a) 0.1 M PBS at pH 6.0, (b) 1.2 × 10−4 M AA, (c) 2.0 × 10−5 M Epi and (d) 2.0 × 10−5 M Epi +1.2 × 10−4 M AA on Au TMBH SAM-modified electrode.

3.9

3.9 Determination of Epi in human blood serum

At the Au-TMBH SAM modified electrode, Epi was not detected in human blood serum. However, when the Epi standard solution was spiked, the presence of AA, Uric Acid and some other interfering substances did not interfere with the determination of Epi. The diluted serum sample was spiked with various concentrations of epinephrine hydrochloride and its DPV was obtained by the modified electrode. The results showed the average recovery in 99.97% (Table 3). The Au-TMBH SAM-modified electrode responds well for the recovery of spiked Epi.

Table 3 Application of the Au-TMBH SAM electrode for the determination of Epi in spiked human blood serum.

Sample	Added (μM)	Found (μM)	Recovery (%)
Serum	0.00	Not detected	–
	1.96	2.01	102.55
	1.92	1.87	97.39

4 Conclusions

In this work, a self-assembled monolayer was prepared on Au electrode surface for the first time. The Au-TMBH SAM electrode was characterized by EIS and CV. The results show that the oxidation of Epi is dependent on pH, and the peak potential of Epi is shifted by −378 mV at the surface of modified electrode. Using chronocoulometry, the diffusion coefficient (D) of Epi in Au-TMBH SAM was estimated. Also, double potential step chronoamperometric experiments was shown two linear segments for the low and high concentration ranges, which the detection limit (3σ) of Epi by this method was found to be 0.19 ± 0.003 μM. The electrochemical studies of epinephrine (Epi) helped us to realize an excellent anti-fouling effect of TMBH film against oxidation products of Epi at pH = 6.0 by successive cyclic voltammetric scans (20 cycles) in comparison with the bare electrode. The DPV studies revealed that no interference could be observed for concentration up to 1.4 × 10⁻⁴ M of AA. High sensitivity, selectivity and reproducibility of the voltammetric responses, and low detection limit (0.29 ± 0.01 μM), together with the ease of preparation, make the proposed modified electrode very useful for accurate determination of Epi in real samples.

Acknowledgments

The authors are grateful to the University of kashan for supporting this work by Grant No. 159195-11.

References

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[Google Scholar]
Sun Y.X., Wang S.F., Zhang X.H., Huang Y.F., . Simultaneous determination of epinephrine and ascorbic acid at the electrochemical sensor of triazole SAM modified gold electrode. Sens. Actuators, B.. 2006;113:156-161.
[Google Scholar]
Wang S., Du D., Zou Q.C., . Electrochemical behavior of epinephrine at l-cysteine self-assembled monolayers modified gold electrode. Talanta. 2002;57:687-692.
[Google Scholar]
Zhang H.M., Zhou X.L., Hui R.T., Li N.Q., Liu D.P., . Studies of the electrochemical behavior of epinephrine at a homocysteine self-assembled electrode. Talanta. 2002;56:1081-1088.
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Show Sections

[1] Bard A.J., Faulkner L.R., . Electrochemical Methods, Fundamentals And Applications. New York: Wiley; 1991. pp. 456–467

[2] Benavente J., Ramos-Barrado J.R., Cabeza A., . Electrical behavior of an inorganic film from ac and dc measurements. J. Colloid Interface Sci.. 1996;180:116-121.
[Google Scholar]

[3] Bockris J.O.-M., Reddy A.K.N., . Modern Electrochemistry. New York: Plenum; 1970. pp. 876–881

[4] Carvalhal R.F., Freire R.S., Kubota L.T., . Polycrystalline gold electrodes: a comparative study of pretreatment procedures used for cleaning and thiol self-assembly monolayer formation. Electroanalysis. 2005;17:1251-1257.
[Google Scholar]

[5] De Souza A.O., Galetti F.C.S., Silva C.L., Bicalho B., Parma M.M., Fonseca S.F., . Antimycobacterial and cytotoxicity activity of synthetic and natural compounds. Quim. Nova. 2007;30:1563-1566.
[Google Scholar]

[6] Ensafi A.A., Rezaei B., Mirahmadi Zare S.Z., Taei M., . Simultaneous determination of ascorbic acid, epinephrine, and uric acid by differential pulse voltammetry using poly (3,3-bis[N,N bis (carboxymethyl)aminomethyl]-o-cresolsulfonephthalein) modified glassy carbon electrode. Sens. Actuators B. 2010;150:321-328.
[Google Scholar]

[7] Ensafi A.A., Taei M., Khayamian T., . Simultaneous determination of ascorbic acid, epinephrine, and uric acid by differential pulse voltammetry using poly(pxylenolsulfonephthalein) modified glassy carbon electrode. Colloids Surf. B. 2010;79:480-485.
[Google Scholar]

[8] Galus Z., . Fundamentals of Electrochemical Analysis. New York: Ellis Horwood; 1976. pp. 273–275

[9] Guo Z., Xing R., Liu S., Zhong Z., Ji X., Wang L., . Antifungal properties of Schiff bases of chitosan, N-substituted chitosan and quaternized chitosan. Carbohydr. Res.. 2007;342:1329-1332.
[Google Scholar]

[10] Inzelt G., Lang G., . Model dependence and reliability of the electrochemical quantities derived from the measured impedance spectra of polymer modified electrodes. J. Electroanal. Chem.. 1994;378:39-44.
[Google Scholar]

[11] Janata J., . Electrochemical sensors and their impedances: a tutorial. Crit. Rev. Anal. Chem.. 2002;32:109-116.
[Google Scholar]

[12] Janek R.P., Fawcett W.R., Ulman A., . Impedance spectroscopy of selfassembled monolayers on Au (III): evidence for complex double-layer structure in aqueous NaClO4 at the potential of zero charge. J. Phys. Chem. B. 1997;101:8550-8558.
[Google Scholar]

[13] Joseyphus R.S., Nair M.S., . Synthesis characterization and antimicrobial activity of transition metal complexes with the Schiff base derived from imidazole-2-carboxaldehyde and glycylglycine. J. Coord. Chem.. 2009;62:319-325.
[Google Scholar]

[14] Jun Y.Y., Beng K.S., . Electrochemical study of monolayers of heterocyclic thiols selfassembled on polycrystalline gold electrode: the effect of solution pH on redox kinetics. Electrochem. Commun.. 2004;6:87-93.
[Google Scholar]

[15] Lasia A., . Electrochemical impedance spectroscopy and its applications. In: White R.E., Conway B.E., Bockris J.O.M., eds. Modern Aspects of Electrochemistry. Vol vol. 32. New York: Kluwer Academic/Plenum Press; 1999. p. :143-248.
[Google Scholar]

[16] Luczak T., . Gold electrodes modified with self-assembled layers made of sulphur compounds and gold nanoparticles used for selective electrocatalytic oxidation of catecholamine in the presence of interfering ascorbic and uric acids. Int. J. Electrochem. 2011
[CrossRef] [Google Scholar]

[17] Mansfeld F., Lorenz W.J., . Electrochemical impedance spectroscopy – applications in corrosion science and technology. In: Varma R., Selman J.R., eds. Techniques for Characterization of Electrodes and Electrochemical Processes. New York: Wiley; 1991. p. :581-647.
[Google Scholar]

[18] Mazloum-Ardakani M., Beitollahi H., Amini M.K., Mirjalili B.F., Mirkhalaf F., . Simultaneous determination of epinephrine and uric acid at a gold electrode modified by a 2-(2,3-dihydroxy phenyl)-1, 3-dithiane self-assembled monolayer. J. Electroanal. Chem.. 2011;651:243-249.
[Google Scholar]

[19] Oesch U., Janata J., . Electrochemical study of gold electrodes with anodic oxide films-I. Formation and reduction behaviour of anodic oxides on gold. Electrochim. Acta. 1983;28:1237-1243.
[Google Scholar]

[20] Protsailo L.V., Fawcett W.R., . Studies of electron transfer through selfassembled monolayers using impedance spectroscopy. Electrochim. Acta. 2000;45:3497-3504.
[Google Scholar]

[21] Raman N., Raja S.J., Sakthivel A., . Transition metal complexes with Schiff-base ligands: 4-Aminoantipyrine based derivatives – a review. J. Coord. Chem.. 2009;62:691-698.
[Google Scholar]

[22] Sabatani E., Rubinstein I., . Organized self-assembling monolayers on electrodes; monolayer-based ultra-microelectrodes for the study of very rapid electrode kinetics. J. Phys. Chem.. 1987;91:6663-6669.
[Google Scholar]

[23] Shervedani R.K., Bagherzadeh M., . Hydroxamation of gold surface via in-situ layer bylayer functionalization of cysteamine self-assembled monolayer: preparation and electrochemical characterization. Electrochim. Acta. 2008;53:6293-6298.
[Google Scholar]

[24] Shervedani R.K., Hatefi-Mehrjardi A., Khosravi Babadi M., . Electrochim. Acta. 2007;52:7051-7060.

[25] Shervedani R.K., Mozaffari S.A., . Preparation and electrochemical characterization of a new nanosensor based on self-assembled monolayer of cysteamine functionalized with phosphate groups. Surf. Coat. Technol.. 2005;198:123-129.
[Google Scholar]

[26] Shervedani R.K., Mozaffari S.A., . Copper (II) nanosensor based on a gold cysteamine self-assembled monolayer functionalized with salicylaldehyde. Anal. Chem.. 2006;78:4957-4963.
[Google Scholar]

[27] Shervedani R.K., Yaghoobi F., Mehrjardi A.H., Barzoki S.M.S., . Electrocatalytic activities of gold-5-amino-2-mercaptobenzimidazole-Mn+ self-assembled monolayer complexes (Mn⁺:Ag⁺, Cu²⁺) for hydroquinone oxidation investigated by CV and EIS. Electrochim. Acta. 2008;53:4185-4192.
[Google Scholar]

[28] Shervedani R.K., Mehrjardi A.H.H., Zamiri N., . Bioelectrochemistry. 2006;69:201-208.

[29] Shervedani R.K., Mehrjardi A.H.H., . Comparative electrochemical behavior of glucose oxidase covalently immobilized on mono-, di- and tetra-carboxylic acid functional Au-thiol SAMs via anhydride-derivatization route. Sens. Actuators. 2009;137:195-204.
[Google Scholar]

[30] Sun Y.X., Wang S.F., Zhang X.H., Huang Y.F., . Simultaneous determination of epinephrine and ascorbic acid at the electrochemical sensor of triazole SAM modified gold electrode. Sens. Actuators, B.. 2006;113:156-161.
[Google Scholar]

[31] Wang S., Du D., Zou Q.C., . Electrochemical behavior of epinephrine at l-cysteine self-assembled monolayers modified gold electrode. Talanta. 2002;57:687-692.
[Google Scholar]

[32] Zhang H.M., Zhou X.L., Hui R.T., Li N.Q., Liu D.P., . Studies of the electrochemical behavior of epinephrine at a homocysteine self-assembled electrode. Talanta. 2002;56:1081-1088.
[Google Scholar]

[33] Zheng J., Zhou X., . Sodium dodecyl sulfate-modified carbon paste in the presence of ascorbic acid. Bioelectrochemistry. 2007;70:408-412.
[Google Scholar]

Applied electrochemical biosensor based on covalently self assembled monolayer at gold surface for determination of epinephrine in the presence of Ascorbic acid

Abstract

Keywords

Self-assembled monolayer

Gold electrode

Electrochemical impedance spectroscopy

Epinephrine

Double potential step chronoamperometric calibration

1 Introduction

2 Experimental

2.1 Chemicals and apparatus

2.2 Synthesis of TMBH

2.3 Electrode preparation

3 Results & discussion

3.1 Electrochemical characterization of the self assembled monolayer of TMBH

3.1.1 Characterization of TMBH in H2SO4 media

3.1.2 Electrochemical desorption in NaOH media

3.1.3 Cyclic voltammetry of TMBH in the presence of [Fe(CN)6]−3/−4

3.1.4 Electrochemical impedance spectroscopy of TMBH in the presence of [Fe(CN)6]−3/−4

3.1.5 Surface titration and evaluation of the surface pKa

3.2 Electrochemical behavior of epinephrine at Au-TMBH SAM electrode

3.3 Study of pH effect

3.4 Study of scan rate

3.5 Electron transfer coefficient of Epi at the surface of Au-SAM modified electrode

3.6 Evaluating of diffusion coefficient & catalytic rate constant

3.7 7. Evaluation of detection limit

3.8 The behavior of Epi and ascorbic acid mixture at the surface of Au TMBH SAM-modified electrode

3.9 Determination of Epi in human blood serum

4 Conclusions

Acknowledgments

References

Suggested read for related articles:

3.1.1 Characterization of TMBH in H₂SO₄ media

3.1.3 Cyclic voltammetry of TMBH in the presence of [Fe(CN)₆]^−3/−4

3.1.4 Electrochemical impedance spectroscopy of TMBH in the presence of [Fe(CN)6]^−3/−4

3.1.5 Surface titration and evaluation of the surface pK_a