CC BY-NC-ND
0Arabian Journal of Chemistry (2013) xxx, xxx-xxx
King Saud University Arabian Journal of Chemistry
www.ksu.edu.sa www.sciencedirect.com
ORIGINAL ARTICLE
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
Received 10 July 2012; accepted 10 November 2012
KEYWORDS
Self-assembled monolayer; Gold electrode; Electrochemical impedance spectroscopy; Epinephrine;
Double potential step chro-noamperometric calibration
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 (a), the ionic exchanging current density (io), catalytic rate constant (kh) 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 iM and 24.9-91.7 iM by a detection limit (3r) of 0.19 ± 0.01 iM 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.
© 2012 King Saud University. Production and hosting by Elsevier B.V. All rights reserved.
1. Introduction
* Corresponding author at: Department of Analytical Chemistry, Faculty of Chemistry, University of Kashan, Kashan, Islamic Republic of Iran. Tel.: +98 3615912395.
E-mail address: s.m.ghoreishi@kashanu.ac.ir (S.M. Ghoreishi). Peer review under responsibility of King Saud University.
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.
1878-5352 © 2012 King Saud University. Production and hosting by Elsevier B.V. All rights reserved. http://dx.doi.Org/10.1016/j.arabjc.2012.11.004
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] ben-zohydrazide (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 anti-fouling 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. Chemicals and apparatus
2-hydroxy-N01-[(E)-1-(3-methyl-2-thienyl) methylidene] ben-zohydrazide (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. 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 sal-icylhydrazone (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), uC=N, 1594 cm-1; Yield = 80%.
2.3. Electrode preparation
The polycrystalline gold disk electrodes were polished using aqueous slurries of alumina (0.3 down to 0.05 im, 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 mVs-1 scan rate in 0.5 M sulfuric acid and 0.000 until -1.400 V with a 100 mVs-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 chronoamperom-etry at the surface of modified electrode. Finally, this electrode was tested for detection of Epi in human blood serum.
3. Results & discussion
3.1. Electrochemical characterization of the self assembled monolayer of TMBH
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 (h) is a key factor, which can be used to estimate the surface state of electrode. The surface coverage can be determined through a
Table 1 Partial surface coverage obtained for TMBH SAM on Au electrode by different methods.
Method Parameters Equation в (%)
CV in H2SO4 CV in NaOH CV in probe Fe+2/+3 EIS in probe Fe + 2/+3 Qbare = 1567.8 & Qmodified = 434.7 цС cm-2 Qbare = 1567.8 & Qmodified = 146.5 цС cm-2 ip = 36.0 & ip = 10.9 цА Rct = 153.9 & Rct = 6932.1 X % в = 100 [1 - (Qmodified /Qbare)] % в = [(3 X Qmodified in NaOH)/(Qbare,in H2SO4/2)] % в = [1 — (ip modified/ip bare)] x 100 % в = [1 — (Rct bare/Rct modified)] • 100 52.0 56.1 69.7 97.8
h The electrode surface coverage. Q: The charge density of surface.
I: The anode or cathode peak current of redox probe in cyclic voltammetry. Rct: The charge transfer resistant in electrochemical impedance spectroscopy.
decrease in the area (Q) associated with gold oxide reduction at 910 mV (Sabatani and Rubinstein, 1987). The results are summarized in Table 1.
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 Auin NaOH is 56.1%.
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 (DEp) 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
E/\ vs.Ag/AgCI
Figure 2 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 mVs-1.
electrode (curve a) in comparison with TMBH electrode (curve b), which the surface coverage (%h) was calculated to be 50.0%. The DEp 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 p-p 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 h using Eq. (1) (Shervedani et al. 2007):
%h =[1 -Opmodified/Cbare)] X 100
Figure 1 Successive cyclic voltammograms obtained for desorption of TMBH from Au-TMBH SAM electrode in 0.5 M NaOH
where ip bare and ip modified are peak currents of redox probe at bare Au and Au-TMBH SAM electrodes under the same conditions. The values obtained for ip and ip were 19.7 and 39.4 iA at pH 3.0, 10.9 and 36.0 iA at pH 6.0 and 4.9 and 39.5 iA at pH 8.0, respectively. Thus, values of 69.7 and 87.6 were estimated for h at pH 6.0 and 8.0. These behaviors were followed more precisely by the EIS studies as follows.
solution. Scan rate: 100 mV s . A = 0.0314 cm2
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 Rct 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 h 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), p-p 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, p-p, 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., haVerage of (cv in H2SO4X (cv in Fe + 2/+3) and (EIS in Fe + 2/+3) - h (cv in NaOH)) can give an insight about the contributions of other factors (e.g., hydrogen bonding, p-p, 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 h.
3.1.5. Surface titration and evaluation of the surface pKa
For determination of surface pKa 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 Rct of the modified electrode was pH dependent. The pH-metric titration curve was constructed using Rct as a function of solution pH (inset of Figure 3A). A surface pKa 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.
Also, the surface pKa 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 ip and DEp 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 pKa. The results support the changes, and thus, modification took place on the Au electrode, i.e. self assembled monolayer of TMBH.
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
Figure 3 (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.
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 p-p 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. 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):
Epa — E
(0.0591 /n) log[(Qx)a/(R)b]-(0.0591m/n)pH (2)
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 (Epa) was shifted to the negative direction by increasing the solution pH. A good linear relation was found between the Epa of Epi and the solution pH in the range of 2.0-8.0 by Epa(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. Study of scan rate
The influence of scan rate (v) on the electrochemical behavior of Epi (20 iM) on the TMBH -modified electrode was investigated. According to Eq (3) for a totally irreversible diffusion controlled process (Bard and Faulkner, 1991):
Ip = 3.01 x 105n[(1 — a)na ]1/2AD1/2Cm1/2
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:
Ipa = 1.7336v1/2 - 0.0328, R2 = 0.9918
And considering (1 - a)na = 0.46 (see below), D = 3.5 x 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 nw 2.0.
Figure 4 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 iM Epi at a sweep rate of 25 mV s_1; (A) Tafel plot derived from linear sweep voltammograms.
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. t1/2 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) x 10-6 cm2 s-1.
The rate constant for the chemical reaction between Epi and the redox sites in Au-TMBH SAM electrode, kh, can be evaluated using chronoamperometry according to the method described by Galus (Galus, 1976):
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 Ep vs. log I using Eq. (4) (Bard and Faulkner, 1991):
Ep — 2.3RT/naaF(log io ) — 2.3RT/naaF(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 (a) is about 0.54. Also, the ionic exchanging current density (io) by average of Tafel intercepts for Epi was found to be 0.14 iA cm~2.
Figure 5 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 iM of Epi. (A) Plots of Q vs. Г1/2 obtained from chronocoulograms a-d. (B) Plot of the slope of the straight lines against Epi concentration.
■1.5 -I-1-1-1-1-1-1—
0.0 10.0 20 0 30.0 40.0 50.0 60.0
Figure 6 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 iM, 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.
Ic/Il = y1/2[p1/2erf(y1/2 ) + (exp(—y)/y1/2)] (5)
where IC is the catalytic current of Au-TMBH SAM electrode in the presence of Epi, IL is the limited current in the absence of Epi, and y = khCbt (Cb is the bulk concentration of Epi) is the argument of error function. In the cases where y exceeds 2, the error function is almost equal to 1.0 and the above equation can be reduced to:
Ic/Il = P1/2y1/2 = P1/2(khCbt)1/2 (6)
where kh and t are the catalytic rate constant (mol-1 L s—*) and time elapsed (s), respectively. The above equation can be used to calculate the rate constant of the catalytic process, kh. Having measured the catalytic current, i.e., Ic, it is possible to carry out the electrode process under identical conditions, but in the absence of Epi, in order to determine IL. From the slope of IC/IL vs. t1/2 plot, the value of kh can be simply calculated for a given concentration of the substrate. The calculated value of kh is (2.04 ± 0.03) x 102mol—1 L s—1 using the slope of IC/IL — t1/2 plot. This value of kh 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. 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 lA L imol-1 in the concentration ranges of 3.3-23.3 iM and 23.3-100.0 iM, respectively. The decrease in sensitivity (slope) of the second linear segment is likely due to kinetic limitation. The detection limit (3r) of Epi was found to be 0.29 ± 0.01 iM. 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 iM 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 iM 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 iM 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 iL 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 electrooxi-dation 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 pM) and high (24.9-91.7 iM) concentration ranges, respectively:
Ipa (iA) = (0.0155 ± 0.001) [epinephrine] (iM) +
(0.2400 ± 0.004) R2 = 0.9944
Ipa (iA) = (0.0088 ± 0.003) [epinephrine] (iM) +
(0.4481 ± 0.002) R2 = 0.9850
The detection limit (3r) of Epi by this method was found to be 0.19 ± 0.003 iM. 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.
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 x 10—5 M Epi is 0.32 iA and after addition of AA up to 1.4 x 10—4M, 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).
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 Detection Working concentration Reference
shift (mV) limit (M) range (M)
Au 2-(2,3-Dihydroxy phenyl)-1, 155 5.1 x 10-7 7.0 x 10-7 5.0 x 10~4 Mazloum-Ardakani
3-dithiane et al. (2011)
Au Homocysteine - 1.0 x 10-7 5.0 x 10~5 8.0 x 10~4 Zhang et al. (2002)
Au Triazole - 1.0 x 10~8 1.0 x 10-7 6.0 x 10~4 Sun et al. (2006)
Au L-Cysteine - 1.0 x 10~8 1.0 x 10-7 2.0 x 10~6 Wang et al. (2002)
Au TMBH 28 1.9 x 10-7 3.3 x 10~6_ 1.0 x 10~4 Proposed method
M: as mole/liter.
Figure 7 DPV curves of (a) 0.1 M PBS at pH 6.0, (b) 1.2 x 10~4M AA, (c) 2.0:
: 10~5 M Epi and (d) 2.0 x 10~5 M Epi
-1.2 x 10 M AA on Au TMBH SAM-modified electrode.
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 (3r) of Epi by this method was found to be 0.19 ± 0.003 iM. 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 x 10 4M of AA. High sensitivity, selectivity and reproducibility of the voltammetric responses, and low detection limit (0.29 ± 0.01 iM), together with the ease of preparation, make the proposed modified electrode very useful for accurate determination of Epi in real samples.
Table 3 Application of the Au-TMBH SAM electrode for the determination of Epi in spiked human blood serum.
Sample Added (iM) Found (iM) Recovery (%)
Serum 0.00 Not detected -
1.96 2.01 102.55
1.92 1.87 97.39
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.
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
Acknowledgments
The authors are grateful to the University of kashan for supporting this work by Grant No. 159195-11.
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