2006 The Japan Society for Analytical Chemistry
Voltammetric Behavior and Determination of 17b-Estradiol at Multi-Wall Carbon Nanotube-Nafion Modified Glassy Carbon Electrode Norifumi TERUI, Bunshi FUGETSU, and Shunitz TANAKA† Section of Integrated Environmental Science, Research Faculty of Environmental Earth Science, Hokkaido University, N10W5, Kita-ku, Sapporo 060–0810, Japan
For the purpose of low cost and sensitive electrochemical detection of 17β-estradiol (E2), a multi-wall carbon nanotube(MWNT)-Nafion modified electrode was fabricated. The MWNT modified electrode shows enhancement for the anodicpeak current of E2 compared with the value obtained using a bare electrode. The anodic peak current measured by squarewave voltammetry after 5 min open-circuit accumulation was proportional to the concentration of E2 over the range of2.5 × 10–7 to 10 × 10–6 M, and a detection limit of 1 × 10–8 M was obtained. (Received December 21, 2005; Accepted March 16, 2006)
copolymer possessing several advantages, such as ion-exchange
characteristics, thermal stability, chemical inertness andmechanical strength, and has been widely used for the MWNT
17β-estradiol (E2) is the most potent steroidal estrogen; it is
modification of electrodes.21 MWNT was dispersed into Nafion
produced endogenously by all mammalian species. Recently
solution via ultrasonication; then the resulting homogeneous
there have been many studies suggesting that estrogens are
dispersion of MWNT was dropped onto a glassy carbon (GC)
being released to aqueous environments such as sewage and
electrode. The MWNT-Nafion modified GC electrode was
river water from wastewater treatment plants. These materials
obtained by evaporating the solvent of the MWNT dispersion.
are suspected of being endocrine-disrupting chemicals that can
The MWNT-Nafion modified GC electrode exhibits
cause hurtful physiological effects in aquatic wildlife.1,2
enhancement for the oxidation peak currents of E2 compared
However, estradiols in environmental sources are present at
with the results obtained with a bare GC electrode, and shows
very low concentrations, and are difficult to detect. In order to
significantly improved sensitivity for the determination of
determine estradiols, various technologies such as GC-MS,3–5
HPLC-MS,6–8 immunoassay,9,10 and electroanalysis11–13 havebeen used.
Carbon nanotubes are novel nanometer-sized materials with
unique properties; these nanotubes have attracted muchattention.14,16
Generally, carbon nanotubes consist of two
categories, single-wall carbon nanotubes and multiwall carbon
MWNT (inner diameter: 5 – 10 nm, outer diameter: 20 – 40
nanotubes (MWNT), and have high electrical conductivity,
nm, length: 0.5 – 50 μm) and 5% (w/w) Nafion solution were
extremely high mechanical strength and relatively high surface
obtained from Aldrich (USA). The standard solution of E2 (1 ×
area. To utilize these unique properties for electrochemical
10–2 M) was prepared by dissolving E2 (Sigma, USA) in
detections, researches have reported carbon nanotube paste
ethanol. All chemicals used were of analytical glade and were
electrodes fabricated by mixing carbon nanotubes with mineral
directly used without further purification. Solutions were
oil,14 and carbon nanotube coated electrodes fabricated by
prepared with deionized water obtained from Milli-Q system
dispersing carbon nanotubes into suitable solvents such as
As for estradiols, Hu et al. fabricated
MWNT modified electrodes based on dispersing MWNTs into
All the electrochemical measurements were carried out with a
water containing oil of turpentine and OP TX-100; they
CV-50W voltammetric analyzer (Bioanalytical Systems, Inc.
reported a high catalytic effect on the oxidation of estradiols
(BAS), USA). The working electrode is a glassy carbon
electrode (3.0 mm diameter, BAS). A three-electrode system
In this research, we fabricated the MWNT-Nafion modified
including the working electrode, a platinum wire counter
electrode and demonstrated direct electrochemical
electrode and an Ag/AgCl reference electrode (BAS) was
measurements by square wave voltammetry with the aim of
employed. All potentials were reported versus Ag/AgCl. The
proposing a simple, low cost and sensitive method for the
pH of buffer solutions was measured using a pH meter, F-22
determination of E2. Nafion is a sulfonated tetrafluoroethylene
† To whom correspondence should be addressed. Preparation of MWNT-Nafion modified GC electrode
The MWNTs were pretreated by refluxing for 10 h in
Square wave voltammograms of 1 × 10–5 M E2 in 0.1 M
Influence of the amount of 1 mg mL–1 MWNT dispersion
phosphate buffer of pH 7.0. The amount of MWNT is 7.5 μL. (a) At
solution on the oxidation peak current of 1 × 10–5 M E2 in 0.1 M
bare GCE without accumulation, (b) at bare GCE after 5 min, (c) at
phosphate buffer of pH 7.0 after 5 min.
Nafion-modified GCE after 5 min, (d) at MWNT-modified GCEwithout accumulation, (e) at MWNT-modified GCE after 5 min.
voltammetry of 1 × 10–5 M E2 at pH 7.0. At a bare GCelectrode, no peak was observed without accumulation (curve ain Fig. 1). After 5 min of accumulation, only an anodic peakcorresponding to the oxidation of E2 at 0.56 V was observed(curve b in Fig. 1). At a Nafion modified GC electrode, no peakwas observed after 5 min of accumulation (curve c in Fig. 1). Incontrast, a distinct anodic peak was observed at MWNT-Nafionmodified GC electrode without accumulation (curve d in Fig. 1), and increased significantly after 5 min of accumulation(curve e in Fig. 1). MWNT has larger specific surface area dueto nanometer-sized structure and a strong adsorptive ability foraromatic hydrocarbons.14,15
the oxidation peak current of E2 is observed at the MWNT-
Influence of pH on the oxidation peak potential and peak
Nafion modified GC electrode. No corresponding reduction
current of 1 × 10–5 M E2 in 0.1 M phosphate buffer at MWNT-
peak was observed on the reverse sweep of cyclic voltammetry.
modified GCE after 5 min. The amount of MWNT is 7.5 μL.
Sadic et al. proposed that the oxidation of E2 consisted of twoelectron transfer steps followed by a chemical reaction (EC-ECreaction mechanism) and finally E2 became a non-electroactive
concentrated HNO3. The 0.1% (w/w) Nafion solution was
prepared by dissolving 5% (w/w) Nafion (Aldrich, USA) into
corresponding reduction peak suggests that the electrode
deionized water. Next, 1 mg of treated MWNT was added to 1
reaction of E2 is an irreversible process due to the accumulation
mL 0.1% (w/w) Nafion solution, then sonicated for several
hours until a homogeneous black dispersion of MWNT wasachieved. Prior to modifications, the GC electrode was polished
with 0.3 and 0.05 μm alumina and cleaned ultrasonically for 1
The effect of the solution pH on the anodic peak current
min. After that, 0 – 20 μL of 1 mg mL–1 MWNT dispersion was
obtained for 1 × 10–5 M of E2 at the MWNT-Nafion modified
pipetted onto the surface of a clean GC electrode, and the
GC electrode after 5 min accumulation was investigated by
solvent was evaporated in a dry oven.
square wave voltammetry. As shown in Fig. 2, no significantpeak current difference was obtained in the range of pH 2.0 –
8.0, and the peak current decreased at pH 10.0. On the other
The MWNT-Nafion modified GC electrode was immersed
hand, a peak potential shift in the positive direction with
into 5 mL of 0.1 M phosphate buffer containing a known
decreasing pH was observed in the pH range of 2.0 to 10.0. The
concentration of E2 in an electrochemical cell. Accumulation
slope of the plots of the peak potential against pH appeared to
was carried out under open-circuit potential while stirring the
be about 60 mV per pH unit. This suggests that equal amounts
solution; the solution was then kept quiet for 10 s, and finally
of proton and electron are involved in the oxidation of E2.11 In
voltammograms were measured. Cyclic voltammograms were
this research, pH 7.0 was selected as the optimal pH.
recorded from 200 to 800 mV at scan rate: 50 mV s–1. Squarewave voltammograms were recorded from 300 to 800 mV at
pulse amplitude: 50 mV, and scan rate: 20 mV s–1.
The effect of the amount of MWNT on the anodic peak
current of 1 × 10–5 M E2 for 5 min accumulation was evaluated. The plots of anodic peak current versus amount of 1 mg mL–1
Results and Discussion
MWNT dispersion solution are shown in Fig. 3. As the amountof MWNT was increased, the oxidation peak current enhanced
greatly to the peak at 7.5 μL. This enhancement of the current
To investigate the electrochemical behavior of E2 at the
indicates that the specific surface area and number of catalytic
MWNT-Nafion modified GC electrode, we used square wave
sites for the oxidation of E2 at the MWNT-modified electrode
Influence of the accumulation time on the oxidation peak
current of 1 × 10–5 M E2 in 0.1 M phosphate buffer of pH 7.0. Theamount of MWNT is 7.5 μL.
Square wave voltammograms of E2 at different
concentrations in 0.1 M phosphate buffer at MWNT-modified GCEafter 5 min. The amount of MWNT is 7.5 μL. (a) 10 × 10–6, (b) 7.5
× 10–6, (c) 5.0 × 10–6, (d) 2.5 × 10–6, (e) 1 × 10–6, (f) 0.5 × 10–6 M.
are increased with an increase for MWNT. When the amount ofMWNT dispersion solution was over 7.5 μL, the oxidation peakcurrent decreased slightly. This indicates that the excess ofMWNT blocks the mass transfer and electron transfer of E2. In
This suggests that the MWNT-Nafion modified electrode
this research, the amount of MWNT dispersion solution was
presented here will be quite useful for the detection of E2. In
future, the electroanalytical methods using the MWNT-Nafionmodified electrode combined with special separation techniques
can be effectively employed for the detection of E2, estradiols
The effect of the accumulation time on the peak current was
and other harmful substance present at very low concentrations
investigated, and the result is shown in Fig. 4. The anodic peak
current increased as the accumulation time became longerwithin a range of 0 – 5 min. When the accumulation time waslonger than 5 min, the increase of anodic peak current was very
slow. This indicates that the MWNT-Nafion modified GCelectrode surface is almost saturated with E2 after the
The work was supported by 21st-Century Center of Excellence
accumulation for 5 min. In this research, the accumulation time
Program (E-01) funded by Ministry of Education, Culture,
The square wave voltammograms of E2 at different
concentrations are shown in Fig. 5. The anodic peak currentwas enhanced linearly as the concentration of E2 increased
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