IMPEDIMETRIC Au-NTA SENSOR FOR LEAD
DETERMINATION
Marina Palcic1, Irena Kerkovic2, S. Milardovic2 and Zorana Grabaric1
1
University of Zagreb, Faculty of Food Technology and Biotechnology,
Pierottijeva 6, 10 000 Zagreb, Croatia
2
University of Zagreb, Faculty of Chemical Engineering and Technology,
Marulicev trg 19, 10 000 Zagreb, Croatia
Summary
A self assembled monolayer (SAM) of cysteamine was prepared on the
surface of gold disc electrode and further modified with nitrilotriacetic
acid (NTA). Sensor’s surface characterization was performed by cyclic
voltammetry (CV) and electrochemical impedance spectroscopy (EIS) in
the presence of potassium hexacyanoferrate (II)/(III) redox pair.
Complexation of NTA with Pb2+ was tested by EIS measurements using
the same redox pair as in the surface characterization. Electrode response
(log ∆Rct) was linearly proportional to log c(Pb2+) with correlation
coefficient R2 = 0.9865 for measurements in buffer pH = 4.60 and R2 =
0.9931 for pH = 7.06 in the concentration range of 0.06-66 µmol/L.
Keywords: impedimetric sensor, lead, NTA, self assembled monolayers
1
Introduction
Thiol monolayers are frequently used for modification of metal electrodes
(gold, silver, copper and platinum) as stable anchor for surface
functionalization. Cysteamine monolayer attached to surface of metal
electrode makes a platform containing large number of free surface amino
groups convenient for further modifications in order to enlarge the
number of other functional groups. Using metal coordination chemistry,
–COOH, –OH-, –PO43-,–NH2 terminal groups were so far used to attach
large number of different metal ions (Cu2+, Ca2+, Ni2+, Pb2+, Zr4+) as
reported in the review by Cao et al. (2009). SAMs can be applied for
sensor preparation for detection of biologically important molecules such
as glucose, dopamine, proteins, cholesterol, antibodies and antigens and
DNA (Davis et al., 2005). SAM’s terminated with nitrilotriacetic acid
complexed with Me2+ are used for selective immobilization of His-tagged
proteins (Sigal et al., 1996), too.
The main purpose of this work was to investigate and characterize gold
electrode modified with cysteamine self-assembled monolayer (Au/CA)
functionalized with nitrilotriacetic acid (Au/CA/NTA) and the possibility
of application in lead determination.
Materials and Methods
Materials and reagents
All chemicals used were of analytical grade and all solutions were
prepared with water from Millipore-MilliQ system (USA).
Cysteamine (HSCH2CH2NH2, CA), Fluka, was used for building SAM
platform. Other reagents used for electrode surface modification, such as
1-ethyl-3(3-(dimethylamino)propyl)carboimide (EDC), nitrilotriacetic
acid (NTA), N-hydroxysuccinimide (NHS) were from Sigma-Aldrich.
Reagents used for preparation of 0.1 M acetic, phosphate and boric buffer
solutions were obtained from Kemika. Standard solution of lead was
prepared from lead nitrate (Merck). Sodium perchlorate as supporting
electrolyte, potassium hexacyanoferrate(II) trihidrate and potassium hexacyanoferrate(III) used as redox probe, were obtained from Merck, too.
2
Instrumentation
Potentiostat 264 A (Princeton Applied Research, USA) connected to a
computer via EG&G-PowerSuite software for data collecting and analysis
was used for CV measurements. Lock-in amplifier 5210 (PAR, USA) was
connected to Potentiostat 264 A for EIS measurements.
Cyclic voltammetry and electrochemical impedance spectroscopy
Both CV and EIS were used for electrode surface characterization,
whereas EIS alone for lead determination. All electrochemical
measurements were carried out in a three-electrode cell. Gold disc
electrode was used as working electrode, Hg|Hg2Cl2| KCl saturated (SCE)
electrode and platinum wire as reference and counter electrodes,
respectively. The applied potential scan rate in cyclic voltammetry was 50
mV s-1. The frequency range for impedance measurements was between
100 mHz and 100 kHz. Small sinusoidal AC signal of 5 mV was applied
to potential of +200 mV vs. SCE. Equivalent circuit parameters were
calculated by fitting the EIS data using appropriate circuit by ZsimpWin
software provided by EG&G. All solutions were deaerated with nitrogen
gas for 10 minutes before measurements and all measurements were
performed at room temperature.
Electrode preparation
Prior to modification of gold disc electrode (1 mm in diameter) with
thiols, surface was chemically cleaned by soaking for 3 minutes in
„piranha“ solution (1:3 (by volume) mixture of 30 % H2O2 and 98 %
H2SO4). Mirror like surface was then obtained by polishing electrode on a
flat pad with SiC powder of different mesh (240, 800 and 1200) and
finally by Al2O3 powder with particle size of 1 and 0.25 µm. After each
polishing, adsorbed particles were removed by immersing electrode in
water and ethanol solutions using ultrasonic bath. Final step in surface
preparation was electrochemical cleaning performed by cycling the
electrode potential in 0.1 M HClO4 at 50 mV/s from 0 to +1.5 V vs. SCE,
until the reproducible voltammograms were obtained.
Electrode surface modification is summarised in Fig.1. First step was
formation of stable SAM containing free amino groups by adsorption of
cysteamine (18 mM ethanol solution) for 24 h in dark at room
temperature. For further modification of Au/CA electrode the solution of
0.002 M EDC, 0.005 M NHS and 0.01 M NTA prepared in phosphate
3
buffer pH = 5 was kept in dark for 3 h in order to activate NTA carboxyl
groups. To obtain Au/CA/NTA modified surface electrode was immersed
in this solution for 24 h in dark at room temperature.
Determination of lead
Lead binding capacity was studied by EIS. After 10 minute of Pb2+
accumulation from standard solutions (c = 0.01 to 66 µM) the
concentration of lead was measured. Two sets of measurements were
performed in order to investigate the influence of pH. One set in acidic
media (acetic buffer pH = 4.60), and the other in neutral media (boric
buffer pH = 7.06). Both solutions contained 0.1 M NaClO4 as supporting
electrolyte and 1 mM K4[Fe(CN)6]/K3[Fe(CN)6] as redox probe.
Results and Discussion
Characterization of Au/CA/NTA electrode
Formation of self-assembled monolayer creates additional barrier on the
electrode, generally leading to a decrease in both, peak current (Ip) and
apparent rate constant, while peak-to-peak separation (∆Ep) increases.
However pH changes in measurement conditions can influence SAM’s
apparent behaviour (Sabatini et al., 1987). Introducing negatively charged
redox probe into the measurement system enables detection of changes in
surface charge.
Au/CA monolayer containing neutrally charged amino groups at pH = 9
caused decrease in Ip more evident in cathodic part of a voltammogram
and increase in ∆Ep of approximately 10 mV (Fig. 2a). The same surface
layer is highly protonated in acetic media at pH = 3, enabling electrostatic
attraction to negatively charged redox couple facilitating approach of
redox probe to electrode surface that is evident in slight Ip increase and
∆Ep diminishing (Fig. 2b).
Further modification of the surface with NTA and formation of
Au/CA/NTA SAM led to another decrease in Ip and broadening of ∆Ep
which is apparent in both acidic and alkaline media. These changes are
more evident in alkaline media on account of NTA carboxyl groups being
highly deprotonated at pH = 9, causing prevalence of negative charge at
electrode surface inhibiting approach of the negative redox couple.
4
When studying influence of electrode surface modification on kinetics of
electrochemical reaction, EIS performed in frequency range between 100
mHz and 100 kHz is the method of choice, mainly because it is a noninvasive technique (Shervedani et al., 2006). Measured data were
processed according to altered Randles’ model shown in Fig.3., where
double layer capacitance is replaced by constant phase element (CPE).
Parameter values of the model are shown in Table 1. with respect to unit
electrode surface (1 cm2).
Electrochemical process under study can be influenced by two major
effects, thickening of barrier between redox probe and electrode and
facilitation or blocking approach of redox pair to electrode surface trough
electrostatic interactions. Later effect is dominant in investigated system,
because cysteamine SAM covered with NTA monolayer isn’t thick
enough to act as insulating barrier. Nyquist plots obtained by EIS
measurements were used to characterize effects that SAMs formed on top
of gold electrode have on electrochemical reaction in question.
Formation of CA monolayer that has neutrally charged terminal amino
groups at pH = 9 caused increase in Rct as shown in Nyquist plot in
Fig.4a., whereas the same surface layer is highly protonated in acetic
media at pH = 3 (Fig.4b.), enabling electrostatic attraction to negatively
charged redox couple significantly facilitating approach of redox probe to
electrode surface that is in agreement to Rct decrease.
Further modification of surface with NTA and formation of Au/CA/NTA
SAM led to another increase in Rct in both acidic and alkaline media.
These changes are more evident in alkaline media on account of NTA
carboxyl groups being deprotonated at pH = 9, causing prevalence of
negative charge at electrode surface to significantly inhibit approach of
the negative redox couple.
In addition to presented results, decrease in double layer capacitance,
which is dependent on gold electrode surface coverage, indicates
formation of stabile film on top of electroactive metal surface. Increase of
CPE parameter (n) also confirms layer formation causing thus increase in
surface homogeneity.
Determination of lead
NTA demonstrates high binding capacity towards metal ions from
solutions because of coordination ability of three carboxyl groups and
nitrogen to metal ions. When NTA is attached to cysteamine one carboxyl
group is engaged, thus the binding of metal is somewhat weaker. The
5
binding of metal is influenced by pH of sample solution used for lead
preconcentration, too. In alkaline media the electrode surface charge
becomes more negative, but at the same time the precipitation of lead as
Pb(OH)2 occures. That is why Pb2+ accumulation was performed in the
solution pH=7.
In order to determine appropriate conditions of EIS measurements,
experiments were performed in two different media, acidic (Fig.5a) and
neutral (Fig.5b). Comparing the two Nyquist plots, change of Rct
contributed to Pb2+ attachment on the top of electrode surface is visible in
both cases. When immersed in neutral boric buffer solution NTA is
deprotonated, causing electrostatic rejection of redox probe. As surface
concentration of Pb2+ increases with each accumulation step, Rct decreases
due to the decrease of negative surface charge.
When the same process is measured in acidic media, unexpected increase
in Rct occurs as surface concentration of Pb2+ increases with each
accumulation step. One of the possible explanation relies on the fact that
at pH = 4.60 NTA surface becomes partially protonated and some of Pb2+
is released in the solution in the vicinity of the electrode surface thus
reacting with OH- and forming Pb(OH)3- that would correlate to Rct
increase, since EIS technique is a type of static measurement.
Linear relation between c(Pb2+)/M in sample, ranging from 60 nM to 66
µM, and measured ΔRct/kΩ is described with Eq.1. with
R2 = 0.9865.
log ΔRct/kΩ = (1.30 ± 0.06) + (0.15 ± 0.01) ∙ log c(Pb2+)/M
(Eq.1.)
The same linear relation described with Eq.2. for c(Pb2+)/M in sample,
ranging from 60 nM to 66 µM, was confirmed for measurements in boric
buffer with R2 = 0.9931.
log ΔRct/kΩ = (2.24 ± 0.01) + (0.15 ± 0.03) ∙ log c(Pb2+)/M
(Eq.2.)
Conclusions
The self assembled layer of cysteamine on gold disc electrode, further
functionalised by NTA, was tested for determination of lead. The binding
of Pb2+ onto the modified electrode was successfully done for a wide
range of tested concentrations using EIS in two different media.
Electrode response (log ∆Rct/kΩ) was linearly proportional to log
c(Pb2+)/M with correlation coefficient R2 = 0.9865 for measurements in
6
acetic buffer pH = 4.6 and R2 = 0.9931 for measurements in boric buffer
pH = 7.06 tested on water solutions with lead content from 0.06 to 66 μM.
Further optimization regarding accumulation time, preconcentration
conditions (buffer composition and pH) and interferences with other
metal ions is required in order to use proposed modified electrode for
detection of lead in various samples.
References
Cao, R. Jr., Diaz-Garcia, A.M., Cao, R. (2009): Coordination compounds
built on metal surfaces, Coordin Chem Rev 253, 1262–1275.
Davis, F., Higson, S.P.J. (2005): Structured thin films as functional
components within biosensors, Biosens Bioelectron 21, 1–20.
Sabatani, E., Rubinstein, I., Maoz, R., Sagiv, J. (1987): Organized selfassembling monolayers on electrodes. 1. Octadecyl derivatives on gold, J.
Electroanal. Chem. 219, 365-371.
Sigal, G.B., Bamdad, C., Barberis, A., Strominger, J., Whitesides, G.M.,
(1996): A self-assembled monolayer for the binding and study of
histidine-tagged proteins by surface plasmon resonance, Anal. Chem. 68,
490-497.
Shervedani, R.K., Mozaffari, S.A. (2006): Impedimetric sensing based on
phosphate functionalised cysteamine self-assembled monolayers, Anal.
Chim. Acta. 562 (2006) 223-228.
7
Fig.1. NTA coupling to Au/CA surface.
8
a)
Au
Au/CA
Au/CA/NTA
3
2
I / A
1
0
-1
-2
-3
-400
-200
0
200
400
600
400
600
E / mV
b)
Au
Au/CA
Au/CA/NTA
3
2
I / A
1
0
-1
-2
-3
-400
-200
0
200
E / mV
Fig.2. Cyclic voltammograms of Au, Au/CA and Au/CA/NTA electrodes
in buffered solution containing 0.1 M NaClO4 and 1 mM
K4[Fe(CN)6]/K3[Fe(CN)6];
a) 0.1 M phosphate buffer pH=9
b) 0.1 M acetic buffer pH=3
9
Fig.3. Randles’ model equivalent circuit, where double layer capacitance
is replaced by CPE.
10
25
a)
Au
Au/CA
Au/CA/NTA
20
Zim / k
Zim / k
15
10
5
5
0
0
5
10
15
-Zre / k
0
0
10
20
30
20
40
50
-Zre / k
20
b)
Au
Au/CA
Au/CA/NTA
10
Zim / k
Zim / k
15
4
2
5
0
0
1
2
0
0
5
10
15
20
3 4 5
-Zre / k
25
6
7
30
-Zre / k
Fig.4. Nyquist plots of Au (solid line), Au/CA (dashed line) and
Au/CA/NTA electrodes before (dotted line). Symbols represent measured
values, whereas lines are values obtained by modeling. Measurements are
performed in buffered solutions containing 0.1 M NaClO4 and 1 mM
K4[Fe(CN)6]/K3[Fe(CN)6];
a) 0.1 M phosphate buffer pH=9
11
b) 0.1 M acetic buffer pH=3
a)
0.01 - 66.7 M Pb
2+
Au/CA/NTA
20
4
-Zim /
k
-Zim /
k
30
10
2
0
0
5
10
Zre / k
0
0
10
20
30
40
50
Zre / k
b)
2+
0.05 - 16.7 M Pb
80
-Zim /
k
60
Au/CA/NTA
40
20
0
0
50
100
150
200
250
Zre / k
Fig.5. Nyquist plots of Au/CA/NTA electrodes before (dashed line) and
after 10 minutes of lead accumulation (solid lines). Dots represent
measured values, whereas lines are values obtained by modeling.
Measurements are performed in buffered solutions containing 0.1 M
NaClO4 and 1 mM K4[Fe(CN)6]/K3[Fe(CN)6];
a) 0.1 M acetic buffer pH=4.6
b) 0.1 M boric buffer pH=7
12
Table 1. Parameters of Randles’ model based on EIS measurements of bare gold electrode (Au), gold electrode after
formation of CA SAM (Au/CA) and after formation of NTA SAM (Au/CA/NTA) taken in alkaline and acidic media.
Parameters of the model are solution resistance (Rs), charge transfer resistance (Rct), Warburg diffusion parameter
(W), double layer capacitance defined trough constant phase element (CPE) and CPE parameter (n). χ2 represents
error in fitting model to measured EIS data.
pH
electrode
n
χ2
Rs / k
Rct / k
Cdl / F
W / ms0.5
9
3
Au
0.18 ± 0.01
1.64 ± 0.02
1.33 ± 0.07
0.85 ± 0.01
0.05 ± 0.01 2.45∙10-4
Au/CA
0.19 ± 0.01
3.46 ± 0.02
0.61 ± 0.07
0.90 ± 0.01
0.05 ± 0.01 6.56∙10-4
Au/CA/NTA
0.17 ± 0.02
11.02 ± 0.02
0.23 ± 0.05
0.89 ± 0.01
0.04 ± 0.02 1.14∙10-4
Au
0.35 ± 0.01
3.88 ± 0.03
1.95 ± 0.06
0.87 ± 0.01
0.07 ± 0.01 3.65∙10-4
Au/CA
0.35 ± 0.01
0.79 ± 0.01
0.56 ± 0.09
0.76 ± 0.02
0.04 ± 0.02 7.89∙10-4
Au/CA/NTA
0.34 ± 0.01
1.79 ± 0.04
0.24 ± 0.10
0.89 ± 0.01
0.04 ± 0.01 7.68∙10-4
13