PAPER www.rsc.

org/analyst | The Analyst

Electrochemical impedance spectroscopy and surface plasmon resonance

studies of DNA hybridization on gold/SiOx interfaces†
Maël Manesse,a Valerie Stambouli,b Rabah Boukherroubc and Sabine Szunerits*a,c
Received 20th March 2008, Accepted 9th May 2008
First published as an Advance Article on the web 1st July 2008
DOI: 10.1039/b804825h

The use of Au/SiOx interfaces for the investigation of DNA hybridization using electrochemical
impedance spectroscopy (EIS) and surface plasmon resonance (SPR) simultaneously is
demonstrated. Standard glass chemistry was used to link single-stranded DNA (ss-DNA) on
aldehyde-terminated Au/SiOx interfaces. The layer thickness and amount of grafted
oligonucleotides (ODNs) were calculated from SPR on the basis of a multilayer system of
glass/Ti/Au/SiOx /grafted molecule. Capacitance and resistance values of the modified interface
before and after hybridization were calculated from EIS data using an equivalent circuit and
allowed the affinity rate constant, K A = 4.07 × 105 M−1 , to be determined. The EIS results were
comparable to those obtained by SPR hybridization kinetics recorded in parallel.

1. Introduction interfaces (e.g. silicon/silicon dioxide).21,23–26 The advantage of

The use of surface-based analytical platforms for the rapid,
sensitive, selective and label-free monitoring of molecular
recognition events has become increasingly popular and has
revolutionized molecular biology. The potential of surface
plasmon resonance (SPR) as a label-free detection scheme,
realized in the early 1980s,1 was able to sense immunoglobulin
antibodies by observing the changes in the critical angle when
the antibodies bound selectively to a gold film. Since then, SPR
has become a very important instrumentation for biological
and medical applications and has been used to study many
kinds of ligand–receptor interactions, including protein–ligand,
antibody–antigen, protein–carbohydrate, protein–DNA, DNA–
DNA and cell adhesion interactions.2–10
Electrochemical impedance spectroscopy (EIS) is, next to
SPR, a useful label-free detection tool for analyzing the
changes in interfacial properties of modified electrodes and
semiconductors induced by the binding of charged biomolecules
on the surface. EIS measures the response (current and its
phase) of an electrochemical system to an applied oscillating
potential as a function of frequency. The detection scheme
is very attractive as EIS can be performed on conductive
polymers,11,12 metallic,13–18 and semiconductor interfaces19–22 as
well as on heterostructures such as semiconductor/dielectric
a
Laboratoire d’Electrochimie et de Physicochimie des Matériaux et des
Interfaces (LEPMI), CNRS-INPG-UJF, 1130 rue de la piscine,
BP 75, 38402 St. Martin d’Hères Cedex, France.
E-mail: sabine.szunerits@enseeg.inpg.fr; Fax: +33 04 76 82 66 30;
Tel: +33 04 76 82 65 52
b
LMGP, Laboratoire des Matériaux et du Génie Physique, INP
Grenoble, Minatec, 3 parvis Louis Néel, BP 257, 38016 Grenoble
Cedex 1, France
c
Institut de Recherche Interdisciplinaire (IRI, USR-3078) and Institut
d’Electronique, de Microélectronique et de Nanotechnologie (IEMN,
CNRS-8520), Cité Scientifique, Avenue Poincaré – B.P. 60069, 59652
Villeneuve d’Ascq, France
† The HTML version of this article has been enhanced with colour
images.
EIS as compared to voltammetric methods is its non-invasive
nature.27 The application of a potential ramp in the case
of cyclic voltammetry can result in the irreversible oxidation
of the molecule linked to the surface. Thus EIS has been
successfully applied for DNA hybridization studies either by
studying the diffusion of redox probes in the buffer solution or
by recording the impedance changes of DNA layers before and
after hybridization.16,18,21,22,25,28–30 However, one problem often
encountered in EIS is the difficulty for the determination of
the uniqueness of the circuit model obtained from experimental
data. The combination with SPR measurements (SPR/EIS)
could help to overcome this hurdle. Indeed, the combination
of SPR with electrochemical investigations is straightforward as
the thin gold films used for the generation of surface plasmon
waves can be simultaneously used as working electrodes.10,31–33
The immobilization of biomolecules on Au substrates is
commonly achieved through the self-assembly of functional
alkanethiols. Even though Au–S bonds are strong, they are prone
to electrochemical and chemical reduction. Thus it becomes
necessary for coating the Au substrates with other materials
to reach a higher stability. A way to take advantage of SPR
spectroscopy for monitoring the course of surface reactions and
the coupling chemistry developed for glass is to coat the noble
metal with a thin layer of SiO2 . Even though silane chemistry
is not well controlled, the formation of organic layers through
covalent bonding leads to stable and reusable devices. Some
attempts on the fabrication of such SPR chips are reported in the
literature.34–36 We have recently reported on the fabrication and
characterization of stable thin films of amorphous silica (SiOx )
deposited on glass slides coated with a 5 nm adhesion layer of
titanium and 50 nm of gold, using the plasma-enhanced chem-
ical vapor deposition (PECVD) technique.37–39 The deposited
silica films (d = 6–60 nm) showed excellent stability in different
media. Silica layers with thicknesses up to 40 nm allowed a
surface plasmon effect while thicker films resulted in the loss of
the SPR properties. The electrochemical characteristics of the
Au/SiOx interface could be improved by decreasing the SiOx

This journal is © The Royal Society of Chemistry 2008 Analyst, 2008, 133, 1097–1103 | 1097
layer thickness to 6.4 nm. This interface showed an enhanced
steady-state current compared to the thicker films. Furthermore,
the Au/SiOx interfaces were successfully applied for polarization
modulation infrared reflection absorption spectroscopy (PM IR-
RAS) structural analysis of DMPC (1,2-dimyristoyl-sn-glycero-
3-phosphocholine) bilayers.40,41 The interest in SiOx coating is
motivated by the ease of surface modification with functional
silanes, being chemically and electrochemically superior in
stability than the Au/thiol system. The linking of DNA on SiOx
surfaces leads to highly stable interfaces, important for multiple
uses of these interfaces for hybridization studies.
In this paper we investigate the DNA hybridization reaction
on Au/SiOx interfaces by SPR and EIS measurements. While
SPR coupled to EIS has been carried out intensively to char-
acterize tethered lipid bilayers,31,32 its advantages for studying
DNA hybridization on SiOx has not been demonstrated. The
grafting of a 15-mer oligonucleotide on a Au/SiOx surface
was achieved using a standard procedure used often for glass
and includes following sequence: (i) reaction of the silicon
oxide layer with 3-aminomethoxysilane (APTMS) to produce an
amine termination, (ii) transformation of the amine to aldehyde
termination by chemical coupling with glutaraldehyde, and
(iii) coupling of amine-terminated oligonucleotides (ODNs) to
the reactive linker. Layer thicknesses and the amount of grafted
ODNs were calculated from SPR on the basis of a multilayer
system of glass/Ti/Au/SiOx /grafted molecule. Capacitance
and resistance values of the modified interface before and after
hybridization were calculated from EIS data using an equivalent
circuit and allowed determination of the affinity rate constant.
2. Experimental
2.1. Materials
Potassium chloride (KCl), potassium hexacyanoferrocyanide
[Fe(CN)6 4− ], potassium hexacyanoferricyanide [Fe(CN)6 3− ],
glutaraldehyde [GA, OHC(CH2 )3 CHO], sodium hydroxide
(NaOH) and phosphate buffered saline (PBS) were ob-
tained from Aldrich and used without further purification. 3-
Aminomethoxysilane (APTMS) was purchased from Gelest Inc.
(France). Saline Sodium Citrate (SSC) 2× was obtained from
Fluka.
The 15-mer pre-synthesized oligonucleotide probes were
purchased from Sigma Proligo and have the following sequence.
Probe: 5 -(NH2 )-(T)5 -GAT AAA CCC ACT CTA; complemen-
tary strand: 5 -CA TAG AGT GGG TTT ATC CCA, non-
complementary strand: 5 - CAT CTC ACT AAC GCG GTCA.
Stock solutions of 10 lM were prepared.
The hybridization buffer was a solution of NaCl (0.5 M),
phosphate buffer solution (0.01 M), ethylenediaminetetraacetic
acid (0.01 M, pH 5.5).
2.2. Preparation of gold/SiOx composite slides
Substrate electrodes were prepared by thermal deposition of
5 nm of titanium and 50 nm of gold onto cleaned glass slides
(76 × 26 × 1 mm, n = 1.58 at k = 633 nm CML, France). Prior
to silica film deposition, the gold samples were first degreased
in isopropanol and acetone in an ultrasound bath at room
temperature, rinsed copiously with Milli-Q water and dried
1098 | Analyst, 2008, 133, 1097–1103
under a stream of nitrogen. The gold slides were then heated in a
plasma chamber at 300 ◦ C at a pressure of 0.005 Torr for 1 h. SiOx
layers were synthesized using plasma-enhanced chemical vapor
deposition in a Plasmalab 800Plus system (Oxford Instruments,
UK). The growth conditions used were as follows: substrate
temperature: 300 ◦ C; gas mixture: SiH4 (3% in N2 ) and N2 O
(the gas flow was 260 sccm and 700 sccm for SiH4 and N2 O,
respectively); total pressure in the reactor: 1 Torr; power:
10 W at 13.56 MHz. Under these experimental conditions, the
deposition rate was 414 Å min−1 and the silica films display
a refractive index (n) of 1.48. We have adjusted the silica film
thicknesses by varying the deposition time. For electrochemical
measurements, only half of the gold-coated interface was coated
with silicon oxide to ensure electrical contact to the gold
interface.
2.3. Silanization of gold/SiOx interface
The silanization reaction was performed using an identical pro-
cess previously described.39 Briefly, the Au/SiOx interfaces were
first cleaned by UV/ozone to remove any organic contaminants
on the surface and to generate surface hydroxy groups. The
silanization was carried out in the liquid phase by reacting the
Au/SiOx interface with 3% APTMS in methanol–water (v/v 95 :
5) for 30 min under sonication. The interfaces were then washed
with methanol, water (two times), and methanol and annealed
for 20 min at 110 ◦ C.
2.4. DNA grafting and hybridization on the APTMS-modified
Au/SiOx interface
Amine-terminated oligonucleotides were linked to aldehyde-
terminated Au/SiOx interfaces, obtained by chemical reaction of
the APTMS-modified surface with 10% glutaraldehyde aqueous
solution for 90 min at room temperature. Fig. 1 shows a
schematic illustration of the surface modification steps. A 100 lL
droplet of 1 lM DNA/PBS (0.1 M) was deposited onto the
aldehyde-terminated surface and left incubating for 3 h at room
temperature. The probes were finally reduced for an hour and
stabilized using an aqueous solution of NaBH4 (0.1 M), which
transforms the resulting imine to amine bonding. The modified
Au/SiOx interfaces were coupled to the SPR prism and the EC-
SPR cell fixed.
Hybridization experiments were performed in the EC-SPR
cell. The SPR cell was filled with a solution of 50 lL of 10 mM
Fe(CN)6 4− + 10 mM Fe(CN)6 3− in hybridization buffer. 5 lL
of complementary DNA (final concentration in cell: 2 lM) was
added to the SPR cell and the hybridization event was monitored
using SPR and EIS measurements simultaneously for 180 min.
After this period, the hybridization was stopped and the cell
was washed with 10 mM Fe(CN)6 4− + 10 mM Fe(CN)6 3− in
hybridization buffer. The denaturation of hybridized DNA was
performed using NaOH (0.05 M) during 10 min followed with
rinsing with deionized water.
2.5. Instrumentation
Electrochemical SPR. Electrochemical Impedance Mea-
surements (EIS) were performed using a Solartron SI 1286
Electrochemical Interface with an SI 1250 Frequency Response
This journal is © The Royal Society of Chemistry 2008
Fig. 1 Schematic illustration of the procedure used to modify the Au/SiOx interface: (1) reaction with 3-aminomethoxysilane (APTMS) to produce
an amine termination, (2) transformation of the amine to aldehyde termination by exposure to 10% glutaraldehyde, (3) oligonucleotide immobilization,
(4) reduction of N=C groups.

Analyzer (Solartron Analytical, Farnborough, UK) connected
to an Autolab ESPRIT SPR Instrument (Eco Chimie, Utrecht,
The Netherlands). EIS experiments were carried out in an
aqueous solution of a mixture of 10 mM Fe(CN)6 4− + 10 mM
Fe(CN)6 3− in KCl (0.01M/PBS) using the following parameters:
amplitude of 10 mV at open circuit potential (OCP) with a
frequency range of 65 kHz to 0.1 Hz. Impedance data were
modeled using ZView2 software.
The electrode cell is the commercial single-channel cell of
the Autolab SPR instrument allowing simultaneous surface
plasmon resonance and EIS measurements to be performed. The
configuration of this equipment is described elsewhere.42,43 In
short, polarized laser light (k = 670 nm) is directed to the bottom
side of the sensor disk via a hemispheric lens placed on a prism
(N-BAF-3 having a refractive index of n = 1.58) and the reflected
light is detected using a photodiode. The angle of incidence is
varied using a vibrating mirror with a frequency of 44 Hz. SPR
curves were scanned on the forward and backward movement
of the mirror and the minima in reflectance determined and
averaged. Fitting of the experimental curves was performed
using Winspall 2.01 software. The instrument is equipped with
an electrochemical open cuvette system of 20–150 lL sample
volume where an Ag/AgCl reference electrode (RE), a platinum
counter electrode (CE) and a fixed contact point to the gold layer
of the sensor chip is incorporated. The active electrode surface
is 0.07 cm2 .
3. Results and discussion
3.1. Electrical characterization of the Au/SiOx sensing
interface
The silica films studied in this work were 9 ± 1 nm thick and
were deposited on thin gold film electrodes using chemical vapor
decomposition of a gas mixture of SiH4 and N2 O in a plasma
reactor at 300 ◦ C.39 Under our experimental conditions, the
deposited films display a refractive index of 1.48 and the film
thickness was controlled by the reaction time (the deposition
rate was 41.4 nm min−1 ). Fig. 2(A) shows a cyclic voltammogram
(CV) for an Fe(CN)6 4−/3− solution recorded on the Au/SiOx

Fig. 2 (A) Cyclic voltammogram of the Au/SiOx interface, (B) EIS of the same Au/SiOx interface and (C) influence of cyclic voltammetry and EIS
measurements on the change of the SPR signal; solution of 10 mM Fe(CN)6 4−/3− in KCl (0.1 M), scan rate (CV) = 20 mV s−1 , DE (EIS) = 10 mV,
frequency range: 65 kHz to 0.1 Hz, E app = OCP.

This journal is © The Royal Society of Chemistry 2008 Analyst, 2008, 133, 1097–1103 | 1099
Table 1 Best parameters for fitting impedance spectra of the different interfaces
Interface Rs /X
Au/SiOx 81.7 ± 4.3
Au/SiOx /APTMS 108.1 ± 3.5
Au/SiOx /APTMS/GA 110.6 ± 5.1
Au/SiOx /APTMS/GA/ODNs 115.2 ± 6.5
Hybridization with comp. ODNs 108.0 ± 2.3
Hybridization with non-comp. ODNs 118.1 ± 4.3
Regeneration 113.2 ± 2.4
interface. The presence of the 9 ± 1 nm thick SiOx layers
limits the diffusion of Fe(CN)6 4− to the modified gold electrode
interface serving as a barrier for electron transfer. CV is not
well adapted for the analytical investigation of semiconducting
and insulating interfaces. Thus EIS was used to characterize the
electrical properties of the Au/SiOx interface. Fig. 2(B) shows
the faradic impedance spectrum of Au/SiOx in the presence
of Fe(CN)6 4−/3− . The EIS spectrum fits well to a Randlers
equivalent circuit [inset in Fig. 2(B)].44 The interfacial layer
at the working electrode (WE) is represented by the parallel
combination of resistance RCT , which models the charge transfer
across the Au/SiOx layer, and capacitance C DL , accounting
for the electrical double layer formed at the interface. The
concentration depletion of the redox species near the interface
is described by the Warburg term Zw . Rs is the solution
resistance. Table 1 summarizes the obtained values. As expected,
EIS measurements are also more favorable to couple to SPR
measurements. Fig. 2(C) shows the change of the SPR signal
during a CV cycle and an EIS frequency scan. The SPR angle
HSPR changes by 60 millidegree in the case of a CV experiment
while no fluctuation was seen in EIS.
3.2. SPR and impedance studies of the modified Au/SiOx
interfaces
The covalent linking of oligonucleotide units follows the pro-
tocol described in Fig. 1. It consists of the formation of
hydroxy groups on Au/SiOx , silanization with amine-terminated
silanes, followed by glutaraldehyde linking of amine-terminated
ODNs to the interface. The surface was stabilized using NaBH4
reduction of the CH=N imine into a CH2 –NH2 amine bond and
deactivating non-bonded CHO functional groups of the glu-
taraldehyde by transforming them into CH2 –OH. The modified
interface shows different EIS characteristics (Table 1). EIS does
not allow direct determination of DNA surface coverage. This
parameter is, however, rather important as the hybridization
capacity depends largely on the density of ODN strands on the
Au/SiOx surface. The DNA surface grafting densities should
be kept below a limiting value for attaining the maximum
hybridization efficiency and thus the highest sensitivity and
selectivity.7 Fig. 3 shows the reflectivity vs. angle spectra for the
unmodified Au/SiOx interface, after modification with APTMS
and GA and after immobilization of 15-mer ODN strands.
The angle where the surface plasmon minimum occurs shifts
from H = 67.98◦ (unmodified) to higher SPR angles using
PBS as the dielectric medium. The angle shift enables a rough
estimation of the thickness of the APTMS and GA layers. An
average refractive index of 1.46 was assumed for both layers45
1100 | Analyst, 2008, 133, 1097–1103
RCT /X C DL /lF a DL
112 ± 2 3.1 ± 0.3 0.91 ± 0.05
647 ± 6 1.5 ± 0.2 0.93 ± 0.05
984 ± 10 1.4 ± 0.5 0.91 ± 0.05
1170 ± 14 1.4 ± 0.1 0.90 ± 0.05
1693 ± 20 1.4 ± 0.3 0.93 ± 0.05
1443 ± 16 1.5 ± 0.4 0.90 ± 0.05
1194 ± 218 1.6 ± 0.2 0.91 ± 0.05
Fig. 3 Reflectivity versus incidence angle curves for a thin gold film
(50 nm) deposited on glass with a 5 nm titanium adhesion layer
coated with 9.5 nm SiOx before (closed circles) after modification
with 3-aminomethoxysilane (APTMS) and GA (closed squares) and
after ODN immobilization (grey squares). Experimental parameters:
symbols; simulated SPR curves: full lines; fitting parameters: n(prism) =
1.58, n(Ti) = 2.36 + i3.112 (d = 5 nm), n(Au) = 0.197 + i3.442 (d = 50 nm),
n(SiO ) = 1.48 (d = 9.5 nm), n(APTMS + GA) = 1.46 + i0.3 (d = 2.1 nm), n(DNA) =
x
1.4 + i0.02 (d = 6 nm), n(PBS) = 1.335.
and a total thickness of ca. 2.1 nm is obtained in that case.
Theoretically, APTMS and GA monolayers are 0.8 and 0.7 nm
thick, respectively, resulting in a total thickness of 1.5 nm. The
experimental value is slightly higher. This could be either due to
APTMS or GA multilayer formation or due to the well-known
difficulty of assuming a correct refractive index for the layers.
Indeed, Au/SiOx interfaces modified with low-density films
will show decreased refractive index values. Covalent linking
of the 15-mer ODN strands results in a further angle shift of
80 millidegrees (120 millidegrees correspond to 100 ng cm−2 ).46
We can thus estimate the DNA surface coverage of ss-DNA as
1.42 × 10−11 mol cm−2 , being ca. 8.57 × 1012 molecules cm−2 .
This corresponds to values reported in the literature.18,47
3.3. Hybridization kinetics with complementary and
non-complementary DNA
The ss-DNA-modified Au/SiOx interface was used for the
detection of biomolecular interaction with complementary
and non-complementary DNA strands. Complementary DNA
(2 lM) was added to the SPR cell and the hybridization event
was followed using EIS and SPR in parallel. Fig. 4(A) shows
the change of the electrochemical impedance characteristics
over 180 min in the form of Nyquist plots. Values for the
solution resistance Rs , the charge transfer RCT across the
Au/SiOx /APTMS/GA/ODN layer and the associated double
layer capacitance C DL were derived by using the equivalent
This journal is © The Royal Society of Chemistry 2008
Fig. 4 Nyquist plots of DNA hybridization in hybridization buffer containing 2 lM fully complementary target DNA with time (A) and
dehybridization in 0.05 M NaOH (B); solution: 10 mM Fe(CN)6 4−/3− in KCl (0.1 M), DE = 10 mV; frequency range: 65 kHz to 0.1 Hz, E app =
OCP; 0 min (●), 5 min (䊊), 15 min (), 30 min (), 60 min (䉬), 90 min (䉫), 120 min (), 160 min (), 180 min (|); (C) DNA hybridization kinetics
followed by EIS; Rct is obtained from the fitting of the impedance spectra in Fig. 4(A) and 4(B) using the equivalent circuit seen in Fig. 2(B), (D)
semilog plot of Rct dissociation data versus time.

circuit shown in Fig. 2(B). As shown in Fig. 4(A), the Nyquist
plot for the DNA-modified Au/SiOx interfaces can be described
as a semicircle near the origin at high frequencies followed by a
linear tail with a slope of almost unity that is dominated by the
interfacial mass transfer of the redox mediator. The solution
resistance Rs , dominant in the limit of high ac modulation
frequency, is almost constant (Table 1). This is expected since
only the interfacial layers are modified during the hybridization
event. The charge transfer resistance RCT is on the other hand
increasing with increasing hybridization time until a steady-state
value is obtained after approx. 180 min. The final RCT value
is about 1.5 times larger than the initial value (Table 1). The
increase in RCT [Fig. 4(C)] follows a Langmuir-like isotherm and
is attributed to the repulsive electrostatic interaction between the
negatively charged redox mediator Fe(CN)6 4−/3− and the increase
in anionic charge induced by duplex DNA formation, due to the
presence of additional phosphate groups in the DNA backbone.
The kinetics of dehybridization of ds-DNA were further
studied by exposing the interface to 0.05 M NaOH [Fig. 4(D)].
The charge transfer RCT decreases with prolonged exposure to
NaOH due to the dissociation of the DNA/DNA complex.
For ODNs of perfect match an accepted model for duplex for-
mation is that of Langmuir model, allowing the determination
of the association kon and dissociation koff rate constants using
eqn (1):7,18,48
RCT (t) = R∞ {1 − exp[−(kon c◦ + koff )t]} (1a)
RCT (t) = R∞ [exp(−koff )t] (1b)
K A = kon /koff (1c)
From the slope of a semilog plot [ln(RCT ) vs. t] of the dehybridiza-
tion event [Fig. 4(D)], koff of 2.2 × 10−3 min−1 was calculated,
which allowed the determination of kon = 8.95 × 102 M−1 min−1 .
An affinity constant K A of 4.07 × 105 M−1 was obtained, being
similar to that reported by Gooding and co-workers,49 but
smaller than that found by Dong’s group.18 Fig. 5(A) shows
the change in the SPR hybridization kinetic measurements (DH
vs. t) determined simultaneously while recording the EIS data
in Fig. 4. During hybridization, the resonance angle increased
by 61.98 mdeg, which corresponds to a surface coverage of
6.63 × 1012 molecules cm−2 and a hybridization efficiency of
77%, calculated from the quotient of the surface coverage of the
target DNA/immobilized DNA. From the SPR sensorgrams,
the association (kon ) and dissociation (koff ) rate constants can be
calculated using a linear transformation of the SPR sensorgram
data according to eqn (2):
ks = kon c + koff (2)
where ks is the slope of a dH/dt versus H plot and is equal to
0.0046. Taking the EIS determined dissociation rate (koff of 2.2 ×
10−3 min−1 ) a kon of 9.30 × 102 M−1 min−1 is obtained. This is
close to the one found by EIS measurements.

This journal is © The Royal Society of Chemistry 2008 Analyst, 2008, 133, 1097–1103 | 1101

Fig. 5 (A) SPR hybridization kinetics of immobilized DNA with its
complementary strand. The data were obtained while recording the
hybridization kinetics with EIS as seen in Fig. 4. (B) Discrimina-
tion of DNA hybridization with the complementary (䊊) and non-
complementary () strands using EIS. For clarity, the Nyquist plots
of the interfaces modified with ss-DNA (●), and after regeneration in
0.05 M NaOH () are shown; solution: 10 mM Fe(CN)6 4−/3− in KCl (0.1
M), DE = 10 mV, frequency range: 65 kHz to 0.1 Hz, E app = OCP.
Furthermore, EIS on Au/SiOx was used to investigate the abil-
ity to discriminate between the interaction of the immobilized
DNA with its complementary and non-complementary strands.
Fig. 5(B) shows the change in the faradic impedance spectra
when reacted with complementary and non-complementary
ODN strands. When incubated in the presence of non-
complementary oligonucleotides, the change of the RCT was
smaller, as compared to exposure to complementary DNA
(Table 1) and allowed discrimination between complementary
and non-complementary reactions. However, hybridization with
non-complementary DNA shows a considerable signal. This is
due to non-specific adsorption of non-complementary DNA to
the Au/SiOx interface. The heavy hydration around mismatched
bases together with the structural distortion of the duplex might
be responsible for impeding, to some extent, the approach of
redox mediator to the electrode interface resulting in increased
RCT values.15
Hybridization and dehybridization cycles were performed on
the same surface several times without any apparent degrada-
tion. This is evidenced by the RCT value of 1194 X obtained after
dehybridization, being close to the RCT value (1170 X) of the
starting interface. The result indicates that the interface can be
reused, which is of high interest for DNA chips.
1102 | Analyst, 2008, 133, 1097–1103
4. Conclusion
The kinetics of hybridization and dehybridization events of
complementary and non-complementary DNA on Au/SiOx
were followed by SPR and EIS simultaneously. The surface
chemistry of glass can be directly adopted for the Au/SiOx
interface resulting in a stable interface with covalently bound
DNA. Indeed, hybridization/dehybridization events could be
performed without significant degradation for at least 20 times.
SPR/EIS allows the determination of all the parameters im-
portant for DNA hybridization simultaneously in one exper-
iment: (i) the shift of the SPR resonance angle allowed the
determination of the surface coverage of immobilized DNA, (ii)
association and dissociation rate constants were obtained by EIS
by recording the development of the charge transfer resistance
with time during the hybridization and dehybridization events,
(iii) SPR hybridization kinetics recorded in parallel allowed
the determination of the hybridization efficiency, and (iv) from
the slope of a dH/dt versus H plot the association and the
affinity constants could be estimated in addition from SPR
measurements. The data validated the results obtained by EIS.
Acknowledgements
The Agence Nationale de la Recherche, the Institut National
Polytechnique de Grenoble (INPG, BQR 2006), the Centre
National de la Recherche Scientifique (CNRS) and the Nord-
Pas-de Calais region are gratefully acknowledged for financial
support.
References
1 B. Liedberg, C. Nylander and I. Lundstrom, Sens. Actuators, B, 1983,
4, 229.
2 R. Georgiadis, K. A. Peterlinz, J. R. Rahn, A. W. Peterson and J. H.
Grassi, Langmuir, 2000, 16, 6759.
3 P. Guedon, T. Livache, F. Martin, F. Lesbre, A. Roget, G. Bidan and
Y. Levy, Anal. Chem., 2000, 72, 6003.
4 M. Mrksich, J. R. Grunwell and G. M. Whitesides, J. Am. Chem.
Soc., 1995, 117, 12009.
5 S. G. Nelson, K. S. Johnston and S. S. Yee, Sens. Actuators, B, 1996,
35/36, 187.
6 K. A. Peterlinz, R. M. Georgiadis, T. M. Herne and M. J. Tarlov,
J. Am. Chem. Soc., 1997, 119, 3401.
7 A. W. Peterson, R. J. Heaton and R. Georgiadis, Nucleic Acids Res.,
2001, 29, 5163.
8 E. A. W. Smith, M. J., Y. Cheng, S. V. P. Barreira, A. G. Frutos and
R. M. Corn, Langmuir, 2001, 17, 2502.
9 A. J. Thiel, A. G. Frutos, C. E. Jordan, R. M. Corn and L. M. Smith,
Anal. Chem., 1997, 69, 4948.
10 N. Zhang, R. Schweiss, Y. Zong and W. Knoll, Electrochim. Acta,
2007, 52, 2869.
11 B. Piro, J. Haccoun, M. C. Pham, L. D. Tran, A. Rubin, H. Perrot
and C. Gabrielli, J. Electroanal. Chem., 2005, 577, 1555.
12 C. Tlili, H. Korri-Youssoufi, L. Ponsonnet, C. Martelet and N. J.
Jaffrezic-Renault, Talanta, 2005, 68, 131.
13 C. Berggren, P. Stalhandske, J. Brundell and G. Johansson, Electro-
analysis, 1999, 11, 156.
14 A. Li, F. Yang, Y. Ma and X. Yang, Biosens. Bioelectron., 2007, 22,
1716.
15 T. Ito, K. Hosokawa and M. Maeda, Biosens. Bioelectron., 2007, 22,
1816.
16 S. Pan and L. Rothberg, Langmuir, 2005, 21, 1022.
17 C. A. Marquette, M. F. Lawrance and L. J. Blum, Anal. Chem., 2006,
78, 959.
18 D. Li, X. Zou, Q. Shen and S. Dong, Electrochem. Commun., 2007,
9, 191.
This journal is © The Royal Society of Chemistry 2008
19 J. Xu, J. J. Zhu, Q. Huang and H. Y. Chen, Electrochem. Commun.,
2001, 3, 665.
20 V. Stambouli, A. Zebda, E. Appert, C. Guiducci, M. Labeau, J. P.
Diard, B. Le Gorrec, N. Brack and P. J. Pigram, Electrochim. Acta,
2006, 51, 5206.
21 W. Cai, J. R. Peck, D. W. van der Weide and R. J. Hamers, Biosens.
Bioelectron., 2004, 19, 1013.
22 A. Zebda, V. Stambouli, M. Labeau, C. Guiducci, J.-P. Diard and B.
Le Gorrec, Biosens. Bioelectron., 2006, 22, 178.
23 C. A. Marquette, I. Lawrance, E. Polychronakos and M. F. Lawrance,
Talanta, 2002, 56, 763.
24 E. Souteyrand, J. P. Cloarec, J. R. Martin, C. Wilson, I. Lawrence,
S. Mikkelsen and M. F. Lawrence, J. Phys. Chem. B, 1997, 101,
2980.
25 J. P. Cloarec, N. Deligianis, J. R. Martin, I. Lawrence, E. Souteyrand,
C. Polychronakos and M. F. Lawrence, Biosens. Bioelectron., 2002,
17, 405.
26 J. P. Cloarec, J. R. Martin, E. Polychronakos, I. Lawrance, M. F.
Lawrance and E. Souteyrand, Sens. Actuators, B, 1999, 58, 394.
27 H. Gu, X. d. Su and K. P. Loh, J. Phys. Chem. B, 2005, 109, 13611.
28 A. Bardea, F. Patolsky, A. Dagan and I. Willner, Chem. Commun.,
1999, 21.
29 C.-Z. Li, Y. Lui and J. H. T. Luong, Anal. Chem., 2005, 77, 478.
30 T. Y. Lee and Y. B. Shim, Anal. Chem., 2001, 73, 5629.
31 S. M. Schiller, R. Naumann, K. Lovejoy, H. Kunz and W. Knoll,
Angew. Chem., Int. Ed., 2003, 42, 208.
32 R. Naumann, S. M. Schiller, F. Giess, B. Grohe, K. B. Hatmann, I.
Karcher, I. Koper, J. Lubben, K. Vasilev and W. Knoll, Langmuir,
2003, 19, 5435.
This journal is © The Royal Society of Chemistry 2008
33 Z. Wang, T. Wilkop and Q. Cheng, Langmuir, 2005, 21, 10292.
34 D. K. M. Kambhampati, J. W. Robertson, M. Cai, J. E. Pemberton
and W. Knoll, Langmuir, 2001, 17, 1169.
35 X. Yang, Q. Wang, K. Wang, W. Tan and H. Li, Biosens. Bioelectron.,
2007, 22, 1106.
36 L. He, E. A. Smith, M. J. Natan and C. D. Keating, J. Phys. Chem.
B, 2004, 108, 10973.
37 S. Szunerits and R. Boukherroub, Langmuir, 2006, 22, 1660.
38 S. Szunerits and R. Boukherroub, Electrochem. Commun., 2006, 8,
439.
39 S. Szunerits, Y. Coffinier, S. Janel and R. Boukherroub, Langmuir,
2006, 22, 10716.
40 I. Zawica, G. Wittstock, R. Boukherroub and S. Szunerits, Langmuir,
2007, 23, 9303.
41 I. Zawica, G. Wittstock, R. Boukherroub and S. Szunerits, Langmuir,
2008, 24, 3922.
42 T. Wink, S. J. Van Zuilen, A. Bult and W. P. van Bennekom, Chem.
Commun., 1998, 70, 827.
43 R. P. H. Kooyman, A. T. M. Lenferink, R. G. eenik and J. Greve,
Anal. Chem., 1991, 63, 83.
44 A. J. Bard and L. R. Faulkner, Electrochemical methods: fundamen-
tals and applications, Wiley, New Jersey, 2nd edn, 2001.
45 H. Ouyang, C. C. triemer and P. M. Fauchet, Appl. Phys. Lett., 2006,
88, 163108.
46 N. Yang, X. Su and W. Knoll, Biosens. Bioelectron., 2007, 11, 2700.
47 C. E. Jordan and R. M. Corn, Anal. Chem., 1997, 69, 1449.
48 J. Liu, S. Tian, P. E. Nielsen and W. Knoll, Chem. Commun., 2005,
2969.
49 E. L. Wong, E. Chow and J. J. Gooding, Langmuir, 2005, 21, 6957.
Analyst, 2008, 133, 1097–1103 | 1103