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Critical View on Electrochemical Impedance Spectroscopy Using the


Ferri/Ferrocyanide Redox Couple at Gold Electrodes
Stephan Vogt, Qiang Su, Cristina Gutierrez-Sanchez, and Gilbert Noll*
Noll Junior Research Group, Organic Chemistry, Chemistry and Biology Department, Faculty IV, Siegen University,
Adolf-Reichwein-Strasse 2, 57068 Siegen, Germany
*
S Supporting Information

ABSTRACT: Electrochemical or faradaic impedance spectros-


copy (EIS) using the ferri/ferrocyanide couple as a redox probe
at gold working electrodes was evaluated with respect to its
ability to monitor consecutive surface modication steps. As a
model reaction, the reversible hybridization and dehybridization
of DNA was studied. Thiol-modied single stranded DNA
(ssDNA, 20 bases, capture probe) was chemisorbed to a gold
electrode and treated with a solution of short thiols to release
nonspecically adsorbed DNA before hybridization with
complementary ssDNA (20 bases, target) was carried out.
Reversible dehybridization was achieved by intense rinsing with
pure water. The experimental procedures were optimized by
kinetic surface plasmon resonance (SPR) and quartz crystal
microbalance with dissipation (QCM-D) measurements to maximize the increase in reectivity or decrease in frequency upon
hybridization before hybridization/dehybridization was also monitored by EIS. In contrast to SPR and QCM-D, repeatable EIS
measurements were not possible at rst. Combined SPR/EIS and QCM-D/EIS measurements revealed that during EIS the gold
surface is seriously damaged due to the presence of CN ions, which are released from the ferri/ferrocyanide redox probe. Even
at optimized experimental conditions, etching the gold electrodes could not be completely suppressed and the repeatability of the
EIS measurements was limited. In three out of four experimental runs, only two hybridization/dehybridization steps could be
monitored reversibly by EIS. Thereafter etching the gold electrode signicantly contributed to the EIS spectra whereas the
QCM-D response was still repeatable. Hence great care has to be taken when this technique is used to monitor surface
modication at gold electrodes.

B esides surface plasmon resonance (SPR)15 or quartz


crystal microbalance (QCM) measurements,2,3,6,7 faradaic
or electrochemical impedance spectroscopy (EIS) using the
on a bare gold surface prior to modication, the subsequent
chemisorption steps became less ecient. To the best of our
knowledge such problems are not mentioned in the vast
ferri-/ferrocyanide redox probe8,9 is a frequently applied majority of papers dealing with the topic. We found only few
analytical technique in order to monitor the stepwise papers dealing with similar drawbacks.1215 In two of these
modication of surfaces with biomolecules such as proteins papers published already in 2000 and 2002, the authors used
or DNA. Often the resulting surfaces are subsequently used for EIS in order to characterize self-assembled monolayers of
dierent kinds of biosensing applications. 35 All three carboxy-terminated alkyl thiols (SAMs) on gold.13,14 The
techniques (an optical, an acoustic, and an electrochemical impedimetric data was in agreement with the interpretation
technique) can be used independently, and which technique is that at extended sample times the thiol SAM was gradually
favored by individual researchers often depends mainly on the removed. A mechanism of etching the gold surface through
availability of the experimental setup. While SPR and QCM can pinholes by CN released from the ferri/ferrocyanide couple
be applied straightforward, the successful performance of EIS
was proposed. This interpretation is in accordance with earlier
has been reported to depend strongly on experimental
papers which report the release of CN ions from ferri/
parameters such as the ionic strength and composition of the
electrolyte.10,11 When we tried to establish EIS using the ferri/ ferrocyanide16 as well as their eect on gold surfaces.17,18
ferrocyanide redox couple as an analytical technique in our However, remarkably in most of the papers dealing with EIS in
laboratory in order to monitor surface modication at gold combination with gold electrodes, the stability of the ferri-/
electrodes, we encountered serious experimental problems. ferrocyanide couple seems not to be an issue. Since etching the
When the same impedimetric measurements were repeated at
the same electrode multiple times, the response curve changed Received: December 20, 2015
continuously, i.e., repeatable measurements were not possible. Accepted: March 18, 2016
Furthermore, when impedance measurements were performed Published: March 18, 2016

2016 American Chemical Society 4383 DOI: 10.1021/acs.analchem.5b04814


Anal. Chem. 2016, 88, 43834390
Analytical Chemistry Article

Scheme 1. (A) Stepwise Surface Modication Procedure,a (B) Corresponding Equivalent Circuit Used for the Determination of
the Charge Transfer Resistance RCT, and (C) Simulated Nyquist Plotsb

a
For EIS the ferri-/ferrocyanide redox couple was added in oxygen-free buer solution before and after each surface modication step, and
impedance spectra were measured. bShows a subsequent increase in charge transfer resistance RCT as expected for the dierent electrode
architectures depicted in part A.

gold electrode by CN would also explain the problems we we show by combining EIS with SPR or QCM, with proceeding
encountered, we decided to carry out a systematic evaluation of measurement duration, serious damage of the gold surface
the method. As model reaction we have chosen the reversible occurs. Apparently this damage is caused by CN released from
hybridization and dehybridization of complementary DNA the ferri/ferrocyanide couple.
strands at gold electrodes. In order to prepare the single
stranded DNA (ssDNA) comprising sensor platform, we
followed a stepwise surface modication procedure as outlined
MATERIALS AND METHODS
The thiol 4-mercapto-1-butanol (MCB) was purchased from
in Scheme 1A. Sigma-Aldrich (Steinheim, Germany), whereas potassium
In the rst step, thiol-modied ssDNA (capture probe) ferricyanide and potassium ferrocyanide trihydrate were from
dissolved in buer solution is adsorbed to the gold surface for a Acros Organics (Thermo Fisher Scientic, Geel, Belgium).
period of about 90 min. Next, a solution of short thiols, i.e., a Tris(2-carboxyethyl) phosphine hydrochloride and 3-mercap-
mixture of mercaptobutanol (MCB) and mercaptopropionic topropionic acid (MPA) were purchased from Alfa Aesar
acid (MPA) in water, is added for a period of 1 h. Subsequently (Karlsruhe, Germany). The thiol-modied DNA (5-HO-
the sensor surface is rinsed and equilibrated with buer (CH2)6-S-S-(CH2)6-AAC-TAC-TGG-GCC-ATC-GTG-AC-3)
solution. For hybridization, the complementary ssDNA (target) and complementary DNA (5-GTC-ACG-ATG-GCC-CAG-
is added in the same buer. As capture probe and target, TAG-TT-3) were ordered from Metabion (Martinsried,
complementary ssDNA strands of 20 bases were used. The Germany). The thiol group was protected with mercaptohex-
probe strand was modied with a 6-mercaptohexyl linker anol (i.e., it was purchased as disulde) and deprotected prior
(including phosphate) at the 5 end. to use. The DNA sequence was chosen in a way that an
We carried out EIS not only as a standalone technique but unambiguous hybridization product is formed.5,20
also in combination with SPR and QCM with dissipation SPR measurements were done using either a setup from
monitoring (QCM-D). In order to optimize the experimental Optrel (Sinzing, Germany) or from Res-Tec (Resonant
conditions, we started with separate SPR and QCM-D Technologies, Framersheim, Germany), each equipped with a
measurements. As we show, SPR and QCM-D are both HeNe Laser which emit light at a wavelength of 633 nm.
reliable techniques, which can be used to monitor DNA Neither ferri- nor ferrocyanide absorb light of this wavelength.
hybridization in a highly repeatable manner. Several hybrid- Both instruments were equipped with homemade ow-cells and
ization/dehybridization steps at the same sensor surface are connected to a potentiostat to perform EIS (model 1025,
possible. In both techniques the change in signal is proportional EG&G, Albuquerque, NM). In order to calculate the optical
to the amount of adsorbed target DNA. Since in previous thickness of the individual DNA layers from the shift in the
studies it was shown that in QCM measurements the adsorbed angular scan curves according to the Fresnel equations, the
mass is overestimated due to solvent molecules trapped in the software Winspall, version 3.0.2.0 (Max Planck Institute of
ssDNA or dsDNA monolayer,2,19 we used for quantitative Polymer Research, Mainz, Germany) was used. QCM-D
analysis solely the SPR data. In contrast to QCM-D and SPR, measurements were performed with a Q-sense E1 instrument
during EIS the repeatability of the experiments was limited. As (Q-Sense, Goteborg, Sweden) which was controlled by the
4384 DOI: 10.1021/acs.analchem.5b04814
Anal. Chem. 2016, 88, 43834390
Analytical Chemistry Article

QSoft 401 acquisition software. If QCM-D measurements were


performed in combination with EIS, a QEM 401 Q-Sense
Electrochemistry Module was used, whereas in all other cases a
QFM 401 Q-Sense Flow Module served as the sensor platform.
The QCM sensor crystals were cleaned in a BioForce ozone
cleaner (BioForce, Ames, IA). For EIS in combination with the
QEM 401 module, a Zahner Zennium electrochemical
workstation (Zahner Elektrik, Kronach, Germany) was used.
The THALES software supplied with this instrument was used
to t EIS data to the Randles circuit (cf. Scheme 1B) equipped
with a constant phase element to account for the surface
roughness of the electrodes used. Commercially available disk
shaped gold electrodes with a diameter of 1.6 mm were
purchased from BASi (BASi, West Lafayette, IN). All EIS
measurements were performed with 5 mV amplitude at the
open circuit potential of the ferri-/ferrocyanide solution. To
exclude any artifacts, the concentration of ferri-/ferrocyanide
was varied from 1 to 10 mM, each. Further chemicals used in
this study and detailed experimental procedures are given in the
Supporting Information.

RESULTS AND DISCUSSION


Surface Plasmon Resonance (SPR). Surface plasmon
resonance (SPR) measurements were carried out in an optical
ow cell using either a commercial Res-Tec or an Optrel SPR-
setup. The freshly cleaned gold surface was modied according
to the procedure shown in Scheme 1A. In Figure 1A a
representative kinetic scan curve is shown. Preparation of the
sensor surface (by adsorption of thiol-modied ssDNA in 20
mM phosphate buer containing 50 mM KCl and 5 mM MgCl2
at pH 7.0 and subsequently a mixture of MCB and MPA in
water) is followed by three hybridization/dehybridization steps. Figure 1. (A) SPR kinetic scan and (B) SPR angular scan curves
After each hybridization step, dehybridization of the target collected during the preparation of a DNA sensor chip, which was
DNA was achieved by extensive rinsing with pure water.2124 subsequently used for the detection of complementary target DNA (5
At the bare gold surface prior to DNA adsorption, after M). Three hybridization/dehybridization steps were carried out.
Adsorption of thiol-modied ssDNA (capture probe) and hybrid-
preparation of the sensor surface, and in between the ization with complementary (target) ssDNA were carried out in 20
hybridization/dehybridization steps, entire angular scan curves mM phosphate buer at pH 7.0 containing 50 mM KCl and 5 mM
were collected as well. The minima of these curves are depicted MgCl2.
in Figure 1B. From the shift in the angular scan curves, the
optical thickness of the individual DNA layers can be calculated
according to the Fresnel equations assuming a refractive index capture probe strands (the calculation is described in detail in
nA of 1.5 for DNA.25 Furthermore, the mass of the DNA lms the Supporting Information). After hybridization with target
before and after hybridization can be calculated using Feijters DNA, an increase in the surface bound mass of 0.43 ng mm2,
equation.26 0.51 ng mm2, and 0.51 ng mm2 was calculated for the three
n nsol individual hybridization steps (0.48 ng mm2 on average).
M = dA A According to the molecular weight of the target DNA (6133 g
dn/dc
mol1), this corresponds to an average increase of 7.5 1012
In Feijters equation dA is the thickness of the layer, and nA and mol cm2. Apparently only about 40% of all capture strands
nsol are the refractive indices of the layer and buer, hybridize with complementary target DNA. It corresponds to a
respectively.26 The quotient dn/dc is the refractive index center-to-center-distance of dsDNA of 4.55 nm (with ssDNA
increment of the adsorbed layer.26 Assuming a value of dn/dc = capture probe strands remaining in between the hybridized
0.175 cm3 g1 for DNA,27 nA = 1.5, and nsol = 1.35, a value of M strands). This degree of hybridization is in the same range as
= 1.2 ng mm2 (or 120 ng cm2) was obtained for the ssDNA reported previously for dsDNA with 15 and 30 base pairs,
layer before hybridization with target. The molecular weight of respectively.32,33
the ssDNA is much larger than that of MCB/MPA (6430 g For comparison also dierent buer solutions with
mol1 106 g mol1). Neglecting the contribution of MCB/ signicantly higher ionic strength (e.g., 100 mM potassium
MPA (due to its lower molecular weight), a surface coverage of phosphate, 1 M KCl, 5 mM MgCl2, pH 7.0) were evaluated
11.2 1010 molecules ssDNA mm2 (corresponding to 11.2 since at higher ionic strength the repulsion between the
1012 molecules cm2 or 18.7 1012 mol cm2) was calculated. negatively charged phosphate backbones of individual ssDNA
This value is in the same order of magnitude as determined by strands should be decreased (leading to an even higher surface
dierent experimental techniques for other thiol-modied DNA coverage of capture probe DNA).9,10,30 However, in our
strands adsorbed to gold10,2831 and corresponds to a center- experiments this did not result in signicant improvements.
to-center distance of about 3 nm between two adjacent ssDNA Also for hybridization with target DNA, the best results (i.e.,
4385 DOI: 10.1021/acs.analchem.5b04814
Anal. Chem. 2016, 88, 43834390
Analytical Chemistry Article

hybridization eciencies of about 40%) were obtained in 20


mM phosphate buer at pH 7.0 containing 50 mM KCl and 5
mM MgCl2. When the KCl concentration was slightly increased
(up to a concentration of 100 mM), the performance was
similar, whereas at signicantly higher salt concentrations the
hybridization eciency decreased. The limited inuence of the
buer composition on the hybridization eciency might be
explained by the fact that the obtained surface coverage 1013
molecules cm2 is already close to the maximum. Since it has
been reported that for lower surface densities (2.0 1012
molecules cm2) hybridization eciencies close to 100% may
be reached,29 we decided to investigate DNA hybridization also
at lower capture probe densities. Therefore, we changed the
procedure for the sensor chip preparation in order to reduce
the surface coverage. Details of the experiments are presented
in the Supporting Information, Figure S-2. By this procedure, a
surface coverage 2.5 1012 molecules cm2 was obtained, i.e.,
the capture probe surface coverage was reduced by a factor of
4.5. At this lower surface coverage hybridization eciencies
close to 100% could be obtained, which might be explained by
less sterical hindrance between individual capture strands and
target. Nevertheless, we refrained from using this low surface
coverage in further experiments, since at this low surface
coverage the increase in reectivity during hybridization with
target was rather small.
Quartz Crystal Microbalance with Dissipation Mon-
itoring (QCM-D). Quartz crystal microbalance with dissipation
monitoring (QCM-D) measurements were carried out using a
commercial Q-Sense E1 setup equipped with a QFM 401 (Q-
Sense Flow Module). Also during QCM-D, best results were
obtained if the adsorption of thiol-modied ssDNA as well as
the hybridization with target DNA were performed in 20 mM Figure 2. QCM-D measurements of DNA hybridization and
dehybridization. Changes of (A) normalized frequency and (B)
phosphate buer containing 50 mM KCl and 5 mM MgCl2 at dissipation factor D versus time. The normalized overtones 513 are
pH 7.0. The ssDNA capture probe surface was prepared shown. The concentration of complementary target DNA was 5 M in
according to Scheme 1A (adsorption of thiol-modied ssDNA 20 mM phosphate buer at pH 7.0 containing 50 mM KCl and 5 mM
in buer was followed by adsorption of a mixture of MCB and MgCl2. Dehybridization was achieved by rinsing with pure water.
MPA in water, data not shown). The surface was left in buer
for overnight equilibration and subsequently used for multiple water molecules are bound to the DNA during hybridization
hybridization/dehybridization cycles. In Figure 2 the normal- under the applied experimental conditions,35 this nding is in
ized frequency (A) and dissipation factor shifts (B) are shown, accordance with comprehensive QCM-D studies referring an
respectively, of multiple hybridization/dehybridization steps increase of the dissipation factor D to an increased amount of
performed on the prepared surface. As it can be seen in Figure surface bound water.36,37
2A, spreading of the normalized frequency overtones indicates Electrochemical Impedance Spectroscopy (EIS). Elec-
that a rather soft layer was formed. In this case, a quantitative trochemical (faradaic) impedance spectroscopy (EIS) using
evaluation using the Sauerbrey equation is not possible.19,34 ferri-/ferrocyanide as a redox probe was carried out at
Analogue to the SPR also during the QCM-D measurements commercially available as well as at homemade ordinary disc
dehybridization could be achieved by extensive rinsing with shaped gold electrodes with diameters between 1 mm and 4
pure water, whereas upon rinsing with buer solution it was not mm. Prior to modication the electrodes were cleaned rst by
possible to remove a signicant amount of target DNA within a mechanical polishing with diamond paste (0.25 m) and then
reasonable time period. In accordance with the SPR measure- by a treatment with hot piranha solution (a 3:7 mixture of
ments it was possible to study a series of hybridization and H2O2 and H2SO4; CAUTION: piranha solution reacts violently
dehybridization events without signicant decrease in perform- with most organic materials and should be handled with care).
ance. Also during the QCM-D studies sensor chips prepared For comparison, in some of the experiments the electrodes
with lower surface coverage of the capture probe strands were were also cleaned by an additional electrochemical polishing
investigated (data not shown). Analogous to SPR, the best procedure (dierent electrochemical polishing procedures were
results (i.e., the strongest decrease in frequency upon examined).10,38,39 Thereafter the electrodes were modied
hybridization) were obtained when the sensor chips were according to Scheme 1A. After each surface modication step,
prepared and hybridization was performed in 20 mM the gold electrodes (serving as working electrode) were placed
phosphate buer at pH 7.0 containing 50 mM KCl and 5 together with a counter and a reference electrode in an
mM MgCl2. Independent of the surface coverage of the capture electrochemical cell with oxygen-free buer solution containing
probe strands and the surface modication protocol, we always equal amounts of ferri- and ferrocyanide (concentrations
observed an increase of the dissipation factor D during between 1 and 10 mM were evaluated), and impedance spectra
hybridization with target. Since it is well-known that additional were collected. From the impedimetric data, the charge transfer
4386 DOI: 10.1021/acs.analchem.5b04814
Anal. Chem. 2016, 88, 43834390
Analytical Chemistry Article

resistance RCT was determined. However, in none of our


experiments, a constant value for the charge transfer resistance
RCT could be obtained. When the impedance measurements
were repeated at the same modied electrode multiple times,
the charge transfer resistance RCT changed continuously (as a
general trend, after an initial increase the values for RCT
decreased with extended measurement periods). This observa-
tion was independent from the electrode size and from the
applied cleaning procedure prior to electrode modication.
There was a second general trend which is worthy to note.
When the impedance measurements were performed also on
the bare gold surface prior to surface modication, the
subsequent adsorption of the thiol-modied ssDNA became
less ecient (RCT values after ssDNA adsorption were smaller
for such electrodes). Furthermore, we did not observe always a
strong increase in the charge transfer resistance RCT after
hybridization with complementary target DNA. In some cases,
the value for RCT did even decrease. Even when we used buer
solution at low ionic strength,10 we could not apply faradaic
impedance spectroscopy with ferri-/ferrocyanide as a redox
probe as a reliable stand-alone technique in order to monitor
DNA hybridization. Furthermore, we have varied the ferri-/
ferrocyanide concentration between 1 and 10 mM, each.
Regarding the poor repeatability of our experiments, there was
no improvement at any concentration.
Combined SPR and EIS Measurements. In order to
elucidate the reason for the instability of the EIS results during
the impedance measurements, we combined SPR and EIS using
a single homemade optical and electrochemical ow cell. When
the gold coated slides were cleaned by electrochemical
polishing, already during the rst cycle, the gold layer was Figure 3. Combined SPR and EIS measurements: (A) SPR kinetic
almost completely destroyed. Therefore, we refrained from scan curve for a ssDNA modied sensor surface undergoing a
using electrochemical polishing in further experiments. hybridization, a dehybridization, and a second hybridization step. In
Apparently the amount of gold, which is cleaved from the between the surface was rinsed with oxygen-free buer (20 mM
surface during this cleaning procedure (e.g., as soluble gold phosphate buer at pH 7.0 containing 50 mM KCl and 5 mM MgCl2),
chloride complex, depending on the polishing procedure),36,40 which also contained the redox probe ferri-/ferrocyanide, 1 mM each,
is rather large. In Figure 3, representative results of combined and electrochemical impedance spectroscopy (EIS) measurements
SPR and EIS measurements on a ssDNA and dsDNA modied were performed. In part B, the corresponding EIS measurements are
sensor chip are shown. Prior to EIS measurements the ssDNA shown.
capture probe layer was prepared in the same way as previously.
As shown in Figure 3, EIS was measured at the ssDNA capture accordance with the change in the SPR signal, EIS is not a
probe modied surface followed by hybridization with target reliable measure for hybridization or dehybridization. Our
DNA. Thereafter a second EIS measurement was performed. observations can be explained by the gold electrode being
Before each EIS measurement the cell was rinsed with oxygen- etched in the presence of CN,18 which is released from
free buer containing the redox probe ferri- and ferrocyanide ferricyanide. This may happen via a mechanism in which CN
(each at a concentration 1 mM) for some minutes (oxygen was is rst adsorbed at the Au surface before a gold complex is
removed by purging with argon for several minutes). After formed, which will then be released from the surface.13,18
dehybridization the experimental procedure was repeated, This mechanism would explain why the adsorption of thiols
whereupon the third and fourth EIS measurements were became less ecient if impedance measurements were
performed. Thereafter two more EIS measurements were performed previously on the bare gold electrode. Furthermore,
carried out. As it can be seen from the SPR kinetic scan curve adsorption of CN might initially lead to an increase in RCT,
depicted in Figure 3A, the hybridization with target DNA as before etching the gold surface will increase the electrode
well as dehybridization by rinsing with water was working roughness and thus the electrode area. This will result in a
properly. From the EIS results depicted in Figure 3B, a small preceding decrease in RCT. In order to prove this assumption,
increase in RCT can be observed for the rst hybridization step. we measured EIS in the presence of ferri/ferrocyanide (1 mM
After dehybridization, the charge transfer resistance RCT each, same buer as before) over a period of 24 h at a bare gold
increased even further. Apparently the changes in the SPR coated SPR-slide. A minimum ow rate was applied in order to
signal and in the charge transfer resistance RCT do not correlate constantly provide a sucient amount of fresh CN (being
with each other but show opposite behavior. After the second released from ferri/ferrocyanide). Before and after 24 h EIS, a
hybridization event, there was again a minor additional increase SPR angular scan curve was collected. From the SPR angular
in the value for RCT, before it started to decrease during further scan curves, the thicknesses of the gold layer before and after
measurements at the same experimental conditions. Since the EIS were calculated. According to this evaluation, the thickness
change in the charge transfer resistance RCT is not in of the gold layer decreased by 2.3 nm during 24 h EIS (for
4387 DOI: 10.1021/acs.analchem.5b04814
Anal. Chem. 2016, 88, 43834390
Analytical Chemistry Article

details see the Supporting Information, Figure S-3). In order to insucient stability of the immobilized DNA strands, we
evaluate the eect of surface roughness, we also studied DNA performed combined QCM-D/EIS experiments on a non-
hybridization/dehybridization by combined SPR and EIS modied QCM sensor slide using the ferri/ferrocyanide redox
measurements on sensor chips with ultraat template stripped couple at a concentration of 5 mM each, as shown in Figure 5.
gold surface.41,42 However, this did not increase the stability of
the sensors during EIS.
Combined QCM-D and EIS Measurements. For
comparison with the SPR and EIS measurements, we decided
to carry out also combined QCM-D and EIS measurements.
For this purpose, a commercially available QCM-D cell
especially designed for electrochemical measurements (QEM
401, Q-Sense Electrochemistry Module) was used. First, the
ssDNA capture probe layer was prepared according to Scheme
1A (in 20 mM phosphate buer at pH 7.0 containing 50 mM
KCl and 5 mM MgCl2). The formation of the ssDNA capture
probe layer was monitored by QCM-D. After rinsing the cell
with oxygen-free buer containing ferri/ferrocyanide, 1 mM
each, three impedance spectra were collected (see Figure 4, red

Figure 5. Normalized frequency of a QCM-D measurement


(overtones 511) performed on a gold coated sensor chip at
continuous ow (0.1 mL min1) in oxygen-free buer containing
ferri-/ferrocyanide, 5 mM each. Every 30 min, impedance spectra were
recorded for a period of 28 min (frequency range 10 mHz to 100
Figure 4. Impedance spectra measured for a gold coated QCM-D kHz). Below a microscopic image of a fresh gold coated QCM sensor
sensor chip, which has been modied rst with ssDNA and short thiols (left) and the QCM-D sensor chip after 26 h of combined EIS/QCM
according to Scheme 1 (red dashed lines) and then hybridized with measurement (right) is shown.
complementary target DNA (black line). For the dsDNA modied
sensor further impedance spectra (gray) were collected over a period Over a period of 26 h, an impedance spectrum was measured
of 60 h (here only every fourth spectrum is shown). Experiments were every 30 min (spanning a frequency range from 10 mHz to 100
performed in oxygen-free buer (20 mM phosphate buer at pH 7.0 kHz), while a minimum ow rate was applied throughout the
containing 50 mM KCl and 5 mM MgCl2), which also contained the experiment to continuously provide fresh ferri/ferrocyanide.
redox probe ferri-/ferrocyanide, 1 mM each.
Within the duration of the experiment, the normalized QCM
frequency increased by more than 900 Hz, whereas the
dashed lines). There was no signicant dierence between the measured charge transfer resistance decreased by about 40%
values of these three impedance spectra, which can be explained compared to its initial value (see the Supporting Information,
by the fact that the electrochemical setup we used for combined Figure S-4 for details). In Figure 5 we also show a microscopic
QCM-D and EIS measurements was measuring signicantly image of a new QCM slide and of the QCM slide used for 26 h
faster than that applied during the combined SPR- and EIS EIS with ferri/ferrocyanide as the redox probe. This experiment
measurements. After hybridization with complementary target was repeated two more times with commercially available brand
DNA and subsequent rinsing with oxygen-free buer spanking new gold coated QCM slides equipped with a
containing the redox probe, a series of impedance spectra chromium or titanium adhesion layer at a lower ferri/
was collected, i.e., one spectrum was measured every 30 min ferrocyanide concentration (1 mM each).
over a period of 60 h. For clarity only every fourth spectrum is Independent of the nature of the adhesion layer underneath
depicted in Figure 4. the gold surface,43 both QCM slides showed a similar increase
As it can be seen from the rst spectrum (shown in black) in normalized frequency (approximately 500 Hz over 24 h for a
measured for the dsDNA modied surface, there was initially an slide equipped with a chromium adhesion layer and
increase in RCT. However, from the series of EIS measurements approximately 440 Hz over 16 h for a slide equipped with a
collected over a period of 60 h it can be seen that with titanium adhesion layer) during combined EIS and QCM
proceeding duration of the experiment, the value for RCT measurements, and serious damage of the surface was visible
decreased continuously. Furthermore, during the experimental under the microscope.
period of 60 h the QCM frequency increased by about 25 Hz Finally we proved whether it was possible to repeatably
(not shown). In order to prove that the observed changes measure hybridization/dehybridization at the same sensor chip
correspond to the etching of the gold surface but not to at optimized experimental conditions, i.e., relatively low
4388 DOI: 10.1021/acs.analchem.5b04814
Anal. Chem. 2016, 88, 43834390
Analytical Chemistry Article

concentration of redox probe, minimized detection times, and At one of the four chips even three repeatable measurements
absence of daylight. In the QCM-D electrochemical ow cell, could be carried out (this series of measurements is shown in
the sensor chip is shielded from light, but as it has been Figure 6, tted values of the RCT, etc. are given in Table S-1).
reported that the decomposition of ferri/ferrocyanide is When hybridization/dehybridization was then further con-
accelerated in the presence of light 400 nm,13,14,44 fresh tinued, the subsequent EIS spectra started to deviate
ferri/ferrocyanide containing buer solutions were prepared for signicantly from the impedance spectra measured during
each EIS measurement and stored in the dark. As shown in previous hybridization/dehybridization steps, whereas the
Figure 6, hybridization and dehybridization were followed by QCM-D response was still in accordance with the data
collected during previous steps. As these experiments were
carried out in the presence of KCl, and it has been reported that
in the presence of KCl cation dependent formation of the
insoluble prussian blue may occur,45 we repeated our
experiments also in potassium-free buer (using NaCl instead
of KCl). However, this did not lead to any improvement of the
repeatability.

CONCLUSIONS
In line with previous critical reports regarding the reliability of
EIS measurements, our results conrm that in contrast to SPR
and QCM-D EIS at gold electrodes, using the ferri/
ferrocyanide redox couple is not a reliable method to monitor
consecutive surface modication steps such as DNA hybrid-
ization/dehybridization, since the gold electrode will be etched
from CN formed by partial degradation of the redox
couple.1215 By optimizing the experimental conditions we
were able to monitor two up to three hybridization/
dehybridization steps by EIS in a repeatable manner, but
even at optimized experimental conditions decomposition of
ferri/ferrocyanide and the etching of the gold surface by CN
cannot be completely suppressed. Therefore, quantitative
measurements are not possible by this technique. Also when
consecutive surface modication steps at gold electrodes
leading to more sophisticated electrode architectures for
biosensor applications shall be followed, EIS using the ferri/
ferrocyanide redox couple may result in partial damage of the
biosensor electrode impairing its performance and should thus
not be used. This is expected also for cyclic voltammetry or for
scanning electrochemical microscopy measurements using the
ferri/ferrocyanide redox couple at gold electrodes. Besides the
Figure 6. QCM measurements (A) and EIS measurements (B) of decomposition of ferri/ferrocyanide, there are additional
three hybridization/dehybridization cycles performed on a gold coated
QCM-D sensor chip with titanium adhesion layer and modied with
limitations of EIS,12 and in contrast to SPR or QCM-D it
ssDNA, MCB, and MPA using the standard protocol as described cannot be applied straightforward. Therefore, we do not expect
before. After injection of ferri-/ferrocyanide (1 mM each), a short time EIS to become a reliable method, even if a stable redox couple
for re-equilibration of the QCM-D signal was left to ensure a as an alternative to ferri/ferrocyanide will be found.


homogeneous distribution of the redox probe in the cell. (A) QCM
normalized frequencies (overtones 313) collected during three ASSOCIATED CONTENT
hybridizations/dehybridization steps. Note that in the QCM-D
electrochemistry ow cell the noise during rinsing is much larger *
S Supporting Information
than in the conventional QCM-D ow cell. (B) Recording the The Supporting Information is available free of charge on the
impedance spectra started at 246 and 306 min for ssDNA and dsDNA ACS Publications website at DOI: 10.1021/acs.anal-
in the rst cycle, at 434 and 518 min in the second cycle, and at 612
and 687 min for in the third cycle, respectively. EIS was measured in a
chem.5b04814.
frequency range between 57 mHz and 100 kHz. Detailed experimental procedures, chemicals and in-
strumentation; SPR measurements of sensors with low
surface coverage of capture probes; and information
QCM-D and two consecutive EIS measurements per step using about combined SPR/EIS and QCM-D/EIS measure-
1 mM ferri-/ferrocyanide, each, were carried out. The presence ments (PDF)


of redox probe containing buer in the ow cell was minimized
to less than 10 min (red curve segments in Figure 6A). Even at AUTHOR INFORMATION
optimized conditions we could not monitor more than two
hybridization/dehybridization cycles repeatably with EIS for Corresponding Author
three out of four investigated sensor chips. *E-mail: noell@chemie.uni-siegen.de.
4389 DOI: 10.1021/acs.analchem.5b04814
Anal. Chem. 2016, 88, 43834390
Analytical Chemistry Article

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S.V. and Q.S. contributed equally. The manuscript was written (29) Peterson, A. W.; Heaton, R. J.; Georgiadis, R. M. Nucleic Acids
through contributions of all authors. All authors have given Res. 2001, 29, 51635168.
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The authors declare no competing nancial interest. (31) Riedel, M.; Kartchemnik, J.; Schoening, M. J.; Lisdat, F. Anal.

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4390 DOI: 10.1021/acs.analchem.5b04814


Anal. Chem. 2016, 88, 43834390