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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
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
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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