Full Paper
Special Issue MATRAFRED
DOI: 10.1002/elan.201400581
Title Monitoring Protamine-Heparin Interactions Using
Microcapillary Impedimetric Sensor
Natalia Abramova*[a, b] and Andrey Bratov[a]
Abstract: We report an impedimetric sensor based on an
interdigitated electrode array with electrode digits located
at the bottom of microcapillaries formed in silicon diox-
ide which is used to monitor protamine-heparin interac-
tions. Modification of the active sensor surface with prot-
amine, a cationic protein used as a high affinity heparin
antagonist, permits to register protamine-heparin com-
plex formation as changes in the surface conductivity de-
termined from the sensor impedance measured in an elec-
trolyte solution. The adsorption curve shows that the de-
tection limit is close to 0.01 U/mL (50 ng/mL) of heparin.
Keywords: Impedimetric sensor · Protamine · Heparin · Surface conductivity
1 Introduction
Heparin, the most charge-dense naturally occurring poly-
anion has been used medically as an intravenous anticoa-
gulant drug since 30ies of last century. During the antico-
agulant therapy and surgery close monitoring of heparin
levels is of vital importance to avoid complications such
as hemorrhage or thrombocytopenia induced by heparin
overdose [1]. The normal heparin concentration during
open heart surgery is between 2 and 8 U/mL, which cor-
responds to 0.8–3.2 mM or 12–48 mg/mL. In the treatment
of post-operative thrombosis and embolism, however, the
therapeutic concentration range of heparin is smaller
(0.2–0.7 U/mL). This implies that any method for heparin
analysis should be able to detect heparin at concentra-
tions as low as 0.2 U/mL [2].
Various chemical sensors for heparin detection have
been developed using appropriate signal transductions
such as spectroscopy [3–5], voltammetry [6, 7], quartz mi-
crobalance [7, 8] and conductometry [9]. The develop-
ment of potentiometric polyion sensors was pioneered by
M. Meyerhoff [10] and P. Bergveld in 1990s [11] and
more sophisticated methods based on application of
pulsed chronopotentiometric polymer membrane elec-
trode (pulstrode) [12, 13] and ion-selective membranes
operated by dynamic electrochemistry protocols [14]
were successfully used for heparin determination later.
More recent developments have focused on the search
for heparin indicator systems which involve an indicator
dye capable of exhibiting a significant switch-on or
switch-off response upon direct interaction with the hepa-
rin polysaccharide. Details about the progress in this area
may be found in a recent review “Heparin sensing and
binding” [15].
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The kinetics of the protamine-heparin complex formation
is rather slow and requires at least 15 min to be accom-
plished. Heparin reaction with protamine is irreversible,
however, it is possible to renew the sensor surface putting
it in contact with protamine solution and thus use the
sensor several times. The future prospects for the pro-
posed sensing strategy are quite wide and can include the
grafting of the sensor surface with modified nanoparticles,
magnetic particles or self-assembled structures based on
small synthetically-accessible molecular-scale building
blocks which can act as protamine mimetics.
During surgical procedure in which heparin was used,
there is an immediate need to neutralize the anti-coagu-
lant effect and allow blood clotting and recovery to
begin. Currently, the only licensed heparin antidote is
protamine sulfate [15]. Commonly, a fixed dose of 1.0–
1.3 mg of protamine for 1 mg (or 100 IU) of heparin is
used for heparin neutralization [16]. Although protamine
is universally used in clinical practice, it induces adverse
effects such as hypotension and idiosyncratic fatal cardiac
arrest [17]. Thus, the determination of both protamine
and heparin and monitoring of their interaction are
highly essential. There is no doubt that significant prog-
ress in heparin and protamine sensing has been reported
in recent years. In many protocol and sensing systems are
used the interaction between this two substances through
electrostatic binding between the cationic arginine groups
of protamine and the anionic heparin. Surprisingly, there
are some contradictions in results reported on kinetic of
this complex formation. The data presented in literature
show the reaction time ranging within 50–100 s [18], 3–
10 minutes [16, 19] or more [4, 8, 20].
Selectivity of protamine-heparin interaction was stud-
ied in many works and was demonstrated that it is possi-
ble to determine heparin concentration in human and
animal serum [2, 4, 7, 18]. The influence from blood elec-
[a] N. Abramova, A. Bratov
BioMEMS Group, Instituto de Microelectrnica de
Barcelona, Centro Nacional de Microelectrnica (CSIC)
Campus U.A.B., 08193 Bellaterra, Barcelona, Spain
*e-mail: natalia.abramova@imb-cnm.csic.es
[b] N. Abramova
Lab. Artificial Sensors Syst., ITMO University
Kronverskiy pr. 49, 197101 St. Petersburg, Russia
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trolyte, serum albumin and g-globulin in diluted samples
is minimal. The presence of heparin-based drugs (enoxa-
parin) or synthetic pentasacharide heparin analogues will
interfere.
Electrochemical impedance spectroscopy (EIS) is
a sensitive tool providing information on various physical
and chemical properties of materials, as well as on inter-
action processes occurring in the bulk or at the surface of
these materials. This method is wildly used to perform in-
vestigations in physical and biological sciences and it
plays an important role in the field of biosensors research
and development. Impedance biosensors lately gained
considerable interest due to their ability to perform label-
free detection and also due to potentially low cost and
ease of miniaturization [21]. A most popular impedimet-
ric transducer [22] is based on an interdigitated electrode
array (IDEA), a device with a pair of comb-like metal
electrodes formed on a planar insulating substrate. Pa-
rameters of IDEA based impedimetric sensors depend on
the electrodes geometry, i.e. dimensions and interspacing.
It is generally accepted [23] that 80 % of the total signal
is enclosed in a certain region close to the electrode sur-
face. Electric field penetrates within the distance equal to
the distance between centers of two adjacent electrodes.
If a monolayer of biomolecules with a typical length of 1–
100 nm is deposited on the electrodes or in the gap be-
tween the IDEA digits the main part of the signal will
depend mainly on the surrounding solution, in which
measurements are carried out and sensitivity of such devi-
ces will be rather low. To enhance the sensitivity of
IDEA sensors we have proposed [24] to separate the
electrode digits with an insulating barrier so that under
applied electric potential difference the main portion of
the current goes not through the surrounding solution but
close to the surface of the barrier modified with receptor
molecules. Changes in the electrical charge distribution at
the solid/liquid interface originating from (bio)chemical
reactions at the surface of the barrier cause respective
changes in conductivity along the barrier surface which is
registered by the device [25].
Fig. 1. Schematic of a 3D-IDE structure. Two adjacent electrode fingers made of TaSi2 are separated by a 4 mm height dielectric bar-
rier made of SiO2. w = 3 mm is the finger width, d = 3 mm is the distance between two fingers and h = 4 mm is the height of the barriers.
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The layer-by-layer (LbL) self-assembly of oppositely
charged polyelectrolytes is one of the simplest ways to
construct multilayer structures [26]. Protamine is a low
molecular weight protein, rich in basic arginine residues,
and has 20 positive charges at physiological pH. The poly-
cationic structure of protamine allows the tight electro-
static binding of polyanionic heparin to form a stable
complex. Modification of the active sensor surface with
protamine permits to register protamine-heparin complex
formation as changes in the surface conductivity deter-
mined from the sensor impedance measured in an elec-
trolyte solution.
2 Experimental
2.1 Sensor Fabrication
The sensor is an interdigitated electrode array (IDEA)
with 216 digits of 3 mm width and 3 mm gap between the
adjacent electrodes. The electrode digits are 1.5 mm long
and the aperture between the adjacent digits is 1.4 mm.
Details on design and fabrication of the device using con-
ventional microelectronic fabrication techniques are pre-
sented elsewhere [27]. In the final fabrication step the
wafer with formed IDEA devices is covered with a 4 mm
thick silicon oxide layer in which trenches are opened
over the electrodes digits and over contact pads by deep
reactive ion etching (DRIE), which permits to obtain
nearly vertical walls. In this way capillaries are formed in
silicon dioxide over the electrode digits. The capillaries
over each electrode opened at their top are 3 mm wide,
4 mm high, and of 162 mm total length. Fabricated device
is schematically presented in Figure 1.
2.2 Chemical Modification of the Sensors
Protamine (sulfate salt from salmon, Sigma) was ad-
sorbed on the silicon oxide surface of the sensors from
10 % PBS solutions (standard phosphate buffer saline
pH 7.4 diluted 1 : 10) of different concentrations. Interac-
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Fig. 2. Schematic presentation of the current paths between the electrodes (A) and possible electrical equivalent circuits (B, C). De-
tails are given it the text.
tion with heparin (sodium salt from porcine intestinal
mucosa, 180 USP units/mg from Aldrich) was registered
by measuring sensor impedance spectra in 1  10 5 M KCl
solution or 10 % PBS solution or by recording the real
part of the impedance at fixed frequency in PBS. To per-
form the layer-by-layer deposition the sensors were im-
mersed into the protamine solution (0.01–5 mg/mL in
10 % PBS) for 10 min, rinsed with a stream of water and
dried with a nitrogen flow. To deposit the next layer sen-
sors modified with protamine were immersed for 10–
20 min into a solution of heparin (0.01–1 U/mL in 10 %
PBS) rinsed and dried. This procedure may be repeated
several times until a desired number of layers is formed.
After each deposition step impedance spectra of sensors
were measured in 1  10 5 M KCl solution. More details
of experimental procedure are presented later in the text.
All experiments were performed at least on three indi-
vidual sensors.
2.3 Impedance Measurements
Characterization of sensors was performed by impedance
measurements in a 100 Hz–1000 kHz frequency range
with a 100 mV (amplitude) voltage excitation using
QuadTech 7600 Plus LCR Meter. Z-Plot/Z-View software
package (Scribner Associates, Southern Pines, NC, USA)
was used for impedance data treatment and an electrical
equivalent circuit fitting.The conductivity of test solutions
was controlled with a commercial conductimeter EC-
Meter GLP 31 + (Crison).
3 Results and Discussion
The studied 3D-IDEA sensor is presented schematically
in Fgure 1.
The system under study may be considered as two
162 mm long parallel in-plane microcapillaries with elec-
trodes at the bottom. Being filled with an electrolyte solu-
tion these two capillaries are interconnected through the
solution bulk. A potential difference applied between two
electrodes causes electrical current to flow between the
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Special Issue MATRAFRED
electrodes from one capillary to another. When a tangen-
tial electric field is applied at the solid/solution interface,
due to a higher concentration of ions within the electrical
double layer, the local electric surface current can be
higher than that typical of the bulk electrolyte solution
(see Figure 2A). Adsorption of charged molecules at the
interface alters the charge distribution and thus the sur-
face conductivity.
Analyzing the possible electrical equivalent circuit of
the microcapillary system presented in Figure 2A and
taking into consideration that the surface conductivity
along the capillary wall may play an important role, we
may distinguish the following components which are pre-
sented in Figure 2B.
RC is the contact resistance introduced by wires and
collector bars of thin film electrodes; CG is the geometri-
cal (stray) capacitance between two interdigitated electro-
des in a water solution; RB is the resistance of the bulk
water solution between two electrodes of the array;
CPEDL is a constant phase element associated with the
capacitance of the electrode – water solution interface.
The solution bulk resistance, RB, is in parallel with the re-
sistance of the surface layer; RSURF, and a capacitive ele-
ment, CSURF, between two resistances. In low conducting
solutions RB and RSURF are of the same order of magni-
tude and it is not possible to distinguish these two ele-
ments from the impedance spectra by model fitting. In
this case the equivalent circuit presented in Figure 2C is
used for spectra fitting in which the resistance RS repre-
sents the contribution of both elements RB and RSURF. In
poorly conducting solution RB may be fixed by experi-
mental conditions so that all changes in RS may be attrib-
uted to surface conductivity changes. In highly conductive
solutions the difference between RS and RSURF may be
several orders of magnitude and in these cases RSURF may
be determined separately [28].
3.1 Sensors Characterization
Figure 3 presents chemical structures of protamine and
heparin. Protamine being a polycation can be very easily
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Fig. 3. Chemical structure of protamine and heparin and LBL deposition scheme.

Fig. 4. Adsorption kinetics of protamine in PBS buffer at different concentration of polycation.

deposited on negatively charged SiO2 surface of the
sensor by layer-by-layer (LBL) method [26] as presented
in Figure 3. Being exposed to solution containing heparin,
acting as a polyanion, surface protamine layer will react
with heparin forming another upper surface layer.
Impedance measurements may be performed in two
different ways, either by registering the impedance spec-
trum in a wide frequency range or by measuring impe-
dance at a fixed frequency [28, 29]. In the first case fitting
the spectrum to an equivalent circuit permits to extract
individual circuit components associated with physico-
chemical processes. In the second, it is possible to moni-
tor changes in real time and thus obtain information on
the kinetics of studied processes. In this way real time
monitoring of the protamine adsorption kinetic on the
sensor surface was performed.
Measurements at fixed frequency of 160 Hz were car-
ried out in 10 % PBS buffer at different protamine con-
centrations. Obtained results are shown in Figure 4. At
low (0.01 mg/mL) protamine concentration 180–200 s are
required to achieved a steady state situation where value
of the real part of impedance does not change in time.
For more concentrated solutions (0.1–5 mg/mL) this time
rises up to 400–500 s. The changes in the base line be-
tween solutions with different concentration of protamine
in the Figure 4 are due to increase of conductivity of solu-
tions with higher protamine concentration.
To check the repeatability of protamine-heparin LBL
deposition and formation of a multilayer structure the
sensors were successively exposed to 10 % PBS buffer sol-
utions of 0.1 mg/mL of protamine (P) and 1 U/mL of hep-
arin (H) during 10 minutes. After each deposition step
the sensors were rinsed thoroughly with water and their
spectra were measured in 1  10 5 M KCl solution with
conductivity of 2.5 mS/cm. Subsequently, RS values in each
case were determined by fitting the spectra to the equiva-

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Fig. 5. RS values obtained by fitting impedance spectra of the IDEA devices measured in 10–5 M KCl solution after the successive
deposition of protamine (P) and heparin (H). In the inset impedance spectra corresponding to the first three points on the graph are
presented.
lent circuit (Figure 2C). Obtained results, typical for LBL
polyion deposition, are presented in Figure 5.
As follows, adsorption of protamine on a silicon diox-
ide sensor surface causes increase of R values. SiO2 at
neutral pH is negatively charged. Mobile cations from so-
lution arrive to compensate this charge increasing the sur-
face conductivity [29]. Protamine layer is positively
charged and anions at the surface are less mobile than
cations which provokes the increase in resistance. Hepa-
rin behaves as a polyanion and its deposition over the
protamine layer compensates its electrical charges and re-
sults in considerable decrease of the resistance as the neg-
atively charged surface attracts more mobile cations. As
the bulk electrolyte conductivity in these experiments was
maintained constant, all the changes may be attributed to
the effect of charges at the solid/liquid interface and their
effect on the surface conductivity RSURF.
Heparin reaction with protamine is irreversible, howev-
er, as we can see, it is possible to renew the sensor surface
putting it in a contact with protamine solution and thus
use the sensor several times. This approach permits to in-
vestigate the adsorption kinetic of heparin from solutions
of different concentrations.
Kinetic curves were measured in 0.04–0.12 U/mL hepa-
rin concentration range during reaction of this polycation
with protamine at the fix frequency and are shown in
Figure 6. In this case after each modification of sensors
with protamine (0.1 mg/mL in 10 % PBS) electrode was
introduced in solution with different concentration of
heparin in diluted PBS with conductivity of 34 mS/cm.
The conductivity of the solution was chosen at such
a level that even at high heparin concentration changes of
overall conductivity were negligible.
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In contrast to publication by Gaus and Hall [19] we
have found that the kinetics of protamine-heparin com-
plex formation is rather slow and requires at least 15 min.
to be accomplished.
These results permitted us to obtain the calibration
curve of sensors in 10 % PBS buffer solutions with differ-
ent heparin concentration starting from very low level of
0.01 U/mL. Before each measurement sensors were treat-
ed during 10 minutes with protamine to renew the sur-
face. Impedance spectra were registered after soaking
sensors during 15 minutes in solution of heparin. RSURF
values were obtained by fitting the spectra to the corre-
sponding equivalent circuit (Figure 2B). Resulting calibra-
tion curve is presented in Figure 7.
Obtained results show that the limit of detection is
really low and changes in measured spectra and consecu-
tive R values were registered at 0.01 U/mL, heparin level
that is 20 times lower than therapeutic concentration of
0.2 U/mL. These results give the possibility to work in di-
luted solutions of real samples reducing possible interfer-
ence by other charged proteins that may be present.
4 Conclusions
Presented microcapillary impedimetric device is shown to
be a useful sensitive tool to control changes in electrical
charges at the surface in contact with electrolyte solution.
Surface charge alterations produced by irreversible sur-
face reactions may be controlled by measuring the device
impedance. The sensors permit to monitor protamine-
heparin reaction in water solutions and thus determine
heparin with the detection limit close to 0.01 U/mL
(50 ng/mL). Though protamine-heparin interaction is irre-
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Fig. 6. Adsorption kinetics curves of heparin in diluted PBS buffer (34 mS/cm) at different heparin concentrations. After contact with
heparin the sensor was treated with protamine to renew the surface.

Fig. 7. Calibration curve obtained from RSURF values determined from impedance spectra measured in 10 % PBS buffer solutions
with different heparin concentration.

versible it is possible to renew the sensor surface by con-
secutive treatment with protamine using the layer-by-
layer deposition methods. Another useful option that
gives the proposed sensor is the possibility of surface re-
actions kinetics studies. Thus it was shown that adsorption
of protamine on silicon dioxide surface reaches saturation
in 3–10 minutes depending on its concentration. On the
other hand, protamine interaction with heparin was deter-
mined to be rather slow and requires at least 15 minutes
to be accomplished.
A new three-dimensional design of an interdigitated
electrode array being very sensitive to charge formation
at a solid/liquid interface offers new possibilities to study
interactions between heparin and non-toxic protamine al-
ternative synthetic compounds that are now under inves-
tigation by many research groups. All these compounds
can be applied using layer-by-layer deposition method on
the surface of interdigitated electrode and their reactions
with heparin may be investigated.
The future prospects for the proposed sensing strategy
are quite wide and can include the grafting of the sensor

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surface with modified nanoparticles, magnetic particles or
self-assembled structures based on small synthetically-ac-
cessible molecular-scale building blocks which can act as
protamine mimetics.
Acknowledgements
This work was partially supported by the Spanish Ministry
of Economy and Competitiveness (Projects: IPT-2011-
1055-900000 and CTQ2011-29163-C03-02) and by the
Government of the Russian Federation, Grant 074-U01.
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Received: October 14, 2014
Accepted: November 7, 2014
Published online: && &&, 2014
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FULL PAPERS
N. Abramova,* A. Bratov
&& – &&
Title Monitoring Protamine-Heparin
Interactions Using Microcapillary
Impedimetric Sensor

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