Biosensors & Bioelectronics 13 (1998) 1061–1068

An immunological Interleukine-6 capacitive biosensor using

perturbation with a potentiostatic step
*
Christine Berggren, Bjarni Bjarnason, Gillis Johansson
Department of Analytical Chemistry, University of Lund, P.O. Box 124, S-221 00 Lund, Sweden

Received 23 February 1998; received in revised form 1 May 1998; accepted 1 May 1998

Abstract

An instrument for potentiostatic capacitance measurements, based on perturbation with a potentiostatic step was used. The capaci-
tive sensor consisted of self-assembled sulfur compounds on gold to which antibodies towards Interleukine 6, Il-6, had been immobil-
ized. The biosensor was part of a potentiostatic three-electrode system with an extra reference electrode. Two different methods
using epoxy- or carbodiimide coupling of the polyclonal antibodies were compared. The antigen Il-6 could be detected from 5 ⫻
10−16 to 5 ⫻ 10−13 M with one immobilization method and to more than 5 ⫻ 10−9 M with the other. No labels were necessary
since the binding of the antigen was detected directly. The insulating properties of the different layers of the biosensor were
investigated.  1998 Elsevier Science S.A. All rights reserved.

Keywords: Capacitive biosensor; Interleukine 6; Immunosensor; Self assembled monolayers

1. Introduction pally different ways. In the first approach two parallel

There is constantly a search for sensitive, fast and easy
analytical methods. Immunosensors are promising as
they show high specificity, selectivity, fast response time
and can be developed for many different substances. The
sensitivity obtained depends on the transducer and on
the affinity of the biological sensing molecules. Several
different direct transducers have been used, such as
potentiometric (Blackburn et al., 1990; Keating and
Rechnitz, 1984; Taylor et al., 1991), piezoelectric (Davis
and Leary, 1989; Muramatsu et al., 1987; Roederer and
Bastiaans, 1983) and optical (Chaiken et al., 1992;
Huber et al., 1992; Johnsson et al., 1991; Löfås, 1995;
Löfås and Johnsson, 1990; Nellen and Lukosz, 1991).
Recently also capacitive transducers have been described
(Ameur et al., 1997; Barraud et al., 1993; Bataillard et
al., 1988; Berggren and Johansson, 1997; Berney et al.,
1997; Billard et al., 1991; Gardies et al., 1989; Gebbert
et al., 1992; Maupas et al., 1996; Saby et al., 1993;
Schyberg et al., 1995; Souteyrand et al., 1994).
Capacitance measurements can be made in two princi-
* Corresponding author. Tel.: + 46-46-222-8170; Fax: + 46-46-222-
4544; E-mail: gillis.johannson@analykem.lu.se
metal plates are used with the biorecognition element in
between. The capacitance of the medium between them
can be measured with a high precision capacitance
bridge. The capacitance is described by the following
equation:
C ⫽ ⑀⑀oA/d (1)
where, ⑀ is the dielectric constant of the material
between the plates, ⑀o is the dielectric constant for vac-
uum, A is the area and d the distance between the plates.
According to the equation the capacitance will increase
when the distance between the two plates decreases. If
the recognition element is immobilized on the electrodes
the sensitivity will increase when the distance decreases.
There will be a mechanical limitation, though in produc-
ing a short and reproducible distance between the two
plates. Interdigitated electrodes can be made with very
short distances between the plates and they have been
used as antibody-based biosensors (Taylor et al., 1991)
with a sensitivity down to 1 ng/ml for human IgG.
Another limitation associated with this approach is that
this method is very sensitive to changes in the bulk sol-
ution. This last problem can be overcome by using a
reference cell, without the recognition element present.

0956-5663/98/$—see front matter  1998 Elsevier Science S.A. All rights reserved.
PII: S 0 9 5 6 - 5 6 6 3 ( 9 8 ) 0 0 0 5 8 - X
1062 C. Berggren et al. / Biosensors & Bioelectronics 13 (1998) 1061–1068
In the second approach the capacitance is measured
at the solution–electrode interface in a potentiostatically
controlled three-electrode system. Ions and dipoles are
ordered outside a metal electrode in such a way that
charges in the metal are balanced, thereby forming the
electrical double layer. As capacitance measurements
give information about the metal–solution interface, a
chemical modification of this boundary should give rise
to a change in capacitance, the size of which will depend
on the nature and coverage of the modification material.
Eq. (1) will still be valid if d is the thickness of the
surface immobilized layer and ⑀ the dielectric constant of
the self-assembled layer including possible contributions
from a remaining Stern layer. The diffuse Gouy and
Chapman capacitance is usually much larger (Janek et
al., 1997) and can therefore be neglected as a first
approximation.
Very thin anodically produced oxide layers can be for-
med on valve metals like tantalum and be functionalized
by silanization (Gebbert et al., 1994, 1992). Silanized
SiO2 semiconductor surfaces on various conducting sup-
ports like gold or silicon have also been used for moni-
toring capacitance changes (Bataillard et al., 1988;
Billard et al., 1991; Gardies et al., 1989; Saby et al.,
1993; Souteyrand et al., 1994). Gold electrodes can be
functionalized through self-assembled monolayers of thi-
ols, sulfides or disulfides (Taira et al., 1993). The final
goal of such attempts is to add a recognition element,
e.g. antibodies, to the surface so that a binding of the
analyte can be detected through the resulting capaci-
tance changes.
This paper describes capacitive immunosensors for Il-
6 using two different immobilization methods both based
on coupling to self-assembled molecules on gold sur-
faces.
2. Materials and methods
2.1. Chemicals
Polyclonal Interleukine-6 (Il-6) antibody and the
recombinant antigen Il-6 (苲 20.3 kDa) both came from
R&D Systems, Minneapolis, MN. 1-3-(dimethylamino)
propyl-3-ethylcarbodiimide hydrochloride (EDC) was
obtained from Fluka AG, Buchs, Switzerland. All other
chemicals used were of analytical grade. All buffers
were prepared with water obtained in a Milli-Q system,
preceded by a reverse osmosis step, both from Millipore,
Bedford MA. Before use they were filtered through a
millipore filter with a pore size of 0.22 ␮m (Millipore,
Bedford, MA) and degassed.
2.2. Preparation of electrodes
Gold rods (Aldrich, 99.99%) with a diameter of 3 mm
were polished with alumina slurries (Struers,
Copenhagen) down to 0.04– 0.05 ␮m. The electrode was
mounted in a Teflon holder and placed in a plasma cle-
aner (Mod. PDC-3XG, Harrich, NY) for 15 min. Two
different procedures were used for antibody immobiliz-
ation.
The first procedure uses carbodiimide activation as
described before (Duan and Meyerhoff, 1994). The elec-
trode was put in a thioctic acid solution (2% w/w in
absolute ethanol) immediately after the plasma cleaning.
After reaction over night, it was thoroughly rinsed with
ethanol and dried under vacuum. It was then reacted with
carbodiimide (EDC, 1% w/w in dry acetonitrile) for 5 h,
thoroughly rinsed and dried. Thereafter the electrode was
dipped into an antibody solution ( 苲 1 mg/ml, Il-6) in
100 mM borate buffer, pH 8.75 and reaction took place
overnight at 4°C. Finally, the electrode was reacted in a
10 mM 1-dodecanethiol buffer solution for 20 min.
The second procedure uses a cysteamine activated
electrode. Immediately after the plasma cleaning the
electrode was dipped into a cysteamine solution (2%
w/w in absolute ethanol) overnight. It was washed and
dried and then reacted in a mixture of 50 ␮l 1,4-butane-
diol diglycidyl ether and 4500 ␮l 150 mM borate buffer,
pH 9 for 24 h. The electrode was thoroughly rinsed and
50 ␮l 150 mM borate buffer, pH 9 containing 苲 2 mg/ml
Il-6 antibody was put as a droplet on top of it. Reaction
took place overnight at room temperature. Finally, the
electrode was reacted in a 10 mM 1-dodecanethiol buffer
solution, pH 7.4, for 20 min.
2.3. Measuring system
The capacitive sensor was part of a potentiostatic
three-electrode system with an extra reference electrode
(see Fig. 1). A Keithley 575 measurement and control
system (Keithley Instruments, Cleveland, OH) was con-
nected between a potentiostat and a computer (486,
33 MHz). The Keithley 575 contained 16-bit A/D and
12-bit D/A converters which could sample or send with
a frequency of 50 kHz. Current readings were made
every 20th ␮s after the application of a potentiostatic
step until 1000 readings had been collected. The poten-
tial was then stepped back to the rest potential and
another 1000 readings were collected. Another potent-
iostatic pulse was applied after 1 s, a time which could
have been shortened if necessary. Averages were formed
from ten pulses. To reduce noise in the system, the
potentiostat was powered from the Keithley instrument,
which in turn was powered from the computer through
a galvanically insulating power supply. This isolates the
noisy computer power from the sensitive analogue parts.
A flow cell with a dead volume of 10 ␮l was used
(see Fig. 1). Four electrodes were mounted in the cell
and connected to the potentiostat. The working elec-
trode, a gold rod, (diameter 3 mm) was placed in a
Teflon holder, before it was inserted into the cell. The
 C. Berggren et al. / Biosensors & Bioelectronics 13 (1998) 1061–1068
Fig. 1. Measuring system including a flow-through cell with a dead volume of approximately 10 ␮l. The buffer is pumped through the cell in
the arrow direction.
auxiliary electrode was a disc shaped platinum foil
(diameter 15 mm) and the reference electrode was a
platinum wire (diameter 0.5 mm) inserted through a hole
in the auxiliary electrode. As the potential of this refer-
ence electrode will drift, its potential had to be compared
to a commercial reference electrode, which was situated
in the outlet flow stream. The computer calculates the
potential which has to be applied to the platinum wire
in order to make it behave as an Ag/AgCl reference. The
platinum reference electrode had been platinized in 20%
hexachloroplatinic acid in 2 N HCl at 4.2 mA/cm2 for
15 min. The rest potential was usually 0 mV vs an
Ag/AgCl reference electrode and the potentiostatic step,
used to perturb the system normally had an amplitude
of 50 mV.
A phosphate buffer, pH 7.0 with various electrolyte
concentrations was used to study the current response.
For the antigen interaction studies a 10 mM phosphate
buffer, pH 7.4 containing 0.5 g/l Tween 20 and 0.5 g/l
sodium azide was used. All experiments were performed
at a flow rate of 0.5 ml/min with an injection loop of
250 ␮l. PEEK-tubings were used for all inlet connec-
tions to prevent air intrusion into the system resulting in
air bubbles on the electrode.
The biosensor could not be regenerated and a new
electrode was therefore prepared for each measurement.
A new cell with disposable gold plates is under way.
1063
3. Results and discussions
3.1. Cell design
It has been observed earlier that a normal Luggin
capillary will distort the planar field between the work-
ing and auxiliary electrodes (Swietlow et al., 1992). A
symmetrical field will give sharper current response
curves for fast rise times. Improved current and potential
shapes (in terms of noise, offset and ringing) were
obtained when the distances between the working and
the auxiliary electrodes were large (Swietlow, 1994).
Unfortunately this resulted in large cell volumes. The
first flow cell used by Swietlow et al. (1992) had a vol-
ume of 5.5 ml and a second redesigned cell 2 ml
(Berggren and Johansson, 1997). A platinum wire elec-
trode can have a small diameter and be placed close to
the working electrode with minimal field distortion. It
has, furthermore, a simpler equivalent circuit than a
Ag/AgCl reference with a salt bridge. This gives an
improved potentiostatic control of the working electrode
potential resulting in suppressed ringing and improved
sharpness of the current transient after a step. As men-
tioned in Section 2, a computerized comparison with a
Ag/AgCl electrode in the outlet corrected the drift of the
platinum electrode. The system behaves as if it had had
a Ag/AgCl reference but with improved high frequency
properties. An additional advantage with a platinum wire
1064 C. Berggren et al. / Biosensors & Bioelectronics 13 (1998) 1061–1068
as the reference electrode is that there is no leakage of
salt bridge solution into the measuring cell.
These changes made it possible to move the auxiliary
electrode very close to the working electrode reducing
the cell volume to 10 ␮l and still have excellent transient
responses. The analytical sensitivity was about the same
in this cell as in the 2 ml cell (Berggren and Johansson,
1997). The mean residence time is of course much larger
in the 2 ml cell but an entering sample is diluted to a
greater extent than in the 10 ␮l cell. With an injection
volume of 250 ␮l, a stable capacitance reading was
obtained after 15 and 10 min, for the respective cells at
a flow rate of 0.5 ml/min. A stable reading indicates that
the analyte substance has been flushed out completely.
The hydrodynamic design of the 10 ␮l cell seems thus
to be less than optimal and a redesign is under way.
3.2. Evaluation of capacitance
The total capacitance of the working electrode surface
is built up from several components. The first is the
capacitance of the self-assembled monolayer (SAM)
including any contributions from a remaining Stern
layer, CSAM. It consists of the thioctic acid or cysteam-
ine, the antibodies as well as the 1-dodecane thiol mol-
ecules. Another part is the diffuse Gouy and Chapman
layer, CGC, which is located outside the SAM- as well
as the antigen layers. When antigens bind they form a
hydrophobic layer in contact with the antibodies of the
SAM, thereby forcing the diffuse layer further out into
the solution. The capacitance of this layer will be CAG.
CAG and CGC will be interdependent if the surface is less
than saturated with antigen. The total capacitance will
then be
1/Cs ⫽ 1/CSAM ⫹ 1/CGC ⫹ 1/CAG (2)
A SAM-layer cannot be expected to have a well-
defined outer surface like those alkanethiols and hydrox-
ythiols which have been studied in the literature (Janek
et al., 1997). The antibodies are bound in a random
orientation with the immobilization methods used here.
Furthermore the surface of an antigen may be irregular
on the atomic scale. The distances between the outer
SAM-surface and the metal will therefore vary along the
surface. The diffuse layer will fold around the antibody
and antigen molecules giving a complex contribution to
the overall capacitance. The analytical signal arises
because additional antigens fill up unoccupied antibody
sites displacing some of the diffuse layer further out into
solution. The capacitance will decrease because a con-
ducting aqueous solution is being replaced by a hydro-
phobic antigen.
The impedances of even the most well-organized self-
assembled layers (Janek et al., 1997) are far from ideal
and the experimental data are therefore translated into
more or less complex equivalent circuits. A certain equi-
valent circuit can be transformed into other models at
a given frequency but because the circuit elements are
frequency-dependent the finally selected representation
will be more or less arbitrary. A four-element model
with two capacitors and two resistors, a Randles’ circuit,
normally gives a better fit to a current–time plot than
a two element model (Swietlow et al., 1992). For self-
assembled sensors with antibodies bound to the surface
a simple RC-model will give a better fit in the relevant
region at the beginning of the transient than a Randles’
circuit (Berggren and Johansson, 1997). Evaluation of
the capacitance was therefore made by assuming that the
system resembled a capacitor and a resistor in series (an
RC-model). However, other models can be fitted to the
transient by non-linear iteration in an off-line program
like Matlab (MathWorks, Natick, MA). In the RC-model
the current transient evoked by a potentiostatic step will
follow Eq. (3):
i(t) ⫽ u/Rsexp( ⫺ t/Rs ⫻ Cs) (3)
where i(t) is the current in the circuit as a function of
time, u is the amplitude of the potential step, Rs is the
dynamic resistance of the self-assembled layer and the
ionic cloud in the solution, Cs is the series capacitance
measured at the working electrode/solution interface and
t is the time elapsed after the potentiostatic step was
applied. Taking the logarithm of the current vs time
results initially in an almost linear curve from which Rs
and Cs can be calculated. The first two readings were
discarded and the next ten current values were collected
and used for the calculation. A correlation coefficient
better than 0.99 was usually obtained. The potential was
stepped back to the rest potential when the current had
attained a steady value, usually after 20 ms. The time
constant for the system (Rs ⫻ Cs) is a measure of how
fast the current will decay. This means that systems with
low values on the capacitance and the resistance are
more difficult to evaluate than those with higher values.
3.3. Current response at different electrolyte
concentrations
The current responses following a potentiostatic step
of 50 mV is shown in Fig. 2 a for a carbodiimide bound
antibody (Il-6) electrode in phosphate buffer, pH 7.0, at
different buffer concentrations. Log(i) vs time is plotted
in Fig. 2(b). Here it can be seen that the simple RC-
model becomes worse as the ionic strength increases.
The time constants and correlation coefficients are given
in Table 1. As the electrolyte concentration is increased
the diffuse ion cloud outside the protein layer will be
compressed resulting in an increased capacitance. It can-
not be excluded that the protein layer itself and its
capacitance also will be affected by changes in the ionic
 C. Berggren et al. / Biosensors & Bioelectronics 13 (1998) 1061–1068 1065

Fig. 2. (a) Current decay curves, at different phosphate buffer concentrations, obtained after a potentiostatic step of 50 mV vs Ag/AgCl. (b) Log(i)
vs time for the current decay curves shown in Fig. 2(a).

Table 1 quencies and the capacitance value will therefore depend

The table shows time constants and correlation coefficients at different on the range used for linearization.
phosphate buffer concentrations, pH 7

Concentration Capacitance Resistance Correlation Time 3.4. Direct detection of Il-6 antigen
(mM) (nF cm2) (⍀ cm2) coefficient constant (R*
C) (␮s)
Interleukines have major roles in inflammatory and
5 2210 331 0.998 731 immuno responses and their levels can be affected by
10 2500 178 0.997 445 e.g. malignancies, psoriasis and autoimmune diseases.
20 2930 100 0.994 295 The natural levels are often below 1 pg/ml (5 ⫻ 10−14 M
100 5260 32 0.967 170 for Il-6), which is the normal detection limit of commer-
250 7060 20 0.948 144
cial ELISA kits. They are therefore suitable targets for
the capacitive sensors with their inherent high sensi-
tivity. Previous work (Berggren and Johansson, 1997)
strength. The capacitance of the ion cloud is in series has shown that the interaction between an immobilized
with the capacitance of the protein layer and the total antibody and an antigen from the solution can be
capacitance, Cs, will therefore also increase. The series detected directly without any labels. All the antibodies
resistance will rapidly be reduced so that the product studied, including the interleukine Il-2, were immobil-
will be diminished resulting in a reduced time constant ized using carbodiimide coupling. Another interleukine,
thus giving a faster current response. This reduces the Il-6, a 20.3 kDa protein formerly also called interferon-
precision of the capacitance evaluation as we are forced ␤2 among other names, was chosen for studies in this
to linearize a very limited part of the curve. There is work. A polyclonal Il-6 antibody was immobilized in the
also a disadvantage of linearizing only a small piece of same way as described before (Berggren and Johansson,
the initial part of the curve as potentiostats, however 1997) and the results are shown in Fig. 3. Data on Il-2
good they may be, always produce some kind of distor- from the previous paper (Berggren and Johansson, 1997)
tion at the beginning of the potentiostatic step. Fitting are also included for comparison. It can be seen that
to fewer points will give another capacitance value than the range for Il-6 extends to lower as well as higher
fitting to a greater portion of the curve because the lin- concentrations of the antigen but with a lower slope. Il-
earity is not perfect. This is due to the fact that the 6 detection is possible at about two orders of magnitude
capacitance is frequency dependent and as a pulse can lower antigen concentration. A reason for the difference
be considered to be a sum of sinusoidal waves with the is probably that a monoclonal antibody was used for Il-
highest frequencies in the beginning. Adding more and 2 while a polyclonal one has been used in this work.
more points will increase the weight of the lower fre- The polyclonal preparation will contain antibodies with
1066 C. Berggren et al. / Biosensors & Bioelectronics 13 (1998) 1061–1068
Fig. 3. Capacitance change vs the logarithm of Il-6 concentration for
an electrode prepared by the carbodiimide method. The capacitance
was measured in a flow system with a carrier of 10 mM phosphate
buffer, pH 7.4 containing 0.5 g/l Tween 20 and 0.5 g/l sodium azide,
at a flow rate of 0.5 ml/min. The capacitance value was taken 15 min
after injection of analyte.
greatly different affinities for the antigen thereby giving
a greater range.
Epoxy immobilization was also studied for Il-6 anti-
body. Fig. 4 shows that an analytically useful response
will be obtained over a rather narrow analyte concen-
tration range compared to that of the carbodiimide
immobilized antibody. Compared with the carbodiimide
immobilization, the epoxy immobilization resulted in
electrodes with larger initial capacitance 9050 vs
4400 nF/cm2. The slopes of the calibration curves were
also different, 370 and 20 nF/cm2/concentration decade,
for epoxy and carbodiimide-immobilized antibodies,
respectively. The detection limits are comparable but
saturation occurs at much lower antigen concentration
for the epoxy immobilized antibody.
No decrease in capacitance was seen when Il-2 was
injected into a system with a Il-6 antibody coated surface
which proves that the biosensor is specific for its antigen.
Fig. 4. Capacitance change vs the logarithm of Il-6 concentration for
an electrode prepared by the epoxy method. Experimental conditions
as in Fig. 4.
The reproducibility from electrode to electrode was
30–40%.
The high capacitances for the epoxy activated elec-
trodes compared to those for the carbodiimide activated
ones are probably due to the larger polarity of the epoxy
chain, see Fig. 5, thereby increasing the value of CSAM,
(Eq. (2)). It is known that incorporation of ⫺ OH groups
in alkanethiols will increase the capacitance drastically
(Janek et al., 1997; Swietlow et al., 1992). The capaci-
tance changes when binding antigen are also greater,
both absolutely and relatively, for the epoxy activated
electrodes. This can be explained by the larger influence
of CAG on Cs when CSAM is large (Eq. (2)). This means
that the capacitance change upon binding an antigen
affect the total capacitance more.
Common methods to regenerate the antibody surface
were previously found to destroy the thiol layer and a
new coating had to be applied for each measurement
(Berggren and Johansson, 1997). Experiments are under
way to use gold sputtered on silica as disposable work-
ing electrodes. It should be possible to use the biosensor
in real biological samples as it was found previously that
serum did not affect the response once it had been
flushed out of the cell.
3.5. Electron transfer through SAM-antibody layers
Electron transfer between a redox couple in solution
and the metallic gold surface can be through holes in
the SAM-antibody layer, through penetration into the
layer or through tunneling across the layer (Becka and
Miller, 1992). Defects or impurity atoms might give
holes but they are supposed to be covered through a col-
lapse where long alkane thiols fold over and isolates the
Fig. 5. The figure shows antibody immobilization to gold, with (a)
the carbodiimide method and (b) epoxy immobilization with 1,4-but-
anediol diglycidyl ether. After antibody immobilization the electrode
is treated with a long hydrocarbon thiol, 1-dodecanethiol, for 20 min.
 C. Berggren et al. / Biosensors & Bioelectronics 13 (1998) 1061–1068
Fig. 6. Cyclic voltammograms of a carbodiimide immobilized poly-
clonal Il-6 antibody electrode obtained in a 5 mM K3Fe(CN)6 solution
at a scan rate of 10 mV/s. All potentials are given vs SCE. (a) An
unmodified gold electrode and (b) a carbodiimide immobilized anti-
body electrode additionally treated with 1-dodecanethiol for 20 min.
site. Cyclic voltammetry in the presence of a fairly
reversible redox couple in solution will display how vari-
ous layers will insulate the metal surface. Fig. 6 shows
that an electrode with carbodiimide immobilized Il-6
antibody, treated with 1-dodecanethiol, blocks most of
the redox processes from a 5 mM K3Fe(CN)6-solution.
A more detailed picture of the blocking has been shown
previously (Berggren and Johansson, 1997). A cyclic
voltammogram for pure gold is included for comparison.
A detailed picture of the blocking of electrodes with
epoxy-bound Il-6 antibody is shown in Fig. 7. Electron
transfer through cysteamine itself gives about half the
currents obtained on pure gold (compare Fig. 6).
Reacting this layer to produce the epoxy compound
reduced the current somewhat more. Big antibody mol-
ecules immobilized on top of the underlying layers had
Fig. 7. Cyclic voltammograms of an epoxy immobilized polyclonal
Il-6 antibody electrode obtained in a 5 mM K3Fe(CN)6 solution at a
scan rate of 10 mV/s. All potentials are given vs SCE. (a) Shows a
gold electrode coated by self-assembled cysteamine; (b) the same after
epoxy activation; (c) as in (b) after immobilization of the Il-6 anti-
bodies and (d) as in (c) after treatment with 1-dodecanethiol for
20 min.
1067
surprisingly little effect, considering the size of the mol-
ecule. The explanations might be that the antibodies are
not closely packed or that the redox couple can penetrate
into the structures. Alkanethiols, finally, reduce the
redox currents very substantially, probably because they
will penetrate down to the gold surface thereby blocking
direct access for the redox couple. A comparison
between the cyclic voltammograms after 1-dodecane-
thiol-blocking in Fig. 6 and Fig. 7 (note the difference
in scales!) shows that the currents are about twice as
large with epoxy- as with carbodiimide immobilization.
The capacitive contributions to the currents are small as
can be seen from the sharpness at the far negative ends.
An enlargement (not shown) shows that the currents are
at maximum at, or slightly later than, the peaks on gold
or cysteamine.
Electron tunneling has been studied for long-chain
hydroxy thiol self-assembled layers (Becka and Miller,
1992; Miller et al., 1991). Although our coatings are less
well-defined and possibly have more remaining holes
than those mentioned above, tunneling might be of
importance for the self-assembled antibody electrodes.
The overpotential of peaks becomes larger as the thick-
ness of the coating increases. The size of the currents
depends also on the selection of test redox couple (Becka
and Miller, 1992).
Plugging with 1-dodecanethiol is of importance from
a practical point of view. The background current is gre-
atly diminished which makes it possible to measure very
small capacity changes. It was shown earlier (Berggren
and Johansson, 1997) that it was possible to detect bind-
ing of antigen to 0.05% of the monoclonal antibodies.
The plugging makes the electrodes fairly insensitive to
interfering substances in the solution.
4. Conclusions
It has been shown that a biosensor for Il-6 can be
prepared using different immobilization methods. The
epoxy activated electrode had a much narrower range
but a higher sensitivity than the electrode activated with
thioctic acid. The reason is that the epoxy activated layer
has a high background capacitance which facilitates
detection of small changes. The detection limit is orders
of magnitude below the presently used clinical tests.
Acknowledgements
This research was supported by grants from the Swed-
ish Natural Research Council.
1068 C. Berggren et al. / Biosensors & Bioelectronics 13 (1998) 1061–1068
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