Sensors and Actuators B 78 (2001) 249±256

Electrochemical impedance spectroscopy as a platform

for reagentless bioaf®nity sensing
Geoffrey Lilliea,*, Peter Payneb, Pankaj Vadgamac
a
Section of Clinical Biochemistry, The University of Manchester, Hope Hospital, Clinical Sciences Building, Eccles old Road, Salford M6 8HD, UK
b
Department of Instrumentation and Analytical Sciences, UMIST, Manchester, UK
c
Interdisciplinary Research Centre in Biomedical Materials, Queen Mary and West®eld College, London, UK

Abstract

Simple reagentless immunosensor formats have been dif®cult to achieve, particularly for electrochemical devices, since antigen/hapten
recognition by an antibody does not directly lead to a reaction cascade. A direct reading electrochemical immunosensor would have major
advantages with respect to speed, de-skilled analysis and the development of multi-analyte sensors.
Electrically conducting polymers, such as poly(pyrrole), allow for the intimate association between a biological recognition element and
a potential reporter polymeric chain. We have polymerised poly(pyrrole), loaded with avidin or antibody to luteinising hormone (LH), on
gold interdigitated electrodes (IDE) and employed two-electrode electrochemical impedance spectroscopy (EIS) to ``visualise'' charge
transfer through the polymer as the basis for a reagentless protocol.
We investigated bulk redox processes in the poly(pyrrole) ®lms by cyclic voltammetry in order to ascertain the redox state of the ®lm
prior to EIS. The redox process of the immobilised protein molecule was identi®ed and allowed the focusing of the EIS studies on polymer
associated with the immobilised bioaf®nity molecule.
The polymer displayed both polaronic and electronic charge transfer during EIS studies. A possible binding-dependent response,
observed as a decrease in peak polaronic phase angle, occurred when a redox cycle was performed on the ®lm following exposure to the
appropriate analyte.
The response of poly(pyrrole) ®lms loaded with avidin to D-biotin and two derivatives was assessed, which was shown to be sensitive to
electrode pre-treatment.
Poly(pyrrole) ®lms loaded with antibody to LH allowed a calibration for LH to be constructed between 1 and 800 IU/l. Importantly, the
®lms were responsive to LH within the clinically relevant range of 1±10 IU/l. # 2001 Elsevier Science B.V. All rights reserved.

Keywords: Direct biosensor; Electrochemical impedance spectroscopy (EIS); Poly(pyrrole); Antibody

1. Introduction was to label antibodies with enzymes; ELISA [2]. As

Direct reagentless biosensors represent the evolution of
biosensor and enzyme-linked immunosorbent assay
(ELISA) technology in which the speed of biosensor
response is combined with the sensitivity of ELISA. Clas-
sically, biosensors have indirectly monitored an analyte via
electrochemical reduction of hydrogen peroxide produced
by an immobilised oxidase enzyme of the analyte [1].
However, oxidase enzymes are limited in the number of
analytes that can be monitored and many do not have
suf®cient af®nity for the analyte to monitor the low levels
required in clinical biochemistry. Antibodies do have appro-
priate af®nity but produce no reaction product. One solution
*
Corresponding author. Tel.: ‡44-161-787-4428;
fax: ‡44-161-787-4336.
E-mail address: glillie_16@yahoo.com (G. Lillie).
traditional ELISA is time consuming, alternate protocols
have been developed to speed up measurements, the natural
conclusion of this being direct reading (or reagentless)
sensor formats [3].
The development of electrochemical reagentless devices
has been problematic due to the dif®culty of interfacing
molecular recognition and electrochemical transduction.
Previously, they have been based on potentiometric/capaci-
tive [4] alterations and a mass change, via acoustic wave [5]
devices, of immobilised antibody ®lms. Sensitivity/fouling
problems and slow response times for both types have been
encountered. In order to overcome this, it requires a more
intimate association between the molecular recognition
element and the transduction technique.
A possible route may be provided by a class of organic
polymers termed the conducting polymers, of which the
most widely used is poly(pyrrole). When exposed to a

0925-4005/01/$ ± see front matter # 2001 Elsevier Science B.V. All rights reserved.
PII: S 0 9 2 5 - 4 0 0 5 ( 0 1 ) 0 0 8 2 1 - 8
250 G. Lillie et al. / Sensors and Actuators B 78 (2001) 249±256
suitable oxidation potential, the monomer units polymerise,
but importantly, a positive charge develops on the polymer
which is cancelled out through the incorporation of oppositely
charged counter-ions, i.e. an electrolyte ion or protein [6].
Once formed, the polymer ®lms can be electrochemically
switched between oxidised (electrically conducting) and
reduced (electrically insulating) states, a process which is
observed by cyclic voltammetry. The electrochemical
switching occurs with alterations in charge, composition
and physical structure [7]. Previously, alterations in the
electrochemical redox properties of polymer ®lms have
shown promise in the development of immunosensors
[8,9]. However, all have targeted high concentration solutes
rather than low concentration, more clinically relevant
analytes, e.g. insulin and peptide hormones.
To improve sensitivity, two-electrode impedance spectro-
scopy was employed in the study of polymer ®lms contain-
ing immobilised protein. Previous research of conducting
polymers by two-electrode electrochemical impedance
spectroscopy (EIS) has shown high sensitivity to gas-phase
molecules [10], and the aim of the research was to study the
application to sensors operating in solution.
In two-electrode EIS, the charge-transfer process of the
polymer is recorded, of which the polymer has two: (1)
polaronic: it represents movement of the delocalised positive
charge (over four pyrrole units); (2) electronic: movement of
electrons along the conduction band of the polymer. In this
study, the polymer ®lms were formed on interdigitated
electrodes (IDE). By the application of a 20 mV RMS
sinusoidal potential between 5 and 13 MHz, two-electrode
EIS was used to ``visualise'' both processes; this was
possible as they occur on different time scales. A Bode plot
is one graphical representation of the impedance data that
can identify charge-transfer processes. Fig. 1 shows an
idealised Bode plot for a single charge-transfer process.
As the redox state of the polymer is ®xed during two-
electrode EIS, cyclic voltammetry was employed primarily
to establish the oxidation potential of the ®lm, and two-
electrode EIS undertaken on polymer ®lms as a platform for
the transduction of bioaf®nity binding.
Fig. 1. Idealised Bode plot for a single charge-transfer process.
2. Experimental
2.1. Instrumentation
The IDE were designed in conjunction with Dr. Graham
Ensell (Southampton University, Southampton, UK) who
also manufactured the devices.
The set-up for three-electrode electrochemistry consisted
of an EG&G potentiostat model 273 (Perkin Elmer, Ten-
nessee, USA) linked to a Gateway G6-350 PC (Gateway,
Somarset, UK) and controlled by EG&G software. Cyclic
voltammetry was performed in a standard three-electrode cell
with an Ag/AgCl reference, a platinum counter electrode.
Two-electrode EIS measurements were performed with a
Hewlett-Packard LF Impedance analyser 4192A (Helett
Packard, Berkshire, UK), which was connected to the
IDE through a test ®xture. The analyser was linked to a
Gateway G6-350 PC and controlled by LabView Software
(GPIB) (National Instruments, Texas, USA).
2.2. Methods
Pyrrole (Sigma, Poole, Dorset) was distilled and then
deoxygenated by passing nitrogen through for 15 min, as
was the distilled water and the sodium dodecyl sulphate
(SDS) (Sigma) solution employed in the polymerisation.
Electropolymerisation of the polymer ®lms was performed
by potential cycling between 0.9 and ‡0.6 V for SDS-
loaded ®lms, and between 0.9 and to ‡0.9 V for Avidin
(Sigma) and antibody to luteinising hormone (LH)-loaded
®lms (Biogenesis, Poole, Dorset, UK). The concentrations
of monomer and SDS or protein employed during the
electropolymerisation are as stated in the results.
Cyclic voltammetry experiments were also performed in a
three-electrode cell with a Ag/AgCl reference and a plati-
num counter electrode. Prior to EIS studies, the ®lm was held
at the required voltage for 1 min, to ensure that the entire ®lm
was set at the desired potential. Polarisation of the polymer
®lm at the two potentials indicated in sequence for 1 min
was employed for redox cycling during the EIS studies.
 G. Lillie et al. / Sensors and Actuators B 78 (2001) 249±256
D-Biotin, biotin amidocaproate sucinimide ester and bio-
tin amidocaproate 3-sulfo ester (all from Sigma) solutions
were made up in 10 ml phosphate-buffered saline solutions
(50 mM Na2HPO4, 50 mM NaH2PO4, 1.5 mM EDTA). The
polymer ®lm was exposed to the appropriate biotin solution
for 15 min. LH was obtained from Biogenesis, and the
antibody-loaded ®lm was exposed to the hormone for
15 min.
3. Results and discussion
3.1. Cyclic voltammetry
Avidin was selected as a model af®nity protein prior to the
study of antibodies, with its robustness and high af®nity for
biotin (Vitamin H) being key reasons [11]. The organic
counter-ion SDS was selected for comparison, as it has
previously been shown to be immobile within poly(pyrrole)
[12].
Cyclic voltammetric deposition of poly(pyrrole) ®lms
was employed for even, stepwise growth of the ®lm over
the digits of the IDE. The polymerisation solution did not
contain additional electrolyte to ensure only the desired
counter-ion was incorporated into the ®lm. This led to a
signi®cant difference in the potentials required for the onset
of polymerisation of the SDS-loaded (‡0.45 V) and protein-
loaded (‡0.7 V) poly(pyrrole) ®lms, respectively. The ano-
dic limit, which was required to achieve a good rate of
polymerisation, was found to be at ‡0.6 and ‡0.9 V for the
SDS- and protein-loaded ®lms, respectively.
Initially, the ®lms were prepared with the criterion that the
polymer had bridged the 15 mm interdigitated electrode gap,
as proven by dry ®lm conductivity at 5 Hz. Empirical
experimentation showed that minimisation of the SDS
Fig. 2. Cyclic voltammogram for the electropolymerisation of a poly(pyrrole) film loaded with avidin (0.5 M pyrrole, 0.25 mg/ml avidin).
251
concentration (17 mM) compared to the high pyrrole con-
centration (1 M) resulted in a bridged IDE after a minimum
of 15 cycles.
0.5 M pyrrole was the maximum concentration that
allowed solubilisation of the protein (0.5 mg/ml), and a
bridged electrode was produced after 50 cycles. However,
this was improved by employing 0.25 mg/ml of avidin,
which yielded a bridged ®lm in 15 cycles. Once again,
the cyclic voltammogram for the avidin-loaded ®lm illus-
trates a stepwise growth of the ®lm (Fig. 2).
Cyclic voltammetry of the as prepared poly(pyrrole) ®lms
loaded with SDS (8 mM) displayed two redox process when
cycled in sodium chloride. This is in contrast to a chloride
®lm that displayed only one redox process (Fig. 3). Previous
research [13] has demonstrated that the redox process with
immobilised counter-ions occurs at more cathodic potentials
than with mobile counter-ions. Thus, the more cathodic
process was attributed to the SDS and the anodic process
to the mobile chloride ion. A similar situation is observed
with the avidin- and antibody-loaded poly(pyrrole) (Fig. 4).
Two redox processes were also observed for the poly-
(pyrrole) ®lms loaded with SDS or avidin when immersed in
phosphate buffer (PB). The immobile counter-ions redox
process was shown to be separate from the mobile process by
only cycling between 0.9 and 0.1 V (Fig. 5). Although,
cycling in electrolyte containing small electrolyte ions can
lead to a ®lm containing mixed dopent ions, this can be
prevented by judicious selection of the potential limits. This
separation of the redox process allowed the focusing of the
EIS studies onto poly(pyrrole) associated with the immobile
counter-ion by holding the ®lm at 0.1 V for 1 min in
phosphate buffer.
The avidin loading within the ®lm was established as
30 mg through ¯uorescence measurement of the decrease in
rhodamine-labelled avidin in the electropolymerisation
252 G. Lillie et al. / Sensors and Actuators B 78 (2001) 249±256

Fig. 3. Cyclic voltammograms for poly(pyrrole) films loaded with chloride and SDS in 50 mM sodium chloride.

solution. For binding studies, the ®lms were exposed to
1 mM (10 ml) of D-biotin. However, no alteration in the
cyclic voltammogram was observed.
3.2. Two-electrode electrochemical impedance
spectroscopy
The Bode plot of a poly(pyrrole) ®lm loaded with SDS
and oxidised to 0.1 V displayed two charge-transfer pro-
cesses when immersed in distilled water (Fig. 6A). These
were assigned to polaronic conduction at low frequencies
and electronic conduction at high frequencies. When
immersed in phosphate buffer, the polaronic conduction
increases due to the facilitation of polaronic charge transfer
by the increase in mobile anions in the solution (Fig. 6B).
Two charge-transfer processes were also observed with the
avidin- and antibody-loaded poly(pyrrole) ®lms (Fig. 7).
Af®nity molecule exposure of the poly(pyrrole) ®lms
loaded with avidin or antibody does not lead to any sig-
ni®cant alteration in the Bode plot. However, following a
redox cycle ( 0.1 V to 0.9 V to 0.1 V), following expo-
sure to the appropriate analyte there is a decrease in the peak
polaronic phase angle. This is illustrated in Fig. 8 for a
poly(pyrrole) ®lm loaded with monoclonal antibody to LH.
The response of avidin-loaded poly(pyrrole) ®lms to
biotin and two derivatives: biotin amidocaproate sucinimide

Fig. 4. Cyclic voltammograms for poly(pyrrole) films loaded with chloride, avidin and antibody in 50 mM sodium chloride.
 G. Lillie et al. / Sensors and Actuators B 78 (2001) 249±256 253

Fig. 5. Cyclic voltammogram for a poly(pyrrole) film loaded with avidin in phosphate buffer.

Fig. 6. Bode plot for a poly(pyrrole) film loaded with SDS in (A) distilled water, and (B) in phosphate buffer.
254 G. Lillie et al. / Sensors and Actuators B 78 (2001) 249±256

Fig. 7. Bode plot for a poly(pyrrole) film loaded with antibody to LH, demonstrating the reagentless protocol.

Fig. 8. Histogram for the change in phase angle of (A) poly(pyrrole) film loaded with avidin to PB, D-biotin and the two derivatives biotin amidocaproate
sucinimide ester (biotin ester) and biotin amidocaproate 3-sulfo ester (biotin sulfo) (n ˆ 6), and (B) poly(pyrrole) films loaded with avidin that were prepared
on recycled electrodes (n ˆ 6).
 G. Lillie et al. / Sensors and Actuators B 78 (2001) 249±256 255

Fig. 9. Histogram for various antibody-loaded poly(pyrrole) films to the indicated analyte (n ˆ 6).

Fig. 10. (A) Calibration for poly(pyrrole) film loaded with poly- and monoclonal antibody to luteinising hormone exposed to PB, LH and bovine serum
albumin (3 mg/ml); (B) calibration for poly(pyrrole) films loaded with monoclonal antibody to LH (n ˆ 6).
256 G. Lillie et al. / Sensors and Actuators B 78 (2001) 249±256
ester (biotin ester) and biotin amidocaproate 3-sulfo ester
(biotin sulfo) was then assessed (Fig. 8A). The increase in
the phase angle for the sensors exposed to phosphate buffer
occurs even without performance of a redox cycle, and may
represent either ion association with the polymer or chemi-
cal oxidation of the ®lm.
The fact that the ®lms appear to show a response to the
analyte following redox cycling would suggest that the
effect is due to a realignment of polymer chains around
the protein following binding. However, similar effects have
only previously been seen for high molecular weight ana-
lytes by cyclic voltammetry [14], and though the underlying
reasons may be the same, such a demonstration for the low
molecular weight analyte D-biotin represents a signi®cant
advance.
A loss of sensitivity was demonstrated if electrodes were
cleaned in formic acid solution. Indeed, the response to the
two derivatives has been lost (Fig. 8B). This occurred even
though the initial Bode plots were identical. This indicates
the importance of the electrode surface and pre-treatment in
such studies. It may also point to alterations in the polymer
growth affecting the permeability of the poly(pyrrole) to the
analytes.
Antibodies to LH (mono- and polyclonal) and avidin
(polyclonal only) were then incorporated into the ®lm,
and the response to their respective analytes was determined
(Fig. 9). The increased af®nity of monoclonal antibodies to
LH in comparison to polyclonal was demonstrated by spik-
ing the polyclonal with a small amount (0.02% by weight) of
monoclonal antibody (Fig. 9). No response was recorded
until 200 IU/l, with saturation occurring at 400 IU/l.
Importantly, a calibration of the monoclonal antibodies to
LH was obtainable between 1 and 400 IU/l (Fig. 10A and B).
4. Conclusions
This study has demonstrated that immobilised protein in a
conducting polymer ®lm can be indirectly recognised by
cyclic voltammetry. Two-electrode impedance spectroscopy
subsequently identi®ed the polaronic and electronic charge
transfer in the polymer associated with the protein.
This provided a platform for the transduction of binding
through measuring peak polaronic phase angle decrease
following exposure to the desired analyte and then a redox
cycle of the ®lm. This suggests an alteration in ®lm struc-
ture around the bioaf®nity molecule following bioaf®nity
binding. Although, it has been found that the electrode
surface is also important, the ability to monitor low molecular
weight analytes directly is an important step in the evolution
of direct electrochemical biosensors. This was further empha-
sised by the ability to monitor leuteinishing hormone.
Acknowledgements
The authors wish to express their gratitude to the EPSRC
for ®nancial support.
References
[1] J. Wang, Amperometric biosensors for clinical and therapeutic drug
monitoring, J. Pharmacol. Biomed. Anal. 19 (1/2) (1999) 47±53.
[2] J. Gervay, Utilization of ELISA technology to measure biological
activity of carbohydrates in relevant disease states, Curr. Med. Chem.
6 (2) (1999) 129±153.
[3] A.L. Ghindilis, et al., Immunosensors: electrochemical sensing and
other engineering approaches, Biosens. Bioelectron. 13 (1) (1998)
113±131.
[4] B. Berggren, et al., Capacitive measurements of antibody±antigen
interactions in a flow system, Anal. Chem. 69 (1997) 3651±3657.
[5] E. Gizeli, et al., Antibody binding to a functionalized supported lipid
layer: a direct acoustic immunosensor, Anal. Chem. 69 (1997) 4808±
4813.
[6] P.N. Bartlett, et al., A review of the immobilization of enzymes in
electropolymerized films, J. Electroanal. Chem. 362 (1993) 1±12.
[7] T.F. Otero, et al., Reinterpretation of poly(pyrrole) electrochemistry
after consideration of conformational relaxation processes, J. Phys.
Chem. B 101 (1997) 3688±3697.
[8] R. John, et al., Development of a polypyrrole-based human serum
albumin sensor, Anal. Chim. Acta 249 (1991) 381±385.
[9] O.A. Sadik, M.J. John, G.G. Wallace, B. Barnett, C. Clarke, D.G.
Laing, Pulsed amperometric detection of thaumatin using antibody-
containing poly(pyrrole) electrodes, Analyst 119 (9) (1994) 1997±
2000.
[10] P.A. Payne, M.E.H. Amrani, K.C. Persaud, Multi-frequency mea-
surements of organic conducting polymers for sensing of gasses and
vapours, Sens. Actuators B 33 (1±3) (1996) 137±141.
[11] Y. Hiller, E.A. Bayer, M. Wilchek, Studies on the biotin binding site
of avidin-minimized fragments that bind biotin, Biochem. J. 278 (2)
(1991) 573±585.
[12] T. Matencio, M.A. Depaoli, R.C.D. Peres, R.M. Torresi, S.I.C.
Detorresi, Ionic exchange in dodecylbenzenesulfonate-doped poly-
pyrrole. Part 1. Optical beam deflection studies, Synth. Metals 72 (1)
(1995) 59±64.
[13] A. Talaie, G.G. Wallace, The effect of the counter-ion on the
electrochemical properties of conducting polymers Ð a study using
resistometry, Synth. Metals 63 (2) (1994) 83±88.
[14] A. Sargent, T. Loi, S. Gal, O.A. Sadik, The electrochemistry of
antibody-modified conducting polymer electrodes, J. Electroanal.
Chem. 470 (2) (1999) 144±156.