America n Journal of Analy tic al Chemistry, 2011, 2, 392-400
doi:10.4236 /ajac.2011.23048 Published Online July 2011 (http://www.scirp.org/journal/ajac)
Copyright © 2011 SciRes. AJAC
Comparison Between DNA Immobilization Techniques on
a Redox Polym er Mat ri x
Vijayalakshmi Velusamy1, Khalil Arshak1*, Catherine F. Yang2, Lei Yu2, Olga Korostynska3,
1Electronic and Computer Engineering Department, University of Limerick, Limerick, Irelan d
2Department of Chemistry and Biochemistry, Rowan University, Glassboro, USA
3School of Physi cs , Dublin Institute of Technology, Dublin, Ireland
4Department of Chemical and Environmental Sciences, University of Limerick, Limerick, Ireland
E-mail: viji.su b h email@example.com
Received February16, 2011; revised April 2, 2011; accepted April 16, 2011
In this paper we report a label-free detection method of unmodified DNA using polypyrrole as a n immobili-
zation matrix by impedance measurement. A probe and a target complementary DNA sequence specific for
the bacterial pathogen, Bacillus cereus are used. Impedance measurements are performed without using ad-
ditional redox probes. The effects of hybridization and non-specific binding are compared when the Probe
DNA molecules were immobilized by two different methods: electrochemical adsorption and entrapment.
The probe DNA immobilized using electrochemical adsorption yielded better hybridization signals com-
pared to that immobilized using the entrapment method. Control experiments were also performed to prove
the specificity of the biosensor in the presence of non complementary DNA. Negligible unspecific binding
with the immobilized probe was observed with the electrochemically adsorbed probe, whereas the entrapped
probe responded to the non complementary target. The performance of the DNA sensor was characterized
using both cyclic voltammetry and impedance spectroscopy techniques and proved to be effective in terms of
specificity of hybridization events.
Keywords: Immobilization, Label Free DNA, Polypyrrole
Biomolecules of interest called bioreceptors/biorecog-
nition elements ca n be generally classified into six diffe-
rent major categories. These categories include antibody
/antigen, e nzymes, nucleic acids/DNA, cellular structures
/cells, biomimetic and bacteriophage (phage). Among
other biorecognition elements, DNA target has received
considerable attention since each organis m ha s its uniq ue
DNA sequences and any self-replicating micro-organism
can be easily identified. Biosensors based on DNA as
bioreco gnition element ar e simple, r apid, and hig hly spe-
cific hence widely used in pathogen detection. In co ntra st
to enzyme or antibodies bioreceptors, nucleic acid rec-
ognition layers can be readily synthesized and regene-
rated. However, there are factors which p lay ke y role s in
the design of DNA biosensors, 1) The immobilization
matrix 2) The method of immobilization and 3) The de-
The choice of suitable substrate/matrix to immobilize
the DNA is a significant factor since the immobilized
single stranded ss-DNA has to be stable on the attached
surface to facilitate the hybridization event. Various ma-
terials being used to modify the electrode (Au, Ag, Pt,
Glassy Carbon, and Indium tin oxide (ITO)) surface and
include carbon nanotubes, conducting polymers, metal
nanoparticles, composites of various electroactive mate-
rials. Conducting polymers (CPs) are preferred among
those materials not only because they can be synthesized
easily but also due to their high stability, excellent elec-
trical properties and suitable to immobilize the bio-
molecules [1,2]. Among the various CPs polyaniline,
polythiophene, and polypyrrole are biocompatible [3-8]
—however, polypyrrole (PPy) is used mostly in biosen-
sors and immunosensors because of its biocompatibility
and the ease of immobilization of various biologically
active compounds . To detect bio-analytes at a phy-
siological pH, b iosensing mat erials must be electroactive
V. VELUSAMY ET AL.
Copyright © 2011 SciRes. AJAC
in neutral environments, unlike polyaniline and poly-
thiophene. To overcome this problem, PPy is attractive
because it can be more easily deposited from neutral pH
aqueous solutions of pyrrole monomers . Most prac-
tical types of PPy have conductivity in the range of 1 -
100 S/cm . PPy can be prepared by various methods
such as chemical polymerization [12-16], electrochemi-
cal polymerization [17,18], spreading [19,20], vapour
phase polymerization , plasma polymerization
[22,23]. The electrochemical polymerization of pyrrole
has been extensively studied as it is simple and reliable
procedure which does not need sophisticated laboratory
equipme nts and reagent s .
One of the critical factors in biosensor design is the
develop ment of im mobiliza tion methodolo gy that strong-
ly stabilizes the DNA on the transducer surface. Integra-
tion of the DNA with the signal transducer is mostly
achieved by immobilizing ss-DNA on the electrode/mo-
dified surface. Different mechanisms of immobilization
techniques are shown in F igure 1.
Mechanisms of immobilization of a label-free umodi-
fied DNA probe on the conducting polymer modified
surface can be divided into two major categories: i) Ad-
sorption and ii) Entrapment.
While immobilization matrix and method are impor-
tant in DNA sensor design, the choice of a suitable de-
tection technique is also of great significance in deter-
mination of overall performance of the DNA biosensor,
in particular with respect to the immobilization and hy-
bridization efficiency of the DNA. The detection tech-
niques employed in the DNA biosensors can be optical
[24-27], electrochemical [28-30] or mass [31,32] based.
Depending on the nature of the target, the detection pa-
rameters can be drawn from the detection of the specific
sequence to confirm the presence of target micro-organ-
isms [33,34], DNA damage [35,36], presence of chemi-
cal  and biological compounds , presence of
toxic  and genotoxic compound s .
In general, compared to the other detection techniques,
the electrochemical based detection method is more ad
Figure 1 . Mechanism of immobilization.
vantageous, not only because it is simple, cost effective
and reproducible but also due to its suitability for minia-
turized real-time handheld use without sacrificing its
sensitivity and specificity. In electrochemical DNA bio-
sensors, detection is based on the variation in the electri-
cal properties of the DNA- modified electrode before and
after hybridization. It is likely that the change may have
resulted from the change of double-layer capacitance,
heterogeneous electron transfer resistance, impedance or
current. DNA sensors based on electrochemical imped-
ance spectroscopy (EIS) detection is a device that tran-
scribe the changes in interfacial properties between the
electrode and the electrolyte induced by DNA hybridiza-
tion, DNA conformational changes, or DNA damages to
an electrical signal . EI S is becoming more and more
popular for electrochemical measurements of molecular
interactions and is also widely used in a variety of bio-
sensing applications. The principle and applications of
EIS to biosensing have been recently summarized in the
literature . The method provides unique advantages
compared to other electrochemica l me t ho ds, such a s hi g h
sensitivity, ease of signal quantification, and ability to
separate the surface binding events from the solution
impedance . Impedance data are normally recorded
in a range of frequencies, using alternating current of
small amplitude, thus the EIS is often referred to as AC
Impedance. Compared to other electrochemical methods,
such as cyclic voltammetry (CV) or differential pulse
voltammetry (DPV), known to characterize molecular
interactions on the surface of electrodes, AC Impedance
is less destructive to the measured biological interac tions
because it is performed in a very narrow range of small
potentials . To cha racterize the DNA immobilized on
the modified electrode surface using EIS, the change in
impedimetric response upon hybridization can be meas-
ured either in terms of kinetics of electron transfer proc-
ess by Faradic impedance measurements or in terms of
alterations of capacitance and molecular layer organiza-
tion, originating from biorecognition events, by non-
Faradic approach .
In this paper, we report the label-free detection of
DNA hybridization using Faradic impedance measure-
ment s . The PPy modified gold electrode surface was
employed to optimize the immobilization of probe DNA
molecules and to resist the non spe cific bindi ng of target
DNA molecules. Further, electrochemical adsorption and
entrapment methods of immobilization are compared
with respect to hybridization with target complementary
and non-c omple menta ry DNA. Here, the present applica-
tion is specifically applied to food quality monitoring
emphasizing on the detection of the bacterial pathogen,
V. VELUSAMY ET AL.
Copyright © 2011 SciRes. AJAC
Table 1 . DNA sequences.
Functio n Seque nce
Probe (5’-3’) A TC GCC TCG TTG GAT
TCG TCA TCC AAC GAG
GC G AT
(5’-3’) AAA ATC GAT GGT AAA
2.1. Material s
Pyrro le a nd M gCl2 solution were purchased from Sig ma-
Aldrich, USA. All the reagents were analytical grade and
used without further purification. Specific sequences for
the Bacillus cereus were designed in our laboratory 
and its synthetic form was purchased from Integrated
DNA technologies, USA. All stock DNA solutions were
prepared using distilled water and a concentration of 1
µg/µl from the stock solution was made using 10mM
Tris-HCl buffer, pH 7.2. The capture probe and the target
probe are 20-mer oligonucleotides and the non comple-
mentary probe 21-mer in length. Table 1 shows the se-
quences of the oligonucleotides used in this work.
A three-electrode cell comprising—a gold (Au) working
electrode (2-mm diameter), a platinum wire counter
electrode and a Ag/AgCl reference electrode were used.
All electrochemical measurements were carried out using
Autolab PGSTAT302N (Eco Chemie, The Netherlands)
with a Frequency Response Analyser (FRA) module in-
terfaced for impedance measure ments.
2.3. Gold Electrode Surface Modification and
Electrochemical Synthesis of Polypyrrole
The Au electrode surface was polished to mirror finish
prior to use sequentially with 1, 0.3, and 0.05 μm α-
Al2O3 paste, and rigorously rinsed with distilled water
following each polish. Prior to surface modification, the
bare electrode was scanned in 0.1M MgCl2 in 10 mM
Tris-HCl buffer (pH 7.2) between –0.3 and 0.8 V until a
reproducible cyclic voltammogram was obtained. Two
different types of methods were employed for immobili-
zation of probe DNA.
The first approach involves incorporation of probe
DNA into the polymer matrix during the growth of the
PPy or during the co-deposition of the pyrrole monomer
which is known as the entrapment method. The second
approach involves the polymerization of the polypyrrole
without the presence of DNA and then i mmobilizing the
probe DNA by electro chemical adsorption.
2.4.1. Immobilization by Entrapment
Immobilization by co-deposition was achieved using
cyclic volta mmetry (CV) for 20 cycles over –0.3 to +0.8
V at a scanning rate of 50 mVs−1. The 2ml solution for
immobilization by co-deposition consists of 0.1M Pyr-
role/0. 1 M MgCl2 so lution and 1 µg of probe DNA.
2.4.2. Immobilization by Electrochemical Adsorption
First, the electrochemical polymerization of PPy was
performed by CV, involved the immersion of an Au disk
electrode into a solution of 0.1 M pyrrole containing 0.1
M MgCl2. A cyclic potential from –0.3 to 0.80 V (versus
Ag/AgCl) for 20 cycles at a scan rate of 50 mVs−1 was
applied. The Au electrode coated with PPy film was then
washed with di stilled water a nd d ried under nitroge n gas .
The so-prepared PPy-coated electrode was characterized
by impedance spectroscopy and CV.
Prior to immobilization, the PPy coated electrode was
transferred to a 20mM Mg Cl2 and 10 mM Tris-HCl
buffer (pH 7.2) for electrochemical oxidation of the PPy
layer at 0.5 V (versus Ag/AgCl) for 600 s. Immobiliza-
tion was thereafter achieved by applying a constant po-
tential of 0.8 V for 600 s and the 2 ml immobilization
solution consisted of 20 mM MgCl2 in10 mM Tris-HCl
buffer (pH 7.2) and 1 µg of probe DNA. The ss-DNA
modified electrode was washed with distilled water to
remove loosely adsorbed DNA and dried under nitrogen
gas. T he ss-DNA/PP y film was later analysed by imped-
2.5. Hybridization of the Target DNA
The hybridization was completed by applying a constant
potential of 0.5 V for 600 s and the 2 ml hybridization
solution consists of 20 mM MgCl2 in 10 mM Tris-HCl
buffer (pH 7.2) and 1 µg of complementary DNA strand.
The same procedure was repeated with
non-complementary DNA as a co ntrol experiment.
2.6. Characterization of the DNA Bio sensor
Both the EIS and CV techniques are used to characterize
the performance of the DNA biosensor. All impedance
measurements were performed at 0.3 V bias potential in
an analysis buffer consisting of 0.1 M MgCl2 in10 mM
Tris-HCl buffer, with a pH of 7.2. AC amplitude of 5
mV was used and the data collected in the frequency
V. VELUSAMY ET AL.
Copyright © 2011 SciRes. AJAC
range of 10 kH z - 100 mHz taking six points per decade.
The CV measurements were performed by applying cy-
clic potential fro m –0.3 to 0.80 V (versus Ag/AgCl) for 6
cycles at a scan rate of 50 m·Vs−1. The analysis bu ffer for
CV measurements consists of 0.1 M MgCl2 in 10 mM
Tris-HCl buffer, with a pH of 7.2.
3. Results and Discussion
3.1. Polymerization of Pyrrole
The electrical conduction of PPy is the result of electron
movement within delocalized orbitals and positive
charge defects known as polarons . In conjugated
polymers other than polyacetylene, electrons added or
removed from the delocalized π-bonded backbone ini-
tially produce polarons (radical ions coupled to a spa-
tially extended distortion of the bond lengths), which
subsequently combine to form dianions or dications
(spinless bipolarons), respectively . The applications
of co nducting polymers in biosensing have been detailed
in a recent review . During the electrop olymerizatio n
of pyrrole, the forming polymer backbone was charged
positively. The positive charge is compensated by anions
from the electrolyte solution via an incorporation of
these anions int o the po lymer film.
3.1.1. Cyclic Voltammetry Characteristics of
Polymerization of Pyrrole
The PPy films were deposited on the working Au elec-
trode by scanning the potential between positive (0.8 V)
and negative (–0.3 V) limits repeatedly for 20 cycles.
The anodic oxidation of PPy, where the anions, Cl– from
MgCl2 compensate the positive charge of the produced
PPy+, enhanced the electrochemical polymerisation of
the PPy film and the formation of PPy film was evident
from the growth of oxidation current after each cycle.
The growth of the PPy can be controlled by varying the
Figure 2. Cyclic voltammograms before (a) and after
polymerization process (b).
applied positive potential, scan rate , number of scans and
also the concentration of monomer/electrolyte ratio.
Figure 2 shows the cyclic voltammogram for the PPy
film prepared by electrochemical polymerization on Au
electrode. After the bare Au electrode was modified with
PPy film, the oxidation current increased significantly.
The increase in oxidation current provides the evidence
to demonstrate the existence of PPy, which is later con-
firmed b y us ing EIS.
3.1.2. Impedance Characteristics of Polymerization of
EIS measurements impedance is generally expressed as a
complex number, which is the combination of the real
mainly from the Ohmic resistance and
the imaginary component,
from the capacitive
reactance. The impedance spectra of the conducting
polymer were used to evaluate film conductivity and the
charge transport at the PPy film/electrolyte interface.
Figure 3 shows the Nyquist diagram for the bare Au
electrode and modified Au electrode (Au/PPy) after po-
lymerization using CV. From the Nyquist plot the im-
pedance (resistance) appears to be substantially larger for
the bare Au electrode (Figure 3(a)) since there is no ac-
Figure 3 . Nyquist pl ots befo re ( 3 a) and aft er pol y merizati on
process (3b) on a gold electrode.
V. VELUSAMY ET AL.
Copyright © 2011 SciRes. AJAC
tive redox species, so no Faradic current. However, the
presence of Faradic current was observed in the PPy
modified electrode (Figure 3(b)) and the resistance is the
charge-transfer resistance of PPy redox reactions on
In general, Faradaic impedance measurements are
performed in the presence of a redox couple in solution
and rely on changes in the barrier to redox conversion
due to the formation of the recognition co mplex itself or
a subsequent complex. The ferri-/ferrocyanide
(Fe(CN)63–/4–) solution is mostly chosen because of its
excellent electrochemical reversibility. However, in this
case, the presence of an additional redox couple is turned
out to be not necessary. Because, conjugated polymers
such as polypyrrole, can be oxidised and reduced which
is in principle a nalogous to the redox species.
3.2. Immobilization and Hybridization of the
Due to intermolecular force between the DNA and PPy,
DN A can fir mly bind on the PPy matr ix. The underlying
principle is t hat the p ositi vel y cha rged PP y can e xc hang e
its negatively charged dopant easily with other negatively
charged species, including biomolecules . Therefore,
PPy provides a unique surface for DNA binding. Due to
its delocalize d electronic structure, the positively charged
sites of PPy are mobile along the chai n axis. T his allo ws
more flexibility towards the binding of DNA’s fixed
negative charge sites and hence it provides a higher af-
finity than surface with fixed positive charges . Hy-
drogen bonding to phosphate oxygen in the DNA back-
bone can also enhance the binding to DNA, and PPy
could provide such hydrogen bonds through its pyrrole
ring nitro gen at om.
Entrapment method is typically adopted when conduct-
ing polymers are employed as immobilization matrix. In
this method the biomolecules are embedded onto the
electrode surface during the growth of the conducting
polymer and therefore, it creates more stable DNA sur-
face. It is a fast and simple procedure, since it involves
co-deposition of pyrrole and ss-DNA together in one
In electrochemical adsorption forces of attraction be-
tween PPy and DNA are due to ion to ion interactions
between the negatively charged DNA and the positively
charged surface. When a positive potential of 0.8 V is
applied, due to electrostatic attraction, the phosphate gr-
oup of the DNA molecule binds to the postively charged
surface. The electrochemical adsorption has several ad-
vantages over physical adsorption or physisorption. In
physisorption forces of attraction are due to Van der
Waals’ forces between the solid surface and the bio-
molecule. The quantity of biomolecules taken up by the
surface depends on several conditions and surface prop-
erties including temperature, pressure and the surface
To detec t the hybridizatio n efficiency and to determine
the ef fective ness of the t wo immobiliza tion methods, the
complementary target DNA was hybridized on the im-
mobilized ss-probe DNA. Both the entrapped and elec-
trostatically adsorbed probes responded to the comple-
mentary DNA target. Figure 4 shows the ac impedances
measured for the Au/PPy/DNA film before and after the
target complementary DNA hybridization and the corre-
sponding Nyquist Plot.
Change in impedance was observed after complemen-
tary DNA hybridization for both the entrapped probe
(Figure 4(a)) and electrostatically immobilized probe
(Figure 4(b)). Figure 4(b) shows the Faradic impedance
spectra after the hybridization event, in which the charge
transfer resistance increased after the capture DNA probe
was immobilized on the PPy film and a further increase
in charge transfer resistance from 2 kΩ to 4.46 kΩ was
Figure 4. Nyquist plots afer hybridization w ith the co mp le -
mentary target DNA (4a) with entrapped probe (4b) with
electrostat ically adsorbed pr obe.
V. VELUSAMY ET AL.
Copyright © 2011 SciRes. AJAC
observed after hybridization. The increase in charge
transfer resistance is due to presence of the DNA probe
at the PPy surface, which blocks the passage of chloride
ions at the PPy/solution interface. This should be attrib-
uted to the repulsive electrostatic interaction which in-
creased the anionic charge along the backbone of the
PPy/ss-DNA and later double the anionic charge by the
forming DNA duplex. Accordingly, results indicate that
the addition of negative charge to the surface of the
PPy/ss-DNA, in the form of complementary oligonu-
cleotide, further blocks the chl orid e ion exc hange which,
yields the increase in impedance after hybridization.
3.3. Specificity of the System
The specificity of this protocol was investigated by
varying the target DNA sequence. A non co mplementary
sequence was used to hybridize with the immobilized
capture DNA probe and only negligible unspecific bind-
ing was observed with the probe immobilized by electro-
static adsorption (Figure 5(b)). Whereas, the entrapped
probe responds to the non-complementary target DNA
which showed an effect on the charge transfer resistance
(Figure 5(a)). Therefore, the electrostatically immobi-
lized DNA sensor demonstrated has more effectiveness
in ter ms of specificity of hybridization events and to co n-
Figure 5. Nyquist plots afer hybridization with the non
complementary target DNA (5a) with entrapped probe (5b)
with electr os tatically adsorbed probe.
firm this, the hybridization events were also character-
ized using cyclic voltammetry. From the cyclic voltam-
mograms results shown in Figure 6(a), a decrease in
oxidation current was observed after hybridization with
complementary target DNA and only a negligible effect
in the chan ge in oxid a tio n cur r ent wa s o bs er ved wit h no n
complementary DNA (Figure 6(b)).
Whatever the method of immobilization, the key per-
formance criterion of a DNA biosensor lays in its effi-
ciency of hybridization. In theory, this should depend on
i) the surface characteristics, ii) the surface coverage of
probe molecules, iii) probe orientation of the surface, and
iv) factors con trollin g the tra nspo rt o f target molec ules to
the surface . Although, entrapped probe leads to a
more stable immobilization by containing the oligonu-
cleotides within the P Py bulk, it i nvariably leads to so me
extend of steric and kinetic barriers to the hybridization
of a macromolecule. In addition, the oligonucleotide
probe can be oxidatively damaged by radical cations
formed during p yrrole p olymerization, leading to its pa r-
Figure 6. Characterization of the hybridization events of
the complementary (6a) and non complementary target
DNA (6b) with the electrostatically adsorbed probe. Cyclic
Voltammograms (a) of the polymerized gold electrode
-Au/PPy, (b) after immobilization of electrost ai cal ly adsorb-
ed probe-Au/PPy/ssDNA (c) after hybridization with com-
plementary DNA target -Au/PPy/dsDNA and (d) after
hybridiz a tio n with no n co mplementary DNA.
V. VELUSAMY ET AL.
Copyright © 2011 SciRes. AJAC
tial degrada tion .
In electrochemical adsorption, by application of a po-
tential to the electro de, the delocalized positive charge of
the oxidized PPy electrostatically attracts the negatively
charged phosphate groups of the DNA. Also, contro lle d
adsorption of DNA onto the PPy surface can be obtained
since the positively charged PPy substrate exerts electro-
static attraction o n the DNA mole cules. T he q uantit y and
strength of adsorption depends on the nature of the anion
dopant (electrolyte, in this case MgCl2), type and the
ionic strength of the buffer used, solution pH, and on the
DNA itself. While the DNA surface coverage can be
controlled, the DNA conformation and orientation on the
surface is difficult to modulate. Studies [52-54] suggest
that electrostatically driven DNA adsorption results in
orientation of the molecules parallel to, rather than per-
pendicular to the surface, with base pairing sites exposed
to the liquid medium. Howev er, the results sho w that t he
orientation is appropriate to target DNA hybridization
and does not limit specificity of the biosensor .
Electrochemically assisted adsorption of DNA on the
PPy modified electrode surface leads to more active and
functional DNA layers. This method takes advantage
over the entrapment method. Therefore, the DNA-PPy
surface interaction is stronger and more stable when a
potential is applied during adsorption. Moreover, the
measurements performed in an inert electrolyte solution
resulted in much higher impedance due to the presence
of redox polymer and were reproducible. Therefore, the
application of redox conductive polymer such as PPy as
an immobilization matrix is an attractive approach in
which the presence of external redox probe or active
species can be totally eliminated. A further study of nu-
cleic acid interaction on transducer interfaces and the
investigation of conformation of DNA on the electrode
surface can generate the insights on the design surfaces
for more effective hybridization.
5. Acknowledgemen t s
This project is funded by Science Foundation Ireland
(SFI) Research Frontiers Programme, ID no: 07RPF-
ENEF500. The presented research work was co funded
by SFI-Short Term Travel Fellowship award.
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