Vol.2, No.6, 374-379 (2009)
SciRes Copyright © 2009 Openly accessible at http://www.scirp.org/journal/JBISE/
Directly immobilized DNA sensor for l abel-free
detection of herpes virus
Phuong Dinh Tam1,2, Mai Anh Tuan 2, Nguyen Duc Chien3
1Hanoi Advanced School of Science and Technology, Hanoi University of Technology, Hanoi, Vietnam; 2International Training Insti-
tute for Materials Science, Hanoi University of Technology, Hanoi, Vietnam; 3Institute of Engineering Physics, Hanoi University of
Technology, Hanoi, Vietnam.
Email: tampd-hast@mail.hut.edu.vn
Received 24 March 2008; revised 12 May 2009; accepted 29 June 2009.
This paper reports the direct immobilization of
deoxyribonucleic acid (DNA) sequences of Her-
pes simplex virus (5’–AT CAC CGA CCC GGA
GAG GGA C–3’) on the surface of DNA sensor
by using the cyclic voltammetric method with
the presence of pyrrole. The potential was
scanned from –0.7 volt to + 0.6 volt, the scan-
ning rate was at 100mV/s. This kind of DNA
sensor was developed to detect Herpes virus
DNA in real samples. The FTIR was applied to
verify specific binding of DNA sequence and
conducting polymer, the morphology of con-
ducting polymer doped with DNA strands was
investigated by using a field emission scanning
electron microscope (FE-SEM). The results sh ow-
ed that output signal given by co-immobilized
DNA/PPy membrane sensor was better than that
given by APTS immobilized membrane sensors.
The sensor can detect as low as 2 nM of DNA
t arget in real samples.
Keywords: DNA Sensor; Hybridization; APTS
The detection of specific DNA/RNA sequences is of
great importance in numerous applications of modern
life science, including identification of medical research
and clinical diagnosis [1,2], controlling the food quality
[3,4], environmental analysis [5,6]. Many methods have
been used for this purpose such as polymerase chain
reaction (PCR) [7,8,9], quartz crystal micro-balance
(QCM) [10,11], fluorescence [12], surface plasmon
resonance [13], microfluidic system [14], cell culture
and real-time PCR, etc. These methods are precise, and
allow a wide, dynamic range of detection. However, they
are complex, costly and time consuming. In addition, it
is impossible to carry the on-site/in-field tests. Thus,
development of a cheap, reliable device allowing rapid
detection is always the challenge for scientists and engi-
neers. In this context, DNA sensor based on electro-
chemical detection is one of the feasible and promising
We reported, in this paper, the direct co-immobiliza-
tion of DNA sequence of Herpes simplex virus and
polypyrrole onto the surface of a sensor by cyclic volt-
ammetry to determine the herpes DNA target sequence
in the sample. The herpes simplex virus (HSV) is an
enveloped double-stranded DNA virus. There are two
distinct forms of HSV, serotype 1 and serotype 2 (HSV-
1 and HSV-2). HSV-2 is the most common cause of
genital herpes, whereas HSV-1 is the most common
cause of facial herpes or cold scores. HSV-1 is transmit-
ted through contact with oral secretions. Diseases caused
by Herpes virus are commonly found in patients in Viet-
2.1. Chemical Reagents
DNA sequences used in this work (Ta ble 1 ) were sup-
plied by Invitrogen Life Technologies Company through
National Institute of Hygiene and Epidemiology of Viet-
nam. Pyrrole was purchased from Merck. Other chemi-
cals are of analytical grade.
2.2. Sensor Fabrication
The microelectrode based DNA sensor was designed and
fabricated at clean room of ITIMS. The sensor consists
of pairs of microelectrodes on the surface of silicon sub-
strate, one of which acts as working sensor and the other
as a reference electrode. The dimension of the inter-
electrodes was 20 µm x 20 µm (Figure 1). The detailed
fabrication process was discussed in [15].
2.3. Cyclic Volttametry Electropolymerization
Electropolymerization was carried out by using IM6EX
P. D. Tam et al. / J. Biomedical Science and Engineerin g 2 (2009) 374-379
SciRes Copyright © 2009 Openly accessible at http://www.scirp.org/journal/JBISE/
Table 1. DNA sequences used in this work.
Types DNA sequences
Non-complementary 5’–AT CAC CGA CCC GGA GAG GGA C–3’
Figure 1. 20 µm x 20 µm microelectrode sensor was fabricated at ITIMS.
(Germany) impedance analyzer at room temperature in
which the micro-sensor acted as working electrode while
auxiliary electrode was a platinum wire. Reference elec-
trode is Ag/AgCl in saturated KCl.
The sensor was first surface cleaned by KCr2O7 in
H2SO4 98% followed by cyclic voltammograms (swept
potential from –1.5V to +2.1V, scan rate: 25mV/s) in
0.5M H2SO4 to activate the surface of the sensors. Fi-
nally, the potential was swept on the working electrode
from –0.7 volt to 0.6 volt versus standard counter elec-
trode (SCE). The scanning rate was 100mV/s.
2.4. Measurement
Differential measurements were realized to determine
the changes in conductance of DNA membrane. An AC
reference signals (10 KHz, 100mV sine wave), generated
by the generator of Lock-in Amplifier SR830, and was
applied on two identical micro-electrodes of DNA sensor.
The output signal was acquired by measuring the voltage
drop on two 1 K resistances by the A and B channels
of the Lock-in Amplifier and processed by a PC through
RS 232 interface. All measurements were performed at
room temperature. In this experiment, five DNA sensors
were used to test the hybridization of DNA sequences.
3.1. The Polymerization of PPy/DNA
Normally, pyrrole is polymerized with the presence of an
anionic dopant which contributes to film conductivity.
Variety of anions can be used as dopant for polypyrrole
(Ppy) polymerization such as Cl-, NO3
-. In this work
- and DNA sequence were used.
According to Wang et al [16], DNA can be considered
as sole counter anion in the electropolymerization proc-
ess at the working electrode. This allowed maximum
possible incorporation of DNA in the conductive poly-
mer throughout the film thickness and full contribution
of oligonucleotides charged phosphates to the polymer
The cyclic voltammograms of synthesized Ppy and
Ppy/DNA film is shown in Figure 2 where the oxidation
of pyrrole monomer leads to the formation of radical
cation, subsequent oxidation of the dimer and coupling
will result in the formation of an insoluble polymer,
positively charged on the surface. This electrochemical
procedure allowed the formation of a copolymer which
Figure 2. Cyclic voltammograms of 0.5mM Ppy doped
0.05µM DNA probe sequence in LiClO4 solution.
Swept potential from –0.7 V to 0.6V, scanning rate is at
P. D. Tam et al. / J. Biomedical Science and Engineerin g 2 (2009) 374-379
SciRes Copyright © 2009 Openly accessible at http://www.scirp.org/journal/JBISE/
is a mixture of polypyrrole and an oligonucleotides that
shows an increasing current along with conducting film
growth which corresponds to incorporation of oligo into
the Ppy film. The film was rinsed and used for detection
of DNA hybridization.
3.2. FTIR Spectrum of Ppy and DNA/Ppy
In this work, the FTIR spectroscopy was used to verify
the existence of polypyrrole and DNA sequence on the
microelectrode surface after the polymerization process.
The infrared spectrum of the DNA/Ppy complexes and
pure Ppy were performed on Niconet 6700 FT-IR ma-
chine with the effective range from 400 cm-1 to 4000
cm-1 at room temperature. As shown in Figure 3, the
absorption band at 1889 cm-1–1629 cm-1 vibration plane
implied G-C and A-T base pairs while the backbone
phosphate group at 1095 cm-1 was perturbed upon Ppy
interaction [17,18].
The absorption band at 1254 cm-1 was assigned to the
biopolaronic species formed in the over oxidation pro-
cess of Ppy [19]. The C-H and N-H bonds were also
observed at 735 cm-1 (for DNA/Ppy film); 734 cm-1 for
Ppy film; 894 cm-1 for DNA/Ppy and 897 cm-1 for Ppy
membrane, respectively. These results show very good
agree- ment with earlier reported work [20].
3.3. Morphology of Conducting Polymer Film
The morphology of sensor surface coated with Ppy film
was studied by FE-SEM. Figure 4 indicated micro-
graphs of polypyrrole doped with LiClO4 (4a) and with
both 0.1 M LiClO4 and 0.05 M DNA sequence (4b)
membrane given by direct electropolymerization method.
In Figure 4(a), the pure PPy doped with LiClO4 was
cauli-flower structure matching other works [21]. This
structure is related to the dopant intercalation in the
polymeric chain. As in Figure 4(b) the DNA strands was
observed as white dots in host polymer membrane. Good
distribution of DNA in PPy membrane makes it advan-
tage for hybridization process of the probe in target solu-
Figure 3. The FTIR spectra of Ppy/DNA and Ppy (upper curve:
Ppy/dopant, lower curve: Ppy/dopant/DNA).
(a) (b)
Figure 4. The FE-SEM of Ppy and Ppy–DNA coated onto microelectrode surface. a) Ppy doped Li-
ClO4; b) © Ppy doped LiClO4 and DNA.
P. D. Tam et al. / J. Biomedical Science and Engineerin g 2 (2009) 374-379
SciRes Copyright © 2009 Openly accessible at http://www.scirp.org/journal/JBISE/
3.4. The Hybridization of DNA Sensor
As above-mentioned, the probe-attached sensor is com-
monly soaked into solution containing target DNA. A
DNA helix sequence is formed on the surface of the
sensor when target/immobilized DNA matching oc-
Such hybridization is detected by changes in the con-
ductance of the conductive membrane on the surface of
sensors leading to the change in output signal of the sys-
tem. In Figure 5, the hybridization illustrated by linear
curve that described the relation between the target DNA
concentration and output signal of the DNA sensor. For
both APTS and Ppy/DNA attachment method, the sensor
can detect as low as 2 nM of target DNA. However, the
intensity of the output signal found to be better when
direct immobilization was used than that given by APTS.
This is explained by the contribution of Ppy and dopant
which improve the conductivity of the membrane
namely enhancing the electric charge transfer within the
Figure 5. DNA sequence hybridization curve at
room temperature, 0.05M probe DNA.
Figure 6. The detection mismatch DNA sequence at
room temperature, 0.05M probe DNA.
3.5. Detection of Mismatches DNA
Sequence Using the DNA Sensor
Mismatch detection is our first trial step for sensor selec-
tivity investigation. In this work, we used two different
mismatched sequences of Herpes virus (Table 1), and
then compared their hybridization signals with those of
fully complementary targets to investigate the effects of
the base pair mismatches. As presented in Figure 6, a
much stronger signal of DNA hybridization containing
fully matched DNA compares to system containing
mismatched DNA was clearly found. This study is still
in progress for statistic conclusion.
The influence of the mismatch positions was investi-
gated by change of the DNA single base pair mismatch
(mis/1) possessed a C (Cytosine) instead of a G (Gua-
nine) at the 7th oligo and the three base pair mismatch
sequence (mis/2) additionally contained CC instead of
AA at 12th and 13th position close to the 5’ end of the
DNA molecule. Figure 6 presents the evident decrease
of signal was observed when probe DNA was hybridized
with two mismatches. It also can be seen that, the signal
reduction of mis/2 was stronger than mis/1. From these
results, it can be deduced that the electrochemical DNA
sensor has enough high sensitivity to detect a single base
pair mismatch DNA at some of positions within the se-
3.6. Hybridization with PCR Amplified
The DNA sequence of herpes virus in real sample was
used in our work. Firstly, the DNA sequences of herpes
virus were amplified by PCR method (20µg/l) and then
divided into 2 parts: 10g/µl DNA sequence for gel
electrophoresis; 10g/µl DNA sequence for DNA hy-
bridization detection using the DNA sensor. The Figure
7 showed results of 92 base pairs fragment–amplified
by PCR method in which lance M indicates the Marker,
Figure 7. Agarose gel electrophoresis of the
PCR products amplified in the thermal cycler.
P. D. Tam et al. / J. Biomedical Science and Engineerin g 2 (2009) 374-379
SciRes Copyright © 2009 Openly accessible at http://www.scirp.org/journal/JBISE/
Figure 8. PCR amplified DNA se-
quence detected by DNA sensor.
lane 1: PCR performed in H2O Enppendorf tubes, lanes
27: the DNA template with various DNA concentra-
tions, and lanes 8, 9 are for the Herpes virus PCR prod-
ucts with 92 base pairs.
Note that, the PCR amplified sample was double
strands, thus, before the performance of DNA sequence
detection, the sample was thermally denatured at 98oC
for 5 minutes and then was quickly decreased to 50oC to
obtain the single DNA strand. Afterwards, our DNA
sensors were immerged into the cell containing the sam-
ple to detect the target. Figure 8 described the relation
between target DNA concentration and output signal of
DNA sensor at room temperature, phosphate buffer solu-
tion with 0.5 µM probe DNA. It can be seen that, output
signal of DNA sensor is linear with target DNA concen-
tration. This result went well that given by PCR method
and matched our purpose to develop the DNA sensor as
pre-diagnostic device.
This paper described the direct immobilization of DNA
strand on the surface of sensor by electrochemical
method. The DNA sensor was used to determine the
Herpes simplex virus DNA in the sample. The results
showed that, the DNA sensor can detect as small as 2
nM of herpes virus DNA concentration at room tem-
perature and the intensity of the output signal is better
than by using APTS attachment method. The influence
of mismatch DNA was determined with decrease of 50%
signal (mis/1) and 75% signal (mis/2) compares to full
matching at 12 nM target DNA concentration. We still
keep doing further research to verify selectivity and sen-
sitivity of the sensors at different measuring conditions.
The work has been supported by Ministry of Education and Training
under research project code B2008-01-175
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