Chemical Papers 69 (1) 202–210 (2015)
DOI: 10.2478/s11696-014-0599-6
ORIGINAL PAPER
Detection of short oligonucleotide sequences of hepatitis B virus
using electrochemical DNA hybridisation biosensor
Sophia Karastogianni, Stella Girousi*
Department of Analytical Chemistry, Aristotle University of Thessaloniki, Panepistimioupoli Thessaloniki 54124, Greece
Received 23 January 2014; Revised 4 April 2014; Accepted 9 April 2014
A novel, sensitive and selective electrochemical hybridisation biosensor was developed for the
detection of the hepatitis B virus (HBV) using a manganese(II) complex as electrochemical indicator
and a DNA probe-modified carbon paste electrode as the biosensor (DNA/CPE). The results showed
that this complex could be accumulated electrochemically the immobilised dsDNA layer rather than
in the single-stranded DNA (ssDNA) layer. On the basis of this, the manganese complex was used as
an electrochemical hybridisation indicator for the detection of oligonucleotides related to HBV. The
hybridisation event was evaluated on the basis of the difference between the reduction signals of the
manganese(II) complex with the probe DNA prior to and post hybridisation with a target sequence
using a differential pulse mode. Several factors affecting the immobilisation and hybridisation of
oligonucleotides as well as the indicator’s accumulation were investigated. Experiments with a noncomplementary and mismatch sequences demonstrated the good selectivity of the biosensor. Using
this approach, the HBV target oligonucleotide’s sequence could be quantified over arange from
0.22 ng L−1 to 5.40 ng L−1 , with a linear correlation coefficient of 0.9994 and the limit of detection
of 0.07 ng L−1 .
c 2014 Institute of Chemistry, Slovak Academy of Sciences
Keywords: Mn(II) complex, differential pulse voltammetry, DNA-hybridisation biosensor, oligonucleotides
Introduction
The detection of DNA hybridisation is of major importance for its application in the diagnosis
of pathogenic and genetic diseases (Castañeda et al.,
2007). Because of their remarkable sensitivity, compatibility, inherent miniaturisation and low cost, modern electrochemical DNA biosensors are extremely attractive.
The hepatitis B virus (HBV) is one of the causative
agents of viral hepatitis and infection with HBV comprises a public health problem of worldwide importance with acute and chronic clinical consequences
(Wright & Lau, 1993), causing acute and chronic hepatitis, cirrhosis and hepatocellular carcinoma (Lee,
1997). ELISA is the main method used for the detection of HBV (Moriya et al., 2002). Labels that provide
a radioactive (Jilbert, 2000), fluorescent (Park et al.,
2000) or chemiluminescent (Young et al., 2002) signal
are the markers most commonly used in detection assays. However, the short lived radioactive labels, the
licensing and safety issues associated with radioactivity, the bleaching of fluorescent markers and the irreproducible response of chemiluminescent labels (in
some cases), the need for complicated pre-treatment
techniques and expensive instruments are the main
problems associated with these techniques. Hence, alternative methods are required with lower detection
limits, higher sensitivity and selectivity, and faster responses.
Electrochemical DNA biosensors are devices that
convert the DNA hybridisation event into an analytical electronic signal to obtain sequence-specific information (Girousi et al., 2012). They can be employed
for determining early and precise diagnoses of infectious agents (Siddiquee et al., 2010a) and for moni-
*Corresponding author, e-mail: girousi@chem.auth.gr
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toring sequence-specific hybridisation events either directly (Paleček et al., 1997; Azizi et al., 2013; Yola
et al., 2014,) or by DNA intercalators (Fojta et al.,
1996; Ziólkowski et al., 2012; Du et al., 2013). Accordingly, DNA electrochemical hybridisation biosensors could be used for the detection of HBV. Some
electrochemical HBV DNA biosensors are labellingfree (Hanaee et al., 2007; Kara et al., 2007; Hassen
et al., 2008; Karadeniz et al., 2008; Caliskan et al.,
2009; Ly & Chob, 2009; Muti et al., 2011; Erdem
et al., 2012) but these biosensors usually need sophisticated or complicated pre-treatment procedures
and expensive electrode modifiers (e.g. silver or gold
nanoparticles) or expensive electrodes (e.g. CNTs).
On the other hand, most indicator-based electrochemical HBV DNA biosensors have used metal complexes
(Zhao & Ju, 2004; Li et al., 2007a, 2007b, 2008; Zhang
et al., 2007; Ding et al., 2008; Liu et al., 2008; Niu
et al., 2009) (e.g. Os, Co, Cd, Cu, etc.) that interact in a different way with ssDNA and dsDNA and
others with methylene blue (MB) (Guo et al., 2007;
Girousi & Kinigopoulou, 2010) but these compounds
are usually expensive, highly toxic and demand laborious and complicated preparation procedures, hence
they are not convenient for practical applications. By
contrast, Mn(II) complexes are much cheaper and less
harmful, but only a few of them have been used (Liu
et al. 2008) for the determination of HBV.
In a recent study, a novel, low molecular mass,
cost-effective, stable, readily prepared and with high
yield, manganese(II) complex i.e. [MnL2 (H3 tea)] (L
= thiophene-2-carboxylic acid and H3 tea = triethanolamine) (I ) was synthesised and structurally
characterised and its DNA-binding properties investigated using differential pulse voltammetry (DPV),
spectrophotometric titrations and fluorimetric competitive studies, which revealed that it was bound to
dsDNA through intercalation (Karastogianni et al.,
2013). Accordingly subsequent study uses this novel
complex as an electrochemical indicator for the determination of short HBV DNA sequences based on the
fabrication of a DNA-hybridisation biosensor based on
a carbon paste electrode (CPE). The new HBV electrochemical biosensor relied on the immobilisation of a
21-base single-stranded (ss) oligonucleotide probe on
the CPE surface and on the electrochemical (DPV)
transduction of the reaction between the short complementary HBV strand (target). The detection of hybridisation was accomplished using I as electrochemical indicator, where strong association with the immobilised double-stranded dsDNA segment led to significantly enhanced voltammetric signals. The hybridisation event was evaluated on the basis of the difference
between the reduction signals of the manganese(II)
complex with the probe DNA prior to and post hybridisation with a target sequence using differential
pulse mode (DPV).
To the best of our best knowledge, this is the first
203
use of this complex as an electrochemical indicator
in the detection of HBV DNA. This new approach is
cheap, simple, fast and has relatively good sensitivity. The results showed that the probe ssDNA immobilised on the CPE surface could be used successfully
for discriminating between a complementary target sequence and a non-complementary strand, indicating
the good selectivity of the assay. The combination of
these features renders this approach suitable as a general platform for the detection of hybridisation events
in DNA biosensors without the need for labelled sequences and a useful tool in the diagnoses of infectious
agents.
Experimental
All reagents were of analytical grade unless stated
otherwise and used as received. Dimethyl sulphoxide (DMSO) and tetra hydrate manganese(II) chloride (MnCl2 · 4H2 O) were purchased from Merck
(USA). Thiophene-2-carboxylic acid was purchased
from Aldrich (WI, USA). Triethanolamine and mineral oil were obtained from Sigma (MO, USA). Ethylene diamine tetraacetic (EDTA, ACS reagent, 99.4–
100.06 mass %) and tris (hydroxymethyl) aminomethane (99.8 mass %, ACS reagent) were obtained
from Sigma–Aldrich (USA). Graphite powder was
purchased from Fluka (USA) (50870, p.a. purity
99.9 % and particle size < 0.1 mm). [Mn(thiophen-2carboxylic acid)2 (triethanolamine)] [MnII (HL1 )2
(H3 tea)] (I ) was prepared as previously reported
(Karastogianni et al., 2013).
All the oligonucleotides related to the HBV gene
were supplied (as lyophilised powder) from Sigma–
Aldrich (USA) with the following sequences: DNA
probe (21-mer base sequence S1 ): 5-GAG-GAG-TTGGGG-GAG-CAC-ATT-3 (MM: 6595); Target DNA
(21-mer base sequence S2 ): 5-AAT GTG CTC CCC
CAA CTC CTC-3 (MM: 6265); One base mismatch
to S1 (21-mer base sequence S3 ): 5-AAT-GTG-GTCCCC-CAA-CTC-CTC-3 (MM: 6305); Non-complementary to S1 (21-mer base sequence S4 ): 5-AACGTG-TGA-ATG-ACC-CAG-TACT-3 (MM: 6443).
All oligonucleotides were dissolved in 10 mmol L−1
Tris-HCl, 1 mmol L−1 EDTA, pH 8.0 (TE buffer) and
kept frozen. In order to prepare the more diluted solutions of nucleotides, a 0.2 mol L−1 acetate buffer
solution (pH 5.0) consisting of 20.0 mmol L−1 NaCl
was applied.
Stock solutions of 3 g L−1 of I were prepared after weighing a certain amount of the compound and
dilution in dimethyl sulphoxide. All aqueous solutions
were prepared with sterilised double-distilled water.
All the experiments were performed at ambient temperature in an electrochemical cell. The electrochemical cells were cleaned with diluted nitric acid and
rinsed with sterilised double-distilled water. Ultrapure
nitrogen was used to de-aerate the solutions by purg-
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ing the dissolved oxygen for 15 min prior to each experiment.
Voltammetric experiments were carried out using a Autolab potentiostat/galvanostat and (Eco
Chimie, the Netherlands) controlled by GPES 4.9.
0005 Beta software. All electrochemical measurements
were carried out at ambient temperature, using a conventional three-electrode cell containing a platinum
wire as a counter and Ag/AgCl/3 mol L−1 KCl as reference electrodes, respectively. A carbon paste electrode of 3 mm inner and 9 mm outer diameter of the
PTFE sleeve was used as a working electrode. The pH
of all solutions was measured using a Consort C830 pH
meter (Consort bvba, Belgium).
The carbon paste electrode was prepared by thoroughly mixing by hand adequate amounts of graphite
powder and paraffin oil of 75/25 mass ratio. A portion of the resulting mixture was packed into the bottom of the PTFE sleeve. The surface was polished to
a smooth finish manually on a piece of weighing paper before use. Electrical contact was established via
stainless steel screws.
According to previous studies (Rice et al., 1983),
the electrochemical pre-treatment produces a more
hydrophilic surface and the concomitant removal of
organic layers. Hence, pre-treatment of the CPE was
performed by imposing +1.7 V vs Ag/AgCl for 60 s
in 0.2 mol L−1 acetate buffer solution (pH 5.0) containing 20 mmol L−1 NaCl without stirring.
The DNA probe oligonucleotide immobilisation on
the surface of the working electrode is an important
parameter of DNA biosensors. This factor affects the
response and performance of DNA biosensors (Siddiquee et al., 2010b). After activation of the CPE surface, the CPE was dipped in 0.2 mol L−1 of acetate
buffer solution (pH 5.0) containing 10 mg L−1 of probe
and 20.0 mmol L−1 NaCl at +0.5 V was applied for
300 s with stirring. The electrode was then washed
with deionised and sterilised water.
Hybridisation was performed by immersing the
probe-modified CPE into a stirred hybridisation solution (0.2 mol L−1 acetate buffer, pH 5.0) containing
5.00 ng L−1 of the target DNA and 20.0 mmol L−1
of NaCl for 240 s without applying any potential to
the electrode. The electrode was then rinsed with sterilised and deionised water. This procedure was applied
for hybridisation of the probe with one base mismatch
(SMB) and non-complementary sequences (NC).
Following hybridisation of probe with the target on the CPE surface, the manganese(II) complex
was accumulated on the hybrid-modified electrode by
immersing the electrode in 0.1 mol L−1 of acetate
buffer (pH 4.6) containing 10 mmol L−1 of KCl and
500 mg L−1 of I for 180 s with stirring and without
applying any potential to the electrode. The electrode
was then carefully rinsed with sterilised and deionised
water. This protocol was used for the accumulation
of I on the bare and probe-modified electrode follow-
ing hybridisation of the probe with complementary
or single-base mismatch and non-complementary HBV
DNA sequences.
The electrochemical behaviour of I was investigated by adsorptive transfer-stripping voltammetry
using the cathodic differential pulse voltammetric
mode in 0.2 mol L−1 of acetate buffer (pH 4.6) containing 10 mmol L−1 NaCl and cathodically scanning
the electrode potential between +1.2 and 0.0 V with
a modulation time of 0.07 s, an interval time of 0.6 s,
a step potential of 6 mV s−1 and a modulation amplitude of 60 mV. The raw data were treated using
the Savitzky and Golay filter (level 2) of the GPES
software, followed by the GPES software moving average base-line correction using a peak width of 0.03.
Repeated measurements were carried out following renewal of the CPE surface by cutting and polishingthe
electrode.
Results and discussion
It should be noted that, in the present work, the reduction peak of I was taken into account, because one
well-defined reduction peak was formed, as compared
with the oxidation procedure where two overlapped
oxidation peaks were present.
Detection of the target DNA was achieved by monitoring the difference between the DPV responses of
I accumulated on the DNA-modified CPE, prior to
and post hybridisation with the target DNA, Fig. 1. A
comparison of the DPV curves of the probe-modified
CPE prior to (curve 2 in Fig. 1) and post hybridisation
with the target (curve 3 in Fig. 1) showed that a significant increase in the reduction signal of I occurred due
to hybridisation of the probe with the target resulting in the probe-target hybrid formation, with which
more molecules of I interacted. The results confirmed
that the interaction mode of DNA and I is mainly
intercalation (Karastogianni et al., 2013).
Taking into consideration that the electrochemical
pre-treatment of CPE is usually required in order to
activate the surface of the electrode (Rice et al., 1983),
the effect of the electrode pre-treatment potential and
pre-treatment time on the probe immobilisation was
studied by monitoring the reduction signal of I The
results provided evidence that the activity of the CPE
reached its maximum value at +1.7 V for 60 s.
The influence of the probe immobilisation potential
was investigated. The measurement was performed
following immobilisation of the probe on the CPE at
different potentials. The results showed that imposing
a positive potential to the CPE favoured the immobilisation of the probe on the electrode and the maximum
voltammetric signal was observed for the immobilisation potential of 0.50 V (data are not shown).
The effect of the probe immobilisation time was
also explored. The results revealed that the signal of
the I signal increased as the immobilisation time in-
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205
Fig. 1. Differential pulse voltammograms for bare CPE (curve 1) without any modification and differential pulse voltammograms
of accumulated I on probe-modified CPE prior to hybridisation (20 mg L−1 probe) (curve 2) and post hybridisation with
20 mg L−1 target (curve 3). (600 mg L−1 of I accumulated on DNA-modified CPE and other experimental and voltammetric
conditions were as described in Experimental).
Fig. 2. Effect of probe deposition time (a) and plots of DPV peak current of accumulated I on probe-modified CPE after hybridisation with 5.0 ng L−1 of target (—), single-base mismatch oligonucleotide (SBM) (· · ·) and non-complementary (NC)
(– – –) vs probe mass concentration (b). (Other experimental and voltammetric conditions were as described in Experimental).
creased to about 300 s and remained constant above
that value, probably due to saturation of the electrode surface (Fig. 2a), hence 300 s was selected for
subsequent experiments. In addition, the effect of the
probe concentration was tested to obtain an optimal
probe density for discrimination of the HBV DNA.
The resulting peak currents of I vs probe concentrations are plotted in Fig. 2b, curve 1. It may be seen
that, by increasing the probe mass concentration up
to 10 mg L−1 , the ratio of DPV peak current of target
to probe signal was increased. At values higher than
10 mg L−1 it decreased to 30 mg L−1 probably because of the decrease in the number of hybrids formed
on the CPE surface due to steric hindrances (Peterson
et al., 2001). Above that value, it remained constant
due to full coverage of the electrode surface. On the
other hand, the ratio of the SBM and NC signals to
the probe (Fig. 2b curves 2 and 3) also increased up to
5 mg L−1 . In the range from 5 mg L−1 to 10 mg L−1 it
decreased. From 10 mg L−1 to 30 mg L−1 it remained
almost constant, while in the range from 30 mg L−1 to
40 mg L−1 it started to increase and after 40 mg L−1
it remained constant. Accordingly, 10 mg L−1 is proposed as a suitable probe immobilisation concentration.
In order to further improve the selectivity of the
biosensor, the effect of the hybridisation time was
tested by monitoring the reduction signal of I after
hybridisation of the probe with the target, SBM and
NC at different hybridisation times (Fig. 3).
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Fig. 3. Column graph of peak current of accumulated I on
probe-modified CPE after hybridisation with target,
single-base mismatch oligonucleotide (SBM) and noncomplementary (NC) at hybridisation time of 240 s.
(Other experimental and voltammetric conditions were
as described in Experimental).
Table 1. Hybridisation time effect on the hybridisation efficiency of the proposed biosensor
Hybridisation efficiency/%
Hybridisation time/s
60
120
180
240
300
600
900
Target
SBMa
NCa
41
51
78
140
134
123
99
–37
–13
–35
–57
–36
–30
–20
–54
–47
–42
–44
–52
–52
7
a) (–) Sign corresponds to the decrease in reduction peak current of accumulated I on probe-modified electrode.
To elucidate this, it would be convenient to use the
difference between the reduction peak current of accumulated I on the probe-modified CPE prior to and
post hybridisation with the target, SBM and NC (Table 1) whereby the hybridisation efficiency could be
calculated. This difference is also related to the above
ratio. As can be seen, the hybridisation efficiency increased with an increase in the hybridisation time for
the probe-target duplex up to 240 s and then it decreased. Meanwhile, the hybridisation efficiency decreased with the increase in the hybridisation time for
the probe-SBM duplex up to 240 s then subsequently
increased. In the case of the probe-NC duplex, the
hybridisation efficiency increased slightly up to 240 s
but only minimally when compared with that of the
target-probe duplex, then decreased after 240 s. Thus,
at 240 s the hybridisation efficiency for the target-
probe duplex achieved its maximum value, while the
probe-SBM duplex and probe-NC duplex had their
minimum values. Accordingly, 240 s was proposed as
the optimum time for hybridisation of the probe with
the target. The effect of the hybridisation potential
was also investigated after hybridisation with the target (data are not shown). The maximum voltammetric
signal was observed when no accumulation potential
was applied.
The influence of the accumulation potential was
investigated. The measurement was performed after
hybridisation of the probe with the target at different
accumulation potentials of I. The potential range was
from –0.5 to +0.5 V and the reduction peak current
of the manganese complex after hybridisation with the
target was increased from –0.5 V to the case where no
potential was applied and subsequently it decreased.
The maximum peak current was observed when no
potential was applied (data are not shown). Furthermore, the accumulation potential of I had no significant effect on the reduction peak current of I after
hybridisation with SBM and NC from –0.5 to 0.0 V
and in the case where no potential was applied, but
for more positive potentials the reduction peak current
of I after hybridisation with SBM and NC started to
increase. This behaviour means that the highest hybridisation efficiency was achieved when no accumulation potential was applied toI. It was also proved that
the manganese complex interacted with dsDNA in the
intercalative mode due to the presence of the free thiophenyl rings in the complex which can be considered as
overlapping with the π–π stacking base pairs of DNA
(Karastogianni et al., 2013). Furthermore, the current
study found that the best interaction with dsDNA occurred when no potential was applied for 300 s. It is
probably this property of the manganese complex that
causes it to interact with the hybrid duplex without
the application of any potential.
In addition, the influence of accumulation time,
potential and mass concentration of I was explored.
Thus, accumulation time was studied after hybridisation with the target, SBM and NC. The reduction signal of I after hybridisation with the target
achieved its maximum value at 180 s, while the reduction peak current of I accumulated on the probemodified CPE after hybridisation with SBM and NC
had its minimum value. Hence, 180 s was chosen for
further measurements. By increasing the accumulation time of I, the selectivity of the sensor (probably
by removing non-specifically adsorbed SBM molecules
or NC) was improved (Fig. 4a). Furthermore, the
effect of the mass concentration of I on the accumulation of I on the probe-modified CPE surface
was investigated. The maximum reduction peak current of I after hybridisation with the target was observed at 500 mg L−1 of I and the reduction peak
current of I after hybridisation with SBM and NC
was very low. Thus, 500 mg L−1 was chosen as the
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Fig. 4. Histograms related to various reduction peak currents of accumulated I on probe- modified CPE prior to hybridisation
(probe) and post hybridisation with 5.0 ng L−1 target, single-base mismatch oligonucleotide (SBM) and complementary
oligonucleotide (NC) at accumulation time of 240 s (a). DPVs of accumulated I on probe-modified CPE under selected
conditions after hybridisation with target at different mass concentrations of target: 0 ng L−1 (curve 1), 0.25 ng L−1
(curve 2), 0.30 ng L−1 (curve 3), 0.49 ng L−1 (curve 4), 0.57 ng L−1 (curve 5), 1.04 ng L−1 (curve 6) and 5.40 ng L−1
(curve 7) (b). Variation in difference between reduction peak current of accumulated I on probe-modified CPE prior to
and post hybridisation with target (∆I = itarget – iprobe ) vs target mass concentration (c) and related calibration graph at
target mass concentration range 0.22–5.4 ng L−1 (d). (Other experimental and voltammetric conditions were as described
in Experimental).
best mass concentration of I in the subsequent studies.
Finally, the diagnostic performance of the proposed DNA electrochemical biosensor was studied under the selected conditions. Hence, inset A of Fig. 4b
describes the variation in the difference between the
reduction peak current of the accumulated I on the
probe-modified CPE prior to and post hybridisation
with target versus the target mass concentration. As
can be seen from inset A of Fig. 4b the difference of
the peak current of I prior to and post hybridisation
with the target increased with increasing the target
mass concentration up to 5.40 ng L−1 . It decreased
from 5.40 ng L−1 to 12.70 ng L−1 and remained constant above that value. Thus, at 5.40 ng L−1 mass
concentration of the target, the maximum capacity of
the probe available on the electrode surface is involved
in the hybridisation event. The decrease in the peak
reduction is either due to conformational changes in
the structure of the hybrid DNA that lead to a steric
positioning of the electroactive duplex DNA residues
at the electrode surface or there is a mixture of the
free and DNA-bound complex on the electrode surface (Tabassum et al., 2005). The DPVs prior to and
post hybridisation with various concentrations of the
target are also shown in Fig. 4b. As shown in inset B
of Fig. 4b, the calibration graph is linear between 0.22
ng L−1 and 5.4 ng L−1 with a correlation coefficient
of 0.9994. The limits of detection and quantification
(cL and cQ ) were calculated by means of 3sb /a and
10sb /a respectively, and the regression equation (in
ng L−1 ): y(∆i) = (635.2 ±6.5)γ target + (436.6 ±14.1)
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Table 2. Analytical features of several electrochemical DNA hybridisation biosensors for determination of HBV
Electrode and its
specification
Method
Limit of detection
Linear range
EIS and DPV
13.25 × 10−3 g L−1
0.01–0.08 g L−1
Erdem et al. (2012)
0.70 × 10−6 g L−1
0.70 × 10−6 –
1.47 × 10−6 g L−1
Hanaee et al. (2007)
EIS and DPV
12.79 g L−1
20–60 g L−1
Streptavidin functionalised magnetic nanoparticles modified gold
electrode
CV and EIS
50 pmol of HBV DNA on
sample of 20 L
–a
SWCNTs modified PGE
DPV and EIS
2.10 × 10−7 mol L−1
50–200 g L−1
MWNT modified SPEs
DPV
96.33 nM HBV DNA on
sample of 40 mL
–a
Ag/MWCNTs modified
GCE
DPV
6.46 × 10−13 mol L−1
GCE
CV and DPV
7.0 × 10−8 mol L−1
GCE
CV and DPV
8.4 × 10−8 mol L−1
GCE
CV and DPV
1.94 × 10−8 mol L−1
GCE
DPV
3.18 × 10−9 mol L−1
GCE
DPV
7.19 × 10−9 mol L−1
GCE
CV and DPV
6.80 × 10−9 mol L−1
Chitosan-modified CPE
DPV
3.0 × 10−10 mol L−1
CPE
DPV
0.07 × 10−9 g L−1 or
1.12 × 10−14 mol L−1
SWCNT-CHIT modified
PGE
Streptavidin-modified gold
Stripping
nanoparticle SPEs
chronopoten-tiometry
GRPox integrated on
single-use PGE
Reference
Muti et al. (2011)
Hassen et al. (2008)
Caliskan et al. (2009)
Karadeniz et al.
(2008)
3.23 × 10−12 –5.31 × 10−9 Niu et al. (2009)
mol L−1
8.82 × 10−8 –8.82 × 10−7
mol L−1
1.49 × 10−7 –1.06 × 10−6
mol L−1
3.96 × 10−7 –
1.32 × 10−6 mol L−1
4.56 × 10−8 –1.25 × 10−7
mol L−1
1.01 × 10−8 –1.62 × 10−6
mol L−1
1.76 × 10−8 1.07 × 10−6
mol L−1
–a
0.22 × 10−9 –
5.40 × 10−9 g L−1 or
3.51 × 10−14 –
8.62 × 10−13 mol L−1
Li et al. (2007a)
Li et al. (2007b)
Ding et al. (2008)
Li et al. (2008)
Zhang et al. (2007)
Liu et al. (2008)
Guo et al. (2007)
This study
SWCNT = single-walled carbon nanotubes, CHIT = chitosan, PGE = pencil graphite electrode, SPEs = screen-printed electrodes,
GRPox = graphene oxide, MWNT = multi-walled carbon nanotubes, GCE = glassy carbon electrode, CPE = carbon paste
electrode, EIS = electrochemical impedance spectroscopy, DPV = differential pulse voltammetry, CV = cyclic voltammetry.
a) Not determined.
where sb and a represent the standard deviation of the
intercept and the slope of the calibration plot, respectively. The limit of detection was 0.07 ng L−1 , while
the limit of quantification was calculated and found to
be equal to 0.22 ng L−1 . The relative standard deviation measured at two level mass concentrations, i.e.
0.5 ng L−1 and 2.5 ng L−1 of the target was 5.50 %
and 5.35 %, respectively, indicating good reproducibility of the detection method. The linear range and its
detection obtained in this work are better than those
in the reported methods, as shown in Table 2. For instance, the limit of detection obtained in this work is
lower than that of 6.80 × 10−9 mol L−1 and 3 × 10−10
mol L−1 for the DNA sequence related to the HBV using MnL2 Cl2 (L = azino-di(5,6-azafluorene)-κ2-N,N )
and MB reported by Liu et al. (2008) and Guo et al.
(2007), respectively (Table 2).
Hybridisation experiments with some non-complementary oligonucleotides were carried out to assess
the selectivity of the DNA sensor. For this purpose,
SBM, NC, a mixture of them and a mixture of the
target, SBM and NC were subjected to this procedure. Fig. 5 shows that the interaction between
these non-complementary oligonucleotides and probe
(10 mg L−1 ) did not lead to any significant increase
in the reduction signal of I due to the absence of significant hybridisation between the probe and the noncomplementary DNAs (compare curves 3 and 4 with
curve 1). The presence of SBM and NC in the mixture solution had a negligible effect on the hybridisation event (compare curve 1 with curve 5). Furthermore, the reduction signals of I for the hybridised target alone (curve 2) in the presence of SBM and NC
were almost the same (compare curve 2 with curve 6).
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Fig. 5. DPVs of accumulated I on probe-modified CPE under
selected conditions prior to hybridisation (curve 1) and
post hybridisation with 5.4 ng L−1 target (curve 2),
SBM (curve 3), C (curve 4), mixture solution of SBM
and NC (curve 5) and mixture solution of target, SBM
and NC (curve 6). (Other experimental and voltammetric conditions were as detailed in Experimental).
This result indicates that only the target could form a
probe/target structure and that the presence of noncomplementary sequences in the mixture solutions had
negligible effect on the hybridisation between the immobilised probe and the target. However, the slight
decrease observed in the reduction signal of I could be
attributed to a partial interaction occurring between
the target and SBM and NC, giving rise to a slight decrease in target availability during hybridisation with
the probe.
Conclusions
The differential pulse adsorptive stripping voltammetric detection of HBV DNA on CPE using a novel
method, readily prepared in the laboratory by simple,
low-cost and user-friendly materials, manganese(II) as
electrochemical indicator, was found to be possible.
The principle of the present strategy was based on the
elevation of the peak current of I on the CPE probemodified electrode, due to hybridisation of the probe
with the fully matched target and almost no significant variation in the peak current of the modified electrode following interaction with non-complementary
HBV oligonucleotides. On the other hand, poor hybridisation efficiency represents a disadvantage for
this procedure because of the multiple sites of binding; probably most of the immobilised DNA probe is
not readily accessible for hybridisation. This drawback
was overcome following the proposed procedure where
under optimal conditions the hybridisation efficiency
was sufficiently satisfactory, since at the optimum hybridisation time (240 s) it was calculated to be 140 %
(Table 1). In addition a variety of washing solutions
such as de-ionised water, Tris-HCl buffer pH 7.0, acetate buffer etc., were tested in order to ensure the
selectivity against mismatch sequences; it was found
209
that washing with de-ionised water in every step had
the lowest effect on the stability of the hybrid thus
formed due to the desorption of the non-covalently
bound hybrid. Furthermore, the efficiency of the hybridisation of these specific sequences of the probe
with the target, SBM and NC using 7-dimethyl-amino1,2-benzophenoxazi-nium Meldola’s Blue (MDB) and
proflavine as electrochemical indicators on CPE was
previously estimated by the current team (Girousi et
al., 2010). In that study, it was evident that hybridisation occurred and was monitored by the changes in the
voltammetric response of MDB and proflavine. The
linear range and its limit of detection were better than
those of the reported methods, as shown in Table 1.
In the meantime, this new biosensor is simple, sensitive and selective within a short manipulation time. In
addition, this sensor has future potential in its application as a general platform for the detection of HBV
DNA in real samples and can serve as a useful tool in
the diagnoses of infectious agents.
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